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Review
Exploring Emergent Properties in Enzymatic Reaction Networks: Design and Control of Dynamic Functional SystemsClick to copy article linkArticle link copied!
- Souvik GhoshSouvik GhoshInstitute for Molecules and Materials, Radboud University, Heyendaalseweg 135, 6525 AJ Nijmegen, The NetherlandsMore by Souvik Ghosh
- Mathieu G. BaltussenMathieu G. BaltussenInstitute for Molecules and Materials, Radboud University, Heyendaalseweg 135, 6525 AJ Nijmegen, The NetherlandsMore by Mathieu G. Baltussen
- Nikita M. IvanovNikita M. IvanovInstitute for Molecules and Materials, Radboud University, Heyendaalseweg 135, 6525 AJ Nijmegen, The NetherlandsMore by Nikita M. Ivanov
- Rianne HaijeRianne HaijeInstitute for Molecules and Materials, Radboud University, Heyendaalseweg 135, 6525 AJ Nijmegen, The NetherlandsMore by Rianne Haije
- Miglė JakštaitėMiglė JakštaitėInstitute for Molecules and Materials, Radboud University, Heyendaalseweg 135, 6525 AJ Nijmegen, The NetherlandsMore by Miglė Jakštaitė
- Tao ZhouTao ZhouInstitute for Molecules and Materials, Radboud University, Heyendaalseweg 135, 6525 AJ Nijmegen, The NetherlandsMore by Tao Zhou
- Wilhelm T. S. Huck*Wilhelm T. S. Huck*Email: [email protected]Institute for Molecules and Materials, Radboud University, Heyendaalseweg 135, 6525 AJ Nijmegen, The NetherlandsMore by Wilhelm T. S. Huck
Chemical Reviews
Cite this: Chem. Rev. 2024, 124, 5, 2553–2582
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Abstract
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The intricate and complex features of enzymatic reaction networks (ERNs) play a key role in the emergence and sustenance of life. Constructing such networks in vitro enables stepwise build up in complexity and introduces the opportunity to control enzymatic activity using physicochemical stimuli. Rational design and modulation of network motifs enable the engineering of artificial systems with emergent functionalities. Such functional systems are useful for a variety of reasons such as creating new-to-nature dynamic materials, producing value-added chemicals, constructing metabolic modules for synthetic cells, and even enabling molecular computation. In this review, we offer insights into the chemical characteristics of ERNs while also delving into their potential applications and associated challenges.
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License Summary*
You are free to share(copy and redistribute) this article in any medium or format and to adapt(remix, transform, and build upon) the material for any purpose, even commercially within the parameters below:
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Attribution (BY): Credit must be given to the creator.
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License Summary*
You are free to share(copy and redistribute) this article in any medium or format and to adapt(remix, transform, and build upon) the material for any purpose, even commercially within the parameters below:
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Copyright © 2024 The Authors. Published by American Chemical Society
1. Introduction
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All key functions of living systems, such as metabolism, reproduction, sensing the environment, adaptation, and homeostasis, are enabled by enzymatic reaction networks (ERNs). In metabolic networks, the activities of many enzymatic reactions are finely tuned to provide responsiveness and controlled behavior. Highly complex emergent properties such as chemotaxis, (1,2) cell division, and the cell cycle (2) are all orchestrated by enzymatic networks. Signaling pathways consisting of cascades of kinases and phosphatases interlock to form complex networks that allow cells to process information from the environment and activate suitable genetic programs. (3−5)
In the field of synthetic biology, enormous progress has been made in appropriating and harnessing some of the enzymatic networks in living cells to create, for example, artificial proteolysis signaling pathways and control intracellular self-assembly. (6−8) Progress in synthetic biology has been reviewed earlier. (9,10) Here, we wish to focus on synthetic enzymatic reaction networks outside a cellular context. These in vitro systems offer the key advantage of precise control over all components. This control enables deep and detailed chemical understanding of the dynamics of ERNs, which is a prerequisite for designing systems with emergent properties. The bottom-up construction of functional ERNs could then be used to create life-like materials, where the emergent dynamics of the networks can be coupled to material properties. Ultimately, ever more complex artificial ERNs may be used in the construction of synthetic cells, composed of multiple networks at the genetic and metabolic level, all compartmentalized within a synthetic compartment.
To achieve these long-term ambitions, the field has started to develop robust methods to construct ERNs and control their temporal and spatial dynamics. In this review, we will provide an overview of the progress to date, discuss challenges ahead (Figure 1), and finish with an overview table listing all enzymes used in enzymatic reaction networks (Table 1). The structure of our review is as follows: we start in section 2 with an overview of the structure of networks by discussing topology, motifs, and kinetics, highlighting approaches to modeling the dynamic properties of ERNs as well as opportunities for computational design of functional ERNs. In section 3, we provide examples of artificial ERNs with nonlinear dynamic behavior. In section 4, we discuss various means to control enzymatic activity via external stimuli such as pH, light, magnetic fields, and sound. Combined, these design and control principles have led to progress in enzymatically powered dynamic materials with life-like properties, which are reviewed in section 5. Having established the key aspects of designing and controlling ERNs, we devote a final set of sections on potential areas of application. In section 6, we review how ERNs can be used in the cell-free synthesis of valuable compounds. In section 7, we summarize the ambitious efforts to build a synthetic cell from the bottom up and highlight new developments where the information processing typically reserved for living systems is harnessed in synthetic ERNs. We conclude the review with a brief summary of the key goals and potential bottlenecks for future research.
Figure 1
Figure 1. Overview of the topics discussed in the review. The image in “building synthetic cell” shows a cartoon representation of a synthetic cell (Credit: Graham Johnson/BaSyC consortium).
Table 1. Overview of Enzymes Mentioned in the Review
| enzymes | Enzyme Commission number | class | mode of action | section | ref |
|---|---|---|---|---|---|
| alcohol dehydrogenase (ADH) | 1.1.1.1 | oxidoreductase | ADH converts primary alcohols to aldehydes | 5.2 | (165) |
| l-lactate dehydrogenase (LDH) | 1.1.1.27 | oxidoreductase | LDH converts pyruvate to lactate and vice versa in glycolysis | 5.2 | (164) |
| l-malate NADP+ oxidoreductase (ME) | 1.1.1.40 | oxidoreductase | ME catalyzes oxidative decarboxylation of l-malate to form pyruvate (reversible) | 4.3 | (105) |
| hydroxymethylglutaryl-CoA reductase (HMGR) | 1.1.1.88 | oxidoreductase | HMGR converts 3-hydroxy-3-methylglutaryl-CoA (HMG-CoA) to mevalonic acid | 6.1 | (208) |
| glucose-6-phosphate dehydrogenase (G6PDH) | 1.1.1.49 | oxidoreductase | G6PDH converts glucose-6-phosphate to 6-phosphogluconolactone in pentose-phosphate pathway | 5.2, 5.3, and 6.1 | (164), (179), (202) |
| hydroxybutyryl-CoA dehydrogenase (Hbd) | 1.1.1.157 | oxidoreductase | Hbd catalyzes conversion of acetoacetyl-CoA to hydroxybutyryl-CoA and vice versa | 6.1 | (205) |
| l-lactate oxidase (LO) | 1.1.3.2 | oxidoreductase | LO is a FMN-containing enzyme that catalyzes conversion of lactate to pyruvate | 5.2 | (164) |
| glucose oxidase (GOx) | 1.1.3.4 | oxidoreductase | GOx catalyzes oxidation of d-glucose to d-gluconic acid by oxygen | 3.1, 3.3, 4.2, 4.5, and 5.1 | (60), (61), (78), (96), (99), (110), (139), (147), (185) |
| choline oxidase (COx) | 1.1.3.17 | oxidoreductase | COx is a flavoprotein, which catalyzes formation of betaine from choline | 5.1 | (147) |
| glyceraldehyde-3-phosphate dehydrogenase (Gap) | 1.2.1.12 | oxidoreductase | Gap converts glyceraldehyde 3-phosphate to 1,3-bisphosphoglycerate during glycolysis | 6.1 | (200) |
| pyruvate dehydrogenase (PDH) | 1.2.4.1 | oxidoreductase | PDH catalyzes the reaction of pyruvate and a lipoamide to give dihydrolipoamide and CO2 and is involved many metabolic pathways like glycolysis and TCA cycle | 6.1 | (200), (201), (203) |
| pyruvate:ferredoxin oxidoreductase (PFOR) | 1.2.7.1 | oxidoreductase | PFOR converts acetyl-CoA to pyruvate in many metabolic cycles including pyruvate metabolism, propanoate metabolism, and butanoate metabolism | 6.2 | (219) |
| d-amino acid oxidase (DAAO) | 1.4.3.3 | oxidoreductase | DAAO converts d-amino acids to 2-oxo carboxylates and is involved in d-amino acid metabolism | 4.2 | (95) |
| sarcosine oxidase (SOx) | 1.5.3.1 | oxidoreductase | SOx catalyzes formation of glycine from sarcosine by oxidative demethylation | 5.1 and 5.2 | (147), (165) |
| NADH oxidase (NoxE) | 1.6.3.4 | oxidoreductase | water-forming NoxE is a flavoprotein that specifically oxidizes NADH, not NADP | 6.1 | (199−202) |
| urate oxidase (UOx) | 1.7.3.3 | oxidoreductase | UOx is involved in the allantoin pathway and converts uric acid to 5-hydroxyisourate | 5.1 | (147) |
| catalase (Cat) | 1.11.1.6 | oxidoreductase | Cat is a peroxidase and is involved in biosynthesis of tryptophan and secondary metabolites | 5.2 | (161−163), (165), (168−170), (184), (185), (187) |
| horseradish peroxidase (HRP) | 1.11.1.7 | oxidoreductase | catalyzes oxidation of various organic substrates by hydrogen peroxide; heme containing glycoprotein with many isoforms | 3.3, 4.2, 4.5, 5.1, and 5.3 | (74−76), (97), (110), (111), (140), (178), (183), (184) |
| NiFe hydrogenase | 1.12.2.1 | oxidoreductase | NiFe hydrogenase oxidizes H2 to H+ (reversible), present in prokaryotes | 3.3 and 4.3 | (79), (106) |
| renillaluciferase (RLuc) | 1.13.12.5 | oxidoreductase | RLuc converts coelenterazine to excited coelenteramide and emits blue light | 5.3 | (176) |
| ferredoxin NADP+ reductase (FNR) | 1.18.1.2 | oxidoreductase | FNR is a flavoprotein involved in photosynthesis | 4.3 | (105) |
| Mo-dependent nitrogenase | 1.18.6.1 | oxidoreductase | Mo-dependent nitrogenase involved in nitrogen fixation, catalyzes ammonia formation from nitrogen | 4.3 | (106) |
| acetyl-CoA acetyltransferase (ACAT) | 2.3.1.9 | transferase | ACAT is present in many metabolic pathways, where it catalyzes formation of acetoacetyl-CoA from acetyl-CoA | 6.1 | (208) |
| PHB synthase (PhaC) | 2.3.1.304 | transferase | Phac catalyzed formation of Bioplastic PHB from 3-hydroxybutyryl-CoA | 6.1 | (202) |
| hydroxymethylglutaryl-CoA synthase (HMGS) | 2.3.3.10 | transferase | HMGS catalyzes formation of HMG-CoA from acetyl-CoA and acetoacetyl-CoA in the mevalonate pathway | 6.1 | (208) |
| glycogen phosphorylase b (GPb) | 2.4.1.1 | transferase | GPb breaks down glycogen to form glucose-1-phosphate and is involved in starch and sucrose metabolism | 5.3 | (179) |
| geranyl diphosphate synthase (GPPS) | 2.5.1.1 | transferase | GPPS forms geranyl diphosphate from the condensation of dimethylallyl diphosphate (DMAPP) and isopentenyl diphosphate (IPP) | 6.1 | (204), (208) |
| hexokinase (HK) | 2.7.1.1 | transferase | hexokinase phosphorylate d-hexose sugars in the presence of ATP, thus playing a very important role in glycolysis | 4.3 and 5.2 | (104), (163) |
| mevalonate kinase (MK) | 2.7.1.36 | transferase | MK phosphorylates mevalonate to mevalonate-6-phosphate | 6.1 | (208) |
| protein kinase A (PKA) | 2.7.1.37 | transferase | PKA initiates phosphorylation of serine residues present in the peptide chain | 5.1 | (145) |
| pyruvate kinase (PK) | 2.7.1.40 | transferase | PK catalyzes the last step of glycolysis by transferring the phosphate group from PEP to ADP | 5.2 | (163) |
| phosphomevalonate kinase (PMK) | 2.7.4.2 | transferase | PMK catalyzes the phosphorylation of mevalonate-6-phosphate to form diphosphomevalonate in the mevalonate pathway | 6.1 | (208) |
| esterase (Est, PLE for pig liver esterase) | 3.1.1.1 | hydrolase | Est catalyzes hydrolysis of the ester bonds of carboxyl esters | 3.1, 4.1, and 4.5 | (59), (60), (65), (93), (110) |
| phospholipase A2 (PLA2) | 3.1.1.4 | hydrolase | PLA2 catalyzes cleavage of phospholipids | 5.3 | (176) |
| acetylcholine esterase (AchE) | 3.1.1.7 | hydrolase | AchE breaks down the ester bond of neurotransmitter acetylcholine to form choline and acetic acid | 4.2 and 5.1 | (99), (147) |
| phosphatase | 3.1.3.1/3.1.3.2 | hydrolase | phosphatases dephosphorylate phosphate esters | 5.1 | (139), (141) |
| amyloglucosidase (AMG) | 3.2.1.3 | hydrolase | AMG breaks down starch to glucose, hence involved in starch metabolism | 4.5 | (110) |
| lactase | 3.2.1.108 | hydrolase | lactase catalyzes the conversion of lactose to galactose and glucose | 5.3 | (183) |
| aminopeptidase (Ap) | 3.4.11.2 | hydrolase | catalyzes cleavage of N-terminal amino acids from peptides and is involved in glutathione metabolism | 3.2 | (67) |
| chymotrypsin (Cr) | 3.4.21.1 | hydrolase | Cr is a serine protease that cleaves peptides on the C terminal of phenylalanine, tyrosine, tryptophan, and leucine amino acids | 3.2 and 5.1 | (70), (73), (146) |
| trypsin (Tr) | 3.4.21.4 | hydrolase | Tr is a serine protease that cleaves peptides on the C terminal of arginine and lysine amino acids | 3.2, 4.1, 4.4, and 5.2 | (42), (67−73), (86), (89), (146) |
| elastase (Els) | 3.4.21.36 | hydrolase | elastase breaks down elastin (responsible for the elasticity of connective tissue) and cleaves after glycine, alanine, and valine amino acids | 3.2 | (73) |
| proteinase K | 3.4.21.64 | hydrolase | proteinase K is a serine protease with a broad spectrum of cleavage site preferences | 5.3 | (175) |
| matrix metalloproteinase 2 (MMP 2) | 3.4.24.24 | hydrolase | MMP 2 is an endopeptidase and cleaves collagens type IV, V, VII, and X; also known as gelatinase A | 5.1 | (145) |
| matrix metalloproteinase 9 (MMP 9) | 3.4.24.35 | hydrolase | similar to MMP 2; also known as gelatinase B | 5.1 | (17) |
| urease (Ur) | 3.5.1.5 | hydrolase | catalyzes the hydrolysis of urea to carbon dioxide and ammonia, which basifies the solution | 3.1, 4.1, 4.2, 5.1, and 5.2 | (51−54), (57−59), (61), (93), (97−99), (110), (157−160), (171), (185), (187), (189) |
| apyrase | 3.6.1.5 | hydrolase | apyrase hydrolyses di- and triphosphate nucleotides to monophosphate nucleotides | 5.3 | (179) |
| oxaloacetate acetylhydrolase (OAH) | 3.7.1.1 | hydrolase | OAH breaks down oxaloacetate to form oxalate and acetate | 6.2 | (219) |
| pyrophosphomevalonate decarboxylase (PMD) | 4.1.1.33 | lyase | PMD catalyzes the last step of the mevalonate pathway, where it converts diphosphomevalonate to isopentenyl diphosphate | 6.1 | (208) |
| carbonic anhydrase (CA) | 4.2.1.1 | lyase | CA catalyzes the equilibrium between carbon dioxide and carbonic acid | 4.3 | (105) |
| fumarase (FumC) | 4.2.1.2 | lyase | FumC converts malate to fumarate (reversible) and is involved in the TCA cycle and pyruvate metabolism | 4.3 | (105) |
| limonene synthase (LS) | 4.2.3.16 | lyase | limonene synthase catalyzes limonene formation from geranyl diphosphate | 6.1 | (200), (208) |
| pinene synthase | 4.2.3.121 | lyase | pinene synthase catalyzes the conversion of geranyl diphosphate to pinene | 6.1 | (200) |
| l-aspartate ammonia-lyase (AspA) | 4.3.1.1 | lyase | AspA converts l-aspartate to fumarate (reversible) and is involved in amino acid metabolism | 4.3 | (105) |
| isopentenyl pyrophosphate isomerase (IDI) | 5.3.3.2 | isomerase | IDI catalyzes the conversion of IPP to DMAPP | 6.1 | (208) |
| phosphoglucomutase (PMG) | 5.4.2.2 | isomerase | PMG catalyzes the isomerization of glucose-1-phosphate to glucose-6-phosphate and is involved in many metabolic pathways including glycolysis and the pentose phosphate pathway | 5.3 | (179) |
| acetate-CoA ligase (ACS) | 6.2.1.1 | ligase | ACS catalyzes acetyl-CoA formation from acetate and CoA in the presence of ATP | 6.2 | (219) |
| pyruvate carboxylase (PYC) | 6.4.1.1 | ligase | PYC catalyzes the carboxylation of pyruvate to form oxaloacetate in the TCA cycle | 6.2 | (219) |
| cytochrome C oxidase | 7.1.1.9 | translocase | cytochrome C oxidase catalyzes the translocation of hydrons and is involved in oxidative phosphorylation pathways | 4.2 | (95) |
| F-type ATP synthase | 7.1.2.2 | translocase | F-type ATP synthase forms ATP from ADP and phosphate (Pi) | 7.1 | (227), (228) |
2. Structural Principles of Enzymatic Networks
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Despite a huge variety in enzymes and enzymatic reactions, many of the enzymatic reaction networks (ERNs) found in living cells feature recurring topologies and motifs. By focusing on these patterns, it becomes clear how certain functionalities arise from specific groupings of enzymes, and similar or alternative networks can be identified.
2.1. Network Topology
At first sight, the enzymatic networks encountered in living systems show an overwhelming complexity. However, statistical network analysis shows that these networks possess a strongly structured topology, where network topology refers to the large-scale logical structure of a network and the statistical properties of its connections. (11) For example, the full metabolism is divided into strongly connected modules and pathways that each have their own function (a schematic example is shown in Figure 2a). (12,13) This modularity is reflected in the statistical description of interactions in ERNs. Generally, these are found to follow scaling relationships (Figure 2b). (14−16) The distribution of reactions per enzyme follows an exponential scaling distribution. This means that many types of enzymes only promote one or a few reactions, while a few enzymes promote many reactions. This characteristic distribution makes the network more robust to failure while remaining efficient. Identifying the most common network topologies for specific functionalities and the most and least prominent enzymes in those networks can lead to insights into plasticity and redundancy features of specific network topologies, (17) which can be exploited by ERNs to function in a range of different environments. (18) A downside to this statistical approach to ERNs is that it only gives a large-scale picture, neglecting any heterogeneity introduced by different types of enzyme reactions and dynamics, and does not distinguish between substrates and effector molecules. Additionally, it does not translate well to the analysis of artificial ERNs, as these networks are often too small to warrant a proper statistical treatment.
Figure 2
Figure 2. (a) A schematic representation of the modular organization of enzymatic networks in the cell. Strongly connected modules with their own specific functions share limited connections to different modules. In this network representation, enzymes are represented by nodes and substrates by edges. (b) An example of typical scaling relationships found in the connectedness of enzymes. Here, dots represent types of enzymes, with colors denoting different modules. k represents the number of connections an enzyme has in the ERN, while P(k) represents the frequency of that number of connections occurring. Inside cellular ERNs, these follow a power-law relationship P(k) ≈ k–1. (c) Examples of network motifs often encountered in ERNs. Again, enzymes are represented by nodes and substrates by edges. Edges with arrows indicate a positive interaction (e.g., a substrate that can be used as a reactant), while a flat end indicates an inhibitory effect. (d). Example of the difference in reaction velocity v as a function of substrate concentration between an enzyme with Michaelis–Menten (MM) kinetics and Hill-type kinetics with substrate affinity KM and turnover number kcat. (e) (Left) Example schematic of a full ERN where all interactions are included. (Right) A reduced model form where only essential interactions are maintained. (f) (Left) Bayesian analysis results in parameter estimate probability distributions instead of point estimates, allowing for nonsymmetric errors and standard deviations. (Right) Bayesian estimates can be used to generate a full probabilistic picture of possible enzymatic behavior.
2.2. Network Motifs
While ERN topology analysis can provide us with information on the dynamics of large and complex systems, zooming in to smaller subunits can help us gain a better understanding of how certain properties arise from the combination of just a few enzymes. Motifs are the smallest functional units, meaning that an individual motif is capable of showcasing complex behavior such as oscillations, adaptation and memory, amplification, and filtering. (19) A classic example is the emergence of ultrasensitivity in a simple network containing a forward and backward reaction catalyzed by two different enzymes and the emergence of adaptation by inhibitory feedback between two different enzymes converting the same substrate, both key motifs in signaling networks. In their classic paper, Goldbeter and Koshland analyzed the kinetics of such systems and explored the control parameters which would show sensitivity. (20,21) In 1997, Barkai and Leibler showed how robust adaptation results directly from network connectivity. (22) Milo and co-workers generalized the concept of these small subnetworks and identified network motifs across a range of different networks─repeated patterns of interactions occurring in large and complex networks at a higher rate than a random network with equivalent topology. (23) Examples of these include simple cascades, positive or negative feedback loops, or bifans (see Figure 2c). Interestingly, network motifs can vary dramatically between different types of networks but may remain largely the same when different networks have the same type of functionality. For example, gene regulation, neurons, and logic circuits often contain feed-forward loops and bifans to enable rapid switch-like behavior, which are different from those found in food webs, electronic circuits used for arithmetic, and social networks. Similarly, in ERNs, different networks with similar behavior are found to often have the same motifs. (24) This can be clearly seen in the design of enzymatic oscillators, which almost exclusively involve delayed negative feedback loops, although more complex motifs may be involved as well. (25) More recently, researchers have shown how different large network topologies can lead to the same effective network motifs capable of, for example, homeostasis and how descriptions of larger networks can be reduced to smaller functional motifs. (26) Network motifs can be used as a starting point for the design of specific behavior, inspired by either behavior shown in already existing ERNs (27,28) or recreating network motifs not found in ERNs but in other types of networks.
2.3. Enzyme Kinetics
Topology and motifs constrain what types of behavior are possible in ERNs, but the kinetics of enzymatic reactions ultimately determine what will happen and how fast. Many enzymatic reactions are traditionally characterized by Michaelis–Menten (MM) kinetics. Michaelis–Menten kinetics assume a two-step mechanism, where the binding of the enzyme to the substrate is reversible and the release of product is not. While a number of assumptions underlie MM kinetics, it is found to be generally applicable even when some of those assumptions do not hold. In the case of cooperative binding, the Michaelis–Menten equation can be extended to the Hill equation (Figure 2d). For multisubstrate enzymes and inhibitory effects, further extensions of the MM equation are possible.
In recent years, further improvements and replacements to MM kinetics have been developed. For example, Piephoff et al. established a generalized form of the Michaelis–Menten equation by explicitly incorporating nonequilibrium effects. (29) They took into account how different enzyme conformations impact the speed of the reaction and automatically corrected for different types of substrate binding and allosteric effects. Alternatively, Rowher et al. have shown that the reversible Hill equation is a good general approximation for a large variety of mechanisms found in enzyme kinetics. (30)
However, kinetics data for individual enzymes or complete ERNs are often limited. Consequently, the data used to obtain kinetic parameters can be insufficient to obtain correct estimates. To warrant against overfitting of data, care should be taken to choose equations that do not contain an excess of parameters. (31) This can be checked for by performing sensitivity analyses on each of the parameters in the kinetic model if a large network proves to be partially unidentifiable; a viable reduced-form network description might still be obtained. By determining which interactions and variables are sloppy and subsequently removing those from the network model, a reduced description shows only those enzymes and interactions that influence the network behavior (schematically shown in Figure 2e). (32) This method has been used effectively to obtain efficient descriptions of cell-signaling networks (33) but has yet to be used for artificial ERNs.
Alternatively, so-called Bayesian inference techniques can be used for parameter estimation. (34) Here, one can use prior knowledge, such as the physically allowed range or probabilistic estimates obtained from previous measurements, to constrain parameter estimations and obtain probabilistic uncertainty intervals and predictions (Figure 2f). (35) Data with non-normal, or unknown, noise distributions can also be incorporated with relative ease by including additional measurement noise terms in the likelihood function. Bayesian inference methods allow for much more accurate quantification of the uncertainties remaining in a model fit. (36) Hierarchical Bayesian models can be used to correctly combine data from different experiments and measurement techniques, taking into account different accuracies, noise profiles, and different subsets of observable parameters, and can even be used to compare the likelihood of different reaction mechanisms. (37)
More recently, techniques to check the identifiability of parameters in enzymatic networks directly from equations have become available. These require the construction of so-called sensitivity matrices directly from the kinetic models in combination with experimental data. (38) When parameters are unidentifiable from a single-input experiment alone, further analysis is required to check if changing the experimental conditions can improve parameter identifiability. (39) More recently, a new approach has been developed to calculate optimal experimental designs for parameter identification. (40) This method uses pulse patterns for input substrates in flow to decorrelate kinetic parameters and improve identifiability. (246) Alternatively, work has been done to decompose a full ERN into separate submodules which are individually identifiable. (41)
Choosing the right analysis method to obtain an accurate kinetic model depends on both the nature and the complexity of the system and the goal behind it. If the goal is optimizing for specific reaction products, it requires a different amount of knowledge than when the goal is to achieve a fundamental understanding of all interactions in an ERN. As shown by the recent developments discussed above, structural identifiability analysis and (Bayesian) parameter estimation highlight the importance of proper uncertainty quantification in increasingly large and complex ERNs.
3. Complex Nonlinear Behavior in Artificial ERNs
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As discussed above, nonlinear dynamics introduced by positive (autocatalytic) or negative (inhibitory) feedback loops are key characteristics of networks exhibiting complex properties. In contrast to the large metabolic and signaling networks found in living systems, synthetic enzymatic networks with complex behavior are typically based on one or just a few feedback reactions. Figure 3a illustrates the general principle behind the design of artificial ERNs with complex nonlinear dynamic behavior: a nonlinear core reaction motif is chosen and combined with additional processes; the actual behavior of this combination is then dependent on the type of reactor that is used. Although the additional processes are typically dependent on the enzymes chosen for the core motif and thus the two are often intertwined, we explicitly separate the two to aid the reader in appreciating the network design and analyzing their dynamics.
Figure 3
Figure 3. Design of artificial ERNs with complex nonlinear dynamic behavior. (a) General design principle for ERNs with complex dynamic behavior. (b) Central reactions and motifs providing core nonlinearity. (c) Additional processes serving to alter the kinetics of the core motifs and connect different nodes. (d) Types of reactor or setup. (e) Examples of complex nonlinear dynamic behavior available in artificial ERNs. Illustrations: (e7) 50 μm polyacrylamide beads with immobilized urease in a solution containing 5 mM acetic buffer, 50 mM urea, and fluorescent dye SNARF-5,6, scale bar 100 μm, own data; (e8) redox forms of the dye ABTS in the solution containing glucose oxidase (GOx) and horseradish peroxidase (HRP). Adapted with permission from ref (78). Copyright 2018 Springer Nature.
The full toolbox of nonlinear core enzymatic reactions described in the literature to date is presented in Figure 3b. The use of autocatalytic core reactions is prominent not only in enzymatic networks but also in organic and DNA networks, (42) and the design principles of autocatalytic networks were recently reviewed by Semenov and Plasson et al.. (43,45) The exploitation of feedback in the engineering of ERNs was reviewed by Bánsági and Taylor. (44,45) The three general ways to autocatalysis in ERNs are shown in Figure 3b1–b3. Figure 3b1 shows autocatalysis by self-activation of an inactive zymogen, with the trypsinogen–trypsin (Tg–Tr) pair as a well-known example discussed in detail in section 3.2. Product autoactivation (Figure 3b2) is another very general mechanism with examples mostly involving urease (section 3.1). The cleavable inhibitor positive feedback is relevant for proteolytic networks (see section 3.2) and shown in Figure 3b3. As we discuss later, individual autocatalytic steps are not a general prerequisite to complex behavior, and autocatalysis can also emerge from closed cycles where the separate steps are linear. (242) The alternative nonlinear cores, namely, those built on substrate competition (Figure 3b4) and switching between enzyme redox forms (Figure 3b5), also deliver nonlinear dynamics, as discussed in section 3.3 using the example of oxidoreductases. Additionally, there are three types of nonlinear cores that are somewhat underexploited. Product inhibition (Figure 3b6), an example of which is the autoinhibitory catalysis of esterase, can be a nonlinear core but has only been found as an additional process in networks comprising a urease core (see section 3.1). The enzyme oligomerization (Figure 3b7) and allosteric effects (Figure 3b8) are well-known ways to achieve nonlinearity, but they have only been demonstrated in theoretical studies and in in vivo networks. (20,21)
Combinations of the core reactions and additional processes yield a range of possible complex behaviors as shown in Figure 3e. While some types are more common and relatively straightforward to obtain (for instance, clock behavior (Figure 3e1) or pulse-like response (Figure 3e2)), some other types are rare (chaos as shown in Figure 3e6 is, to the best of our knowledge, only obtained in peroxidase–oxidase reaction (section 3.3)).
In order to provide the reader with a readily accessible overview, we grouped the synthetic ERNs with complex nonlinear behavior according to the central enzymatic reaction around which the network is built. For all of the discussed networks, we refer to the motifs they contain and behavior types they produce, as per Figure 3.
3.1. Networks Based on the Urea–Urease Reaction
In general, enzymes producing acid or base are a fruitful ground for designing systems with complex dynamics due to the nonlinear kinetics intrinsic to pH-reactive systems. (48−50) A simple and well-studied enzymatic network that produces nonlinear behavior is the urea–urease network. The enzyme urease (Ur) is found in many bacteria and plants. It catalyzes the hydrolysis of urea into carbon dioxide and ammonia, resulting in an increase in pH. The enzyme has a standard bell-shaped activity–pH profile with an optimum around 7.5. (51) When starting from a lower pH (usually buffers of pH 3.5–5.0 with low buffer capacity), the enzyme basifies the solution, thereby overcoming the buffer and increasing its own activity, up to pH 7.5. As the reaction proceeds further to even higher pH values, the Ur activity drops again and the final pH of about 8.5 is that of ammonia/ammonium buffer. This process creates an autocatalytic sigmoidal signature in pH vs time, and the time of the steep transition from low to high pH state is addressed in the literature as “clock time” (Figure 3e1). (51,52) The increase in Ur activity due to the Ur reaction is an example of product autoactivation (Figure 3b2).
Compartmentalization and coupling to diffusion enrich the range of complex behaviors obtained in Ur networks. Effectively, the simple combination of the Ur nonlinear core (Figure 3b2) with transport phenomena (Figure 3c4) yields five types of complex behavior (Figure 3e1–e7), particularly because diffusion coefficients for H+ and other species are very different. Ur-loaded millimeter-sized particles exhibit quorum-sensing-like autoactivation (similarly to Figure 3e7), which is a consequence of autocatalysis and diffusion front propagation. (53) Miele et al. demonstrated the collective synchronized behavior of Ur-loaded microvesicles. (52) Muzika et al. showed sustained oscillations in a flow reactor with differential influx of urea and H+, (54) building on earlier work on bistability, (55) and noise-induced irregular oscillatory switching. (56) Oscillations were also obtained in a system with Ur-loaded lipid vesicles by combining the clock reaction inside of the vesicle with the influx of urea and H+ from the bulk phase. (57) Finally, oscillations in packed arrays of Ur-loaded beads with influx of urea and acid from the bulk solution were predicted theoretically but not yet experimentally proven. (58)
An alternative approach to obtain richer dynamic behavior from the urea–urease network is combining it with antagonistic acidifying enzymes (Figure 3c3). Heinen et al. showed that a urea–urease/ester–esterase network produces pulse-like pH responses under batch conditions with, first, a Ur activity-related increase followed by an esterase (Est) activity-induced decrease in the pH (see Figure 4d for network topology). The kinetic profile of the response can be finely tuned by changing the buffer and substrate composition. (59) Compartmentalization of Ur and Est units in a two-layered gel aided in programming of transient acidic pH flips. (60) This latter work also addressed the lack of nonlinearity of the Est response (as compared to Ur), which originates in the shape of the Est pH–activity profile and its autoinhibitory nature (Figure 3b6). An alternative acidifying enzyme is glucose oxidase (GOx), converting glucose to δ-gluconolactone, hydrolyzing spontaneously into gluconic acid. Wang achieved dynamic pH switching in response to the addition of urea and glucose in a system with Ur and GOx incorporated in pH-responsive polymersomes. (61) While Ur is very fast, all of the acidifying enzymes explored thus far exhibited certain limitations on rates. We hypothesize that coupling the urea–urease network to faster acidifying counterplayers would yield more robust dynamic behavior and possibly oscillating systems. The pH transition caused by the Ur-based ERNs can be coupled to a wide range of chemical and physical processes, (62) such as polymerization and sol–gel transitions, (59,63) gel growth, (64,65) and precipitation of CaCO3, (66) that all can serve as handles for functionalities as discussed in section 5.
Figure 4
Figure 4. Overview of enzymatic reaction networks that are controlled by different external stimuli. (a) Schematic representation of the trypsin oscillator where the enzyme activity is regulated by a photocleavable inhibitor. Adapted with permission from ref (86). Copyright 2020 John Wiley and Sons. (b) Photoswitchable inhibitor that can reversibly control the enzyme activity in an out-of-equilibrium system using light. Adapted with permission from ref (88). Copyright 2020 licensed under CC 4.0 American Chemical Society. (c) DASA-based polymersomes enable reversible control over the accessibility of substrate toward the active site of an enzyme by light. Adapted with permission from ref (93). Copyright 2022 licensed under CC 4.0 Springer Nature. (d) Ur–Est-based network where Ur increases the pH and Est decreases the pH. Adapted with permission from ref (65). Copyright 2021 John Wiley and sons. (e) Haber–Bosch process where NH3 can be generated from H2 and N2 using enzymes, hydrogenase, and nitrogenase by applying an electrochemical potential. Adapted with permission from ref (106). Copyright 2017 John Wiley and Sons. (f) Cascade network that is spatiotemporally controlled by sound. Adapted with permission from ref (111). Copyright 2022 licensed under CC 4.0 Springer Nature.
3.2. Networks Based on Proteases
A general route to autocatalysis is to exploit the formation of certain proteases from their inactive precursors (zymogens or proenzymes), as shown in Figure 3b1. Semenov et al. constructed a bienzymatic oscillatory network with a core reaction of trypsin (Tr)-mediated trypsinogen (Tg) activation modified with a delayed negative feedback loop via an aminopeptidase-activated trypsin proinhibitor (combination of motifs Figure 3b1, 3c1, and 3c2; full network topology is depicted in Figure 4a). (67) Further investigations of this trypsin oscillator included studies on the boundary conditions of the oscillatory regimes using a range of slightly different inhibitors in the negative feedback loop (68) and synchronization with external temperature oscillations. (69) Exploiting protease zymogen activation as a core step, Helwig et al. demonstrated adaptation to an external stimulus in an incoherent type 1 feed-forward loop network motif, comprising the chymotrypsinogen–chymotrypsin (Cg–Cr) pair and Tr. (70) The network responded with a peak to a persistent external stimulus (influx of Tr) and adapted over time, bringing the response Cr activity close to the original baseline, a behavior type in Figure 3e3. The possibility to use autocatalytic trypsinogen activation for signal enhancing was demonstrated in a diffusion-reaction system with trypsin and its inhibitor in a gel. (71) Finally, Kriukov et al. showed in a Tr–Tg network with an additional inhibition step that the combination of autocatalytic activation and flow leads to hysteresis, and the inhibition can be used to control the network dynamics in a history-dependent manner. (72)
A different and novel type of autoactivation in enzymatic systems was introduced by Pogodaev et al. using slow substrates (cleavable inhibitors) for proteinases Tr, Cr, and elastase (Els), as shown in Figure 3b3. (73) The reactions started in highly inhibited states, where the slow substrates occupied the active sites of the enzymes. With time, the peptides were cleaved into low-inhibitory fragments, thereby liberating more of the free enzymes, leading to an autocatalytic increase in enzyme activity. It was also demonstrated that in mixtures with Cr, Tr, and their corresponding slow substrates, crosstalk occurred, while Cr and Els remained orthogonal under these conditions, which opens roads to modular construction of modified networks. By converting a cleavable peptide inhibitor into a phosphate-modified proinhibitor, the behavior of the network was coupled to the activity of alkaline phosphatase, which acted as an initiator of the flip response in Cr activity. This is another example of using the motif of proinhibitor activation (Figure 3c1 and 3c2)) to connect behaviors of two enzymes. (73)
3.3. Networks Based on Oxidoreductases
In oxidoreductase networks, the nonlinearity often originates from the switching between redox and coordination forms of the enzymes (Figure 3b5).
The earliest example of an in vitro enzymatic oscillatory reaction is the peroxidase–oxidase (PO) reaction, first described by Yokota and Yamazaki in 1965. (74) Oscillations occur during the horseradish peroxidase (HRP)-catalyzed oxidation of NADH (reduced nicotinamide adenine dinucleotide) by oxygen in the presence of additives such as methylene blue and 2,4-dichlorophenol in a semibatch stirred reactor with an influx of NADH and diffusion of oxygen from a gas mixture and no outlet as shown in Figure 3d3. The PO reaction has a complex mechanism comprising multiple redox and coordination forms of the heme cofactor of HRP and very rich dynamics that include normal, period-doubled, and mixed-mode oscillations, quasi-periodicity, and chaos, shown in Figure 3e5 and 3e6. (75,76) It is hypothesized that many more new dynamic states of the PO reaction are still to be discovered using experimental parameter combinations that go beyond the current regimes, with the pH being an especially important control parameter. (76) We refer the reader to more specialized literature for further details. (75,77)
Recently, HRP was combined with GOx to produce tunable pulse responses in a stirred reactor (Figure 3e2) and form spatiotemporal patterns (Figure 3e8) in a nonstirred thin layer experiment. Remarkably, this example of complex dynamics is not comprised of individual steps of autocatalytic activation or inhibition, with the nonlinearity originating only in delayed feedback loops and substrate competition (Figure 3b4). (78) A unique aspect of the behavior of this network is the spontaneous formation of patterns of convection flows when not stirred.
Another enzyme exhibiting autocatalytic kinetics is hydrogenase, a metalloenzyme catalyzing the reaction H2 = 2H+ + 2e–. The mechanism of autocatalysis differs from the simple acid–base activation/deactivation shown for urease and is thought to be connected to redox forms. (79) The hydrogenase reaction has been observed to produce reaction fronts and oscillations in a thin layer reaction–diffusion experiment via a mechanism that is not yet fully understood. (80)
3.4. Summary
In summary, synthetic in vitro ERNs with complex behavior have exploited a limited set of core reactions but demonstrated a large range of types of complex behavior. As the recent finding with hydrogenase demonstrates, identifying novel experimental realizations of theoretical network motifs is still a fruitful area of research. Expanding the types of enzymes and combining multiple enzymes into more complex networks are necessary to broaden the scope of accessible network motifs and to realize the theoretical motifs not found in real networks yet. Currently, only two out of seven EC classes of enzymes (oxidoreductases and hydrolases) are used as core reactions in complex ERNs.
An important open question is the design of an ERN which would sustain oscillations under batch conditions (stirred or nonstirred), an enzymatic analogue of the Belousov–Zhabotinsky (BZ) reaction. (247) The oscillators described in the sections 3.1–3.3 rely on fluxes of reagents (by means of flow reactors or transport processes between compartments). In contrast to the BZ reaction, all enzymatic networks reported thus far consume all “fuel” in a single reaction cycle under well-stirred conditions, thus making it impossible to sustain oscillations in batch. In general and as highlighted by the nonautocatalytic HRP/GOx network example, it is still an open fundamental question what network motifs are required to yield a certain desired type of nonlinear behavior. A strong coupling between experimental and theoretical studies is needed in this regard, especially when incorporating diffusion and other transport phenomena.
4. Influence of External Stimuli on ERNs
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It is difficult to engineer the properties of complex enzymatic reaction networks as their dynamic output requires all reaction rates to be adjusted to each other, while the formation of byproducts or the emergence of hidden interactions could prevent or disrupt the dynamic behavior of the system. (81) Fine-tuning rates by controlling the activity of individual enzyme activities is therefore desired. Multiple methods were developed to gain control over enzyme activity by applying an external stimulus such as light, pH, redox potential, heat, magnetic field, and sound. (81−83) In this section, we will focus on the use of external stimuli to control enzymatic reaction networks that consist of two or more enzymes.
4.1. Light
Light irradiation in combination with photocleavable or photoswitchable molecules allows for selective, rapid, and spatiotemporal control over enzyme activities. (81,84,85)
Pogodaev et al. used ultraviolet (UV) irradiation of a photocaged irreversible inhibitor to obtain control over the oscillations of the Tr oscillator discussed in section 3.2 and presented in Figure 4a. (86) The oscillations were dependent on the duration and timing of UV light, demonstrating that external stimuli can introduce extra complexity without disrupting the rest of the system. However, for many enzymatic systems it is desirable to obtain reversible control over the enzymatic activity. The groups of Feringa, König, and Szymanski have reported many examples where photoresponsive molecules are used to reversibly inhibit specific enzymes upon light irradiation. (83,85,87) For this, photoswitchable molecules such as azobenzenes, spiropyrans, diarylethenes, and donor–acceptor Stenhouse adducts (DASAs) could be used. (85)
The method to use photoswitchable molecules in combination with light is known as the chromophore-warhead strategy, where one of the two conformations acts as a stronger inhibitor. The warhead, a functional group that is known to inhibit the enzyme, can be covalently attached to a chromophore that can change their 3D structure upon light irradiation. (85) Using this strategy, Teders et al. recently designed an azobenzene-based inhibitor for Tr and Cr. (88,89) The activity of these enzymes could be reversibly adjusted under flow conditions upon different light intensity, duration, and wavelength of light, see Figure 4b. These systems exhibited an ultrasensitive response, illustrating potential for using light as an input for controlling complex dynamics in ERNs.
In addition to directly photoswitching molecules that interact with the enzyme (via the active site or allosteric interactions), one could alter the kinetics by controlling the accessibility of the substrate toward the enzyme, as demonstrated by encapsulating enzymes in vesicles or polymersomes and controlling the transport of substrate using light. (90−93) The latter example, presented in Figure 4c, was based on polymersomes containing a DASA dye in the polymer shell, allowing switching between semipermeable states. (93) These DASA polymersomes were filled with Est and then mixed with Ur containing polymersomes that were permeable and light insensitive. Upon green light irradiation, the Est–DASA polymersomes became semipermeable, which resulted in the conversion of ethyl acetate into acetic acid by Est. A concomitant pH decrease from pH ≈ 8 to pH ≈ 7 activated urea hydrolysis by Ur, which resulted in a pH rise. The pH increase by Ur resulted in the deprotonation of a pigment that absorbs green light in the protonated form. Therefore, the absorption of green light stopped, and the Est activity could be reactivated.
4.2. pH
All enzymes have a specific pH value at which their activity is maximized. (94,95) In pH-dependent networks, the pH can be altered by addition or generation of acid or base by another enzyme. Often used combinations contain Ur, Est, GOx, or HRP as sources of acid or base. (48,59,96) As described by Che et al., the addition of chemical fuel can influence the biocatalysis of pH-sensitive polymersomes loaded with HRP or Ur. (97) The polymersomes shrank in a high-pH buffer due to deprotonation, whereby they became impermeable and thereby inactivate the enzyme. Addition of hydrochloric acid (HCl) and urea (fuel) resulted in a decrease of pH, swelling of the polymersomes, and activation of the enzymes. The depletion of urea resulted in shrinkage of the polymersomes and a decrease in the enzymatic activity. The activation cycle could be repeated multiple times by addition of acid and fuel to create a pH-dependent system. Maity et al. constructed an Ur/Est loop by encapsulating Ur and Est in different hydrogel beads that formed pH fronts. (65) As described in section 3.1, the conversion of urea by Ur results in a pH increase which is counteracted by the conversion of ethyl acetate into acetic acid by Est, see Figure 4d. The same pH-dependent enzymatic reaction network was used to create enzymatic logic gates. (98)
Wang et al. developed a DNA-based hydrogel that contained GOx, acetylcholine esterase (AchE), and Ur to alter the pH. (99) GOx and AchE activity resulted in a decrease in pH, while the hydrolysis of urea by Ur increased the pH. Different enzyme compositions allowed control over the pH and thereby the stiffness of the hydrogel. Control over enzymatic batch processes was obtained with acid-producing enzymes and poly(methacrylic acid) (PMAA)-functionalized gold particles. First, these particles were dispersed in the reactor, and reaction-induced pH changes were allowed to take place. When the buffer capacity was exceeded, protonation of the PMAA led to aggregation of the functionalized gold nanoparticles, which could be resuspended upon addition of fresh buffer and substrate. (100) Instead of changing the pH of the bulk solution, Zhang and co-workers showed that the enzyme kinetics of immobilized enzymes could also be influenced by pH changes in the microenvironment. (95) The local pH around cytochrome C, which is most active under acidic conditions, could be lowered by immobilization on negatively charged high-density polyelectrolytes that can attract ions of opposite charge. The throughput of a cascade with immobilized cytochrome C and d-amino acid oxidase was increased 10-fold by lowering the local pH of cytochrome C in an alkaline environment where d-amino acid oxidase is most active.
4.3. Electrochemical Control
The redox potential can be used as an external stimulus to control the activity of enzymes. (101−103) Mallawarachchi et al. showed that the accessibility of the substrate toward the active site of hexokinase (HK) entrapped in an electroresponsive hydrogel was altered upon application of an electrochemical potential due to the reversible contraction and expansion of the hydrogel. (104) The reaction kinetics of enzymes can also be modified by trapping enzymatic cascades in a porous conducting metal oxide electrode material. This was done by Morello et al. to reversibly recycle a nicotinamide cofactor that was generated with an enzymatic cascade consisting of ferredoxin NADP+ reductase, l-malate NADP+ oxidoreductase, fumarase, l-aspartate ammonia-lyase, and carbonic anhydrase. (105) It was shown that the reaction direction and rate of ferrodoxin NADP+ reductase could be regulated based on the electrochemical potential which allowed control over the synthesis of aspartic acid from pyruvic acid or the reverse reaction. Furthermore, Milton et al. used a combination of nitrogenase and hydrogenase enzymes to electrify the production of NH3 from N2 and H2, which is depicted in Figure 4e. In this work, methyl viologen was used as an electron transfer agent between the enzymes and the electrodes to produce NH3 and an electrical current at the same time. (106)
4.4. Heat
Heat can be used as a parameter to influence the dynamics of ERNs since the rate constants of enzymes are dependent on temperature. (107,108) As mentioned in section 3.2, Maguire et al. studied the influence of temperature on the Tr oscillator. They investigated the effect of temperature perturbations under conditions close to the so-called tipping point, the boundary between the oscillatory and the steady state regime. (68) Close to the boundaries of the stable oscillatory regime (between 15 and 38 °C), sensitivity to short temperature perturbations increased, causing the recovery time to stable oscillations to increase as well. In another study, they investigated the use of small temperature oscillations to regulate the periodicity of the Tr oscillator. (69) For temperature oscillations with an amplitude of just 3 °C, the periodicity of stable oscillations could be shifted to match the externally induced periodicity through a process known as phase locking or synchronization. Outside the phase-locking regime, quasi-periodic behavior was observed. Since the temperature can have a significant influence on the dynamic behavior of a network, a method to locally adjust the temperature was developed by Zhang et al. This involved the embedding of enzymes on platinum nanoparticles decorated with thermoresponsive copolymers, allowing local heating by irradiation with near-infrared light. The polymer–enzyme hybrids formed aggregates in solution below a certain temperature and disassembled above that temperature, thereby adjusting the accessibility of the substrate toward the active site of the enzyme. In general, the formation of aggregates resulted in a decreased enzymatic activity compared to the disassembled state of the particles. (108) Thermoresponsive polymers were also used by Gobbo and co-workers to reversibly regulate enzyme activity by contractions of a polymer–protein-based protocell upon temperature changes. (109)
4.5. Other Control Factors
The Katz group presented an enzymatic system where a magnetic field was used to induce local pH changes, yielding reversible control over the enzyme activity. (110) Here, two types of enzyme-modified magnetic nanoparticles that contained amyloglucosidase (AMG) and Ur or Est were used. Upon applying a magnetic field, the nanoparticles with AMG and Ur displayed aggregation. Due to the aggregation, the enzymes were in closer proximity to each other and therefore showed higher enzyme activity. The AMG activity was monitored by a cascade reaction with GOx and HRP.
Furthermore, sound can be used as an external stimulus to control the dynamics of enzymatic cascades. Gradients and patterns of vibrations in an aqueous medium form upon application of sound, see Figure 4f. Dhasaiyan et al. found that at the minima of the vibration, gas dissolution was lower than that at the maxima of the vibration. This was exploited in an enzymatic cascade dependent on dissolved oxygen. First, GOx-FAD was reduced to GOx-FADH2 by glucose consumption. The dissolved oxygen was used to produce H2O2 that can be used by HRP to oxidize ABTS (2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid). ABTS is a colorful compound that was used to visualize gradients in the enzyme activity of the cascade, see Figure 4f. (111) Please note, the compartmentalization of enzymes or the immobilization of enzymes on different types of support can enhance the reaction rate and therefore the total output of an enzymatic cascade. For more information about compartmentalization, (120−122) in DNA nanocages, (123−128) immobilization in metal organic frameworks (MOFs), (129−133) or immobilization of enzymes on other supports, (112−119) we would refer to other reviews.
5. Designing “Life-Like” Systems Using ERNs
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In previous sections, we discussed how ERNs can exhibit intricate, complex features such as feedback loops, bistability, ultrasensitivity, or adaptive behavior. Employing these complex features of enzyme-catalyzed reactions to trigger morphological transformations and modulate the physical properties could lead to the development of so-called life-like systems. In this section, we will delve into specific examples that highlight the design of complex systems utilizing enzymatic networks.
5.1. Dynamic Materials
Enzyme-catalyzed reactions typically occur under mild conditions and show high substrate specificity, making enzymes an attractive choice for the design of new dynamic materials. To make the link to materials, researchers studied the influence of enzymatic reactions on polymer chains in order to induce self-assembly or disassembly or other morphological reorganizations. (134,135) For example, Amir et al. reported the use of acid phosphatase to cleave phosphate moieties of hydrophilic monomers, resulting in the formation of an amphiphilic diblock copolymer of polyethylene glycol (PEG) and poly(4-hydroxystyrene) that assembled into spherical micelles. (136) In another example, the autocatalytic nature of urea-Ur was harnessed to control base-catalyzed thiol-Michael addition reaction for time-lapse hydrogelation. (63) Enzyme-catalyzed reactions have been utilized to initiate polymerization of monomeric building blocks. (137,138) Mao et al. used acid phosphatase and GOx to make hydrogels out of supramolecular and polymeric networks. (139) A similar strategy was later implemented to design printable hydrogels using GOx and HRP. (140) In this study, an acrylic acid-modified hydrogelator (NapFFK-acrylic acid) was used as a monomer, undergoing self-assembly to form hydrogels (gel-I, Figure 5a). GOx converted glucose to gluconic acid and subsequently reduced O2 to H2O2. Next, HRP utilized H2O2 to catalyze the formation of acetylacetone (AcAc) radicals via oxidation of AcAc. These radicals initiated polymerization of poly(ethylene glycol) methacrylate (PEGMA) with acrylic-modified hydrogelators, resulting in a cross-linked hydrogel (gel-II, Figure 5a). Gel-II showed very good mechanical properties with an approximately 16 times higher storage modulus compared to gel-I (without cross-linking). Klemperer et al. utilized a similar strategy to design cytocompatible bioinks with interpenetrating polymer network (IPN) formed under mild and aerobic conditions. (138)
Figure 5
Figure 5. Overview of dynamic materials designed with enzymatic reaction networks. (a) Enzymatic polymerization of hydrogels. Adapted with permission from ref (140). Copyright 2016 licensed under CC 3.0 Royal Society of Chemistry. (b) Schematic representation of cross-link degradation of a hydrogel controlled by an enzymatic network. Adapted with permission from ref (146). Copyright 2017 John Wiley and sons.
Moreover, enzyme-mediated de-crosslinking or cleavage has been exploited to install stimuli responsiveness in the presence of enzymes. Hydrogels containing enzyme-cleavable groups were used for designing responsive materials susceptible to enzymes like proteases, lipase, Est, and phosphatase activities. (141−144) Yang et al. demonstrated for the first time an enzyme-regulated reversible gel–sol transformation. They utilized kinase–phosphatase switch to regulate assembly of peptide-based hydrogelator (Nap-FFGEY). The addition of kinase and adenosine 5′-triphosphate (ATP) led to phosphorylation of hydrogelator, disrupting self-assembly, while phosphatase reversed this process, restoring the self-assembly property. (141) Interestingly, micelles formed from polymer–peptide block copolymers modified with substrates for different enzymes (protein kinase A (PKA), protein phosphatase-1 (PP1), and matrix-metalloproteinases MMP-2 and MMP-9) showed different morphologies and aggregation behavior upon enzymatic reactions. (145) In addition, Postma et al. reported a systematic approach in designing adaptive matter by integrating reaction networks with materials. (146) They designed a polyacrylamide (PAAm)-based hydrogel containing two orthogonal types of cross-links, which could either be enzymatically degraded or formed (Figure 5b). Tr rapidly cleaved the first cross-links, thus forming a liquid phase. Simultaneously, Tr slowly cleaved a copolymerized thioester (cross-link precursor), and these newly exposed thiol groups reacted rapidly with linker (poly(ethylene glycol)-bis-maleimide) to form a new gel. Upon addition of Tg and Cg, Tr was formed autocatalytically from Tg and thus speeding up the degradation and activation processes of the first and second cross-links, respectively. Tr also rapidly converted Cg into Cr, which induced even faster cleavage of copolymerized thioester. Finally, to set a threshold for activating the entire network, soybean trypsin inhibitor (STI) protein was introduced, which deactivated the enzymatic network by inhibiting Tr activity (Figure 5b).
Enzyme-catalyzed reaction products can also be used as triggering stimuli which influence polymeric assemblies to exhibit dynamic functions. For example, oxidases like GOx, sarcosine oxidase (SOx), choline oxidase (COx), and urate oxidase (UOx) generate H2O2 upon oxidation of their substrates. Hydrogels containing both hydrolases (AchE) and oxidases displayed a gel–sol transition in the presence of hydrolase substrate (acetylcholine) by the cascade generation of H2O2. (147) In earlier sections, we have discussed how enzymes like Ur, GOx, and Est are capable of controlling the pH of the system environment. Walther and co-workers have demonstrated how enzyme-mediated pH feedback can be introduced to design stimuli-responsive smart materials. For example, Heuser et al. integrated Ur-mediated pH feedback to a pH-responsive photonic gel composed of polystyrene-b-poly(2-vinylpyridine) (PS-b-P2VP) diblock copolymers to control swelling of the photonic hydrogel, which in turn controls transient optical reflection. (148) Moreover, enzyme-mediated pH change was used to design temporal materials where gelation is controlled by a change in pH via enzymatic reactions. (59,65) To demonstrate pH-dependent transient gelation, Heinen et al. used a hybrid hydrogel made of DNA-containing polyacrylamide copolymer with pH-responsive DNA cross-linker (i-motif). This pH-responsive unit formed a tetraplex intramolecularly at low pH and as a result did not take part in cross-linking (sol state). With increasing pH, the unreactive tetraplexes unfold and induce hybridization with the copolymerized strand, resulting in a gel state. A sol–gel–sol transition was observed by the antagonistic effect of Ur-mediated pH increase and pH decrease by Est. (59) In short, enzyme-catalyzed chemical reactions have a diverse range of applications, from enabling cross-linking polymerization under mild conditions to directly controlling material properties to install responsiveness.
5.2. Enzyme-Powered Motile Systems
The inspiration for designing motile systems stems from various biological motor proteins which operate via conversion of chemical energy into mechanical motion by the hydrolysis of ATP. Besides these motor proteins, enzymes have shown increased diffusion during substrate turnover and can impart forces (ca. 10 pN) quite comparable with biological motor proteins. (149,150) In addition, an asymmetric distribution of reactant and product molecules during enzyme catalysis can create local gradients of concentration or an electrical field, which can lead to enhanced diffusion of enzymes. (151) However, active movement of enzymes in the presence of substrates is part of an ongoing debate, and we refer to other literature sources for a more thorough discussion. (152,153) A number of research groups have worked on endowing small micro/nanosized particles with enzymes in order to create motile systems. (154−156) These particles achieved motility by enzyme-catalyzed chemical reactions. For example, Ur can show ionic self-diffusiophoresis due to the generation of a local electric field from diffusivity differences between the oppositely charged ions (NH4+ and CO32–) formed from urea. (157) Ur has been extensively used as a catalytic engine to design self-propelling motors. (158−160) On the other hand, oxygen bubble generation from H2O2 by Cat or a pair of enzymes like GOx/Cat can propel microswimmers opposite to bubble formation. (161,162) One promising system is based on stomatocytes, bowl-shaped particles formed by deformation of polymersomes by osmotic pressure. During the deformation process, enzymes can be encapsulated in the nanocavity that is formed when the shell folds in on itself, and the enzymatic activity of the encapsulated enzymes provides a force that can propel these stomatocytes. (163) For example, a metabolic network of six enzymes was compartmentalized in stomatocytes, which was responsible for converting glucose (fuel) into motion (Figure 6a). This network started with an ATP-mediated activation module containing HK and pyruvate kinase with phosphoenolpyruvate (PEP, phosphate donor) and glucose (energy source). Pyruvate (the product of the first cycle) triggered the pyruvate–l-lactate cycle where l-lactate dehydrogenase (LDH) consumed pyruvate. But, l-lactate oxidase (LO) catalyzed the reverse reaction. NADH was regenerated by the conversion of glucose-6-phosphate by glucose-6-phosphate dehydrogenase (G6PDH) into 6-phosphogluconolactone. This resulted in net H2O2 production, which was converted to molecular oxygen by Cat. (164) Here, ATP determined the concentration of NADH, thus regulating the entire network. In another example, amyloid microphases loaded with alcohol dehydrogenase (ADH), SOx, and Cat exhibited microscopic motility. These self-assembled structures contained imidazole moieties, which hydrolyzed the 6-methoxy naphthyl alcohol ester of N-methyl glycine (starting substrate) to 6-methoxynaphthyl alcohol and N-methyl glycine. Subsequently, ADH converted 6-methoxynaphthyl alcohol to 6-methoxynaphthaldehyde (a fluorescent compound), while the cascade of SOx and Cat utilized N-methyl glycine to generate oxygen bubbles, responsible for the observed motility. (165) Increased diffusive movement of free enzymes and enzyme-coated particles toward higher substrate concentration has been reported and extensively studied. (150,166,167) The Wilson group designed Cat-powered PLGA (poly(lactic-co-glycolic acid) micromotors as chemotactic drug delivery vehicles, which can follow the H2O2 gradient produced by macrophage cells. (168) Joseph et al. demonstrated how coupling enzyme cascades (GOx and Cat) with asymmetric polymeric vehicles could be used to design potential chemotactic drug delivery vehicles for crossing the blood–brain barrier. (169)
Figure 6
Figure 6. Overview of enzyme-powered motile systems designed with enzymatic reaction networks. (a) Self-propulsion of stomatocytes by generating oxygen from glucose using an encapsulated metabolic enzymatic network. Adapted with permission from ref (162). Copyright 2022 American Chemical Society. (b) Illustration demonstrating expansion and contraction of a spring made of calcium alginate by antagonistic interaction of two enzymes, i.e., Ur and GOx. Adapted with permission from ref (169). Copyright 2021 Springer Nature.
Aside from enzyme-powered self-propulsion, oscillatory movement was achieved in self-assembled organoclay/DNA semipermeable microcapsules containing Cat and GOx. In the presence of H2O2, Cat produced oxygen bubbles which were taken up by GOx in the presence of glucose. The generation and consumption of oxygen bubbles was responsible for sustained oscillatory movement. The system was able to achieve multiple oscillations under a continuous flow of glucose and H2O2. (170) In other recent work by the same group, the interaction between protocells and their environment was explored by designing microactuators capable of chemically induced spring-like compression and relaxation. (171) Helical filaments of calcium alginate containing Ur-loaded proteinosomes were fabricated using microfluidics. When these coiled hydrogels were exposed to urea, carbonate ions were produced and started chelating the calcium ions (Ca2+) from the hydrogel cross-links. This Ur-mediated removal of Ca2+ ions led to the release of the stored elastic potential energy, which in turn induced extension of the helical filaments. To contract these extended filaments, they were exposed to proteinosomes containing GOx. In the presence of glucose, gluconic acid was produced, which dissolves CaCO3 and releases Ca2+. This protocell-mediated Ca2+ flux in turn restored cross-links in the calcium alginate matrix which led to a slow retraction of the extended filaments (Figure 6b). These examples highlight current endeavors to utilize enzymes to control motility of artificial systems, which can be useful in designing intelligent sensors and novel therapeutic vehicles. (189)
5.3. Communication in Compartmentalized Bioreactors
Compartmentalization is an effective strategy to segregate enzymes catalyzing sequential reactions and can be helpful to increase productivity by concentrating substrates and avoiding unwanted side reactions. (120,172,173) This spatial segregation, coupled with interactions between compartments with distinct chemistries, can further lead to the emergence of life-like properties in artificial systems like predator–prey behavior, (174−176) chemical signaling, (177−181) and chemostructural feedbacks. (171,182) In this context, Elani et al. designed a multicompartment vesicle-based platform to spatially segregate reaction processes where chemical signals can transverse from one compartment to another. A three step cascade by lactase, GOx and HRP was conducted where each enzyme catalyzed step was isolated in distinct compartment. (183) Wang et al. developed methodologies to design microarrays of giant unilamellar lipid vesicles (GUVs) containing GOx and HRP to facilitate controlled chemical signaling in the presence of melittin, a pore-forming peptide. (178) Buddingh’ et al. demonstrated chemical communication between giant vesicles through allosteric signal amplification. They used glycogen phosphorylase b (GPb) which switched to a high-activity state upon binding adenosine 5′-monophosphate (AMP). The sender vesicle contained apyrase which produced AMP in the presence of ATP (Figure 7a). The produced AMP diffused to the receiver vesicle, where a small ERN comprising phosphoglucomutase (PMG), G6PDH, GPb, and glycogen was compartmentalized. As AMP acts as allosteric activator of GPb, it induced conversion of glycogen to glucose-1-phosphate, followed by the conversion of glucose-1-phosphate to glucose-6-phosphate by PMG, and subsequent generation of NADH by G6PDH (Figure 7a). This allosteric amplification of the initial weak signal from the sender vesicles, achieved through GPb activation, resulted in a robust response (high NADH output) in the receiver vesicles. Remarkably, the strong signal amplification engineered into this pathway allowed long distance (5 mm) communication between senders and receiver vesicles. (179)
Figure 7
Figure 7. Overview of communication in compartmentalized bioreactors facilitated by enzymatic reaction networks. (a) Communication between sender and receiver GUVs where the sender produced AMP which allosterically activated ERN within receiver GUVs. Adapted with permission from ref (179). Copyright 2020 licensed under CC 4.0 Springer Nature. (b) Cartoon representation of artificial response–retaliation behavior among protocell communities. In the presence of glucose, GOx containing protease-sensitive proteinosome (P) released acid (2) which disassembled pH-sensitive protease containing coacervate (CT) and released protease (3). Coacervate CK recaptured protease and eventually destroyed P. Adapted with permission from ref (175). Copyright 2019 John Wiley and sons.
Qiao et al. achieved artificial response–retaliation behavior using ternary protocell populations. These populations included proteinase K-sensitive GOx-containing proteinosomes (P) that released H+ in the presence of glucose. (175) These also consisted of small pH-sensitive proteinase K-containing polypeptide (poly-d-lysine)/adenosine 5′-diphosphate (ADP) coacervates (CT) and pH-resistant positively charged polymer (poly(diallyldimethylammonium chloride), PDDA)/polysaccharide (dextransulfate, DS) coacervate droplets (CK) that adhered to negatively charged proteinosomes via electrostatic interactions (Figure 7b). Proteinase K was initially sequestered in CT coacervates, but the addition of glucose led to gluconic acid release from P, lowering the pH and causing disassembly of the coacervates CT. The released proteinase K was then sequestered by the proteinosome-adherent coacervates CK. Finally, proteinosomes were destroyed by proteinase K, and only CK remained. Expanding on the idea of communication between compartments, Chakraborty et al. demonstrated how a bioluminescent signal triggered prey–predator behavior in GUVs. Renillaluciferase (RLuc) containing GUVs (sender) produced blue light in the presence of coelenterazine, activating iLID and Nano proteins in the outer membranes of both sender and receiver GUVs. The interaction between iLID and Nano under blue light mediated the adhesion between the GUVs. GUV–GUV adhesion allowed transfer of Ca2+ from the sender to the receiver through unblocked α-hemolysin (α-HL) pores. Ca2+ activation of phospholipase A2 (PLA2) in prey GUVs led to phospholipid cleavage and collapse of receiver GUVs. (176) A recent study from the same group showcased bidirectional communication in GUVs through similar chemiluminescence-triggered adhesion, enabling exchange of H2O2 from the sender and Ca2+ from the receiver. Interestingly, GUVs separated when the signaling molecule production ceased. (180) Liu and Zhang et al. designed a three-layer tubular prototissue comprising concentrically arranged agarose hydrogel layers containing GOx, HRP, and CAT containing coacervates as the outer, middle, and inner layers, respectively. Glucose and hydroxyurea were added specifically to the exterior side of the model prototissue. Through inward diffusion, glucose was first processed by GOx containing a hydrogel layer, leading to H2O2 production. HRP in the middle layer converted hydroxyurea in the presence of H2O2 into nitric oxide (NO). Excess H2O2 was then consumed by Cat in the final layer, such that NO became the main output, which was further used to inhibit blood coagulation in samples located within the device’s internal lumen. (184)
Interestingly, enzymes fixed in a microchamber can act as “chemical pumps” in the presence of specific substrates. Density differences between the substrates and the enzyme-catalyzed products give rise to solutal buoyancy, in turn generating convective flows of the enclosed fluids. (185,186) This system can be understood as follows: when the density of the enzyme-catalyzed products exceeds that of the reactants, the fluid becomes denser, sliding down and away from the patch like an outward pump. Conversely, if the product density is lower, the fluid rises up, falls back down due to convective flows, and moves toward the enzyme patch, forming an inward pump. The fluid flow velocity is dependent on the enzymatic reaction rates. These micropumps have been utilized as proof-of-concept delivery vehicles to transport small molecules like insulin in the presence of a glucose stimulus or act as sensors to detect toxins that hamper enzymatic reactions. (185,187) Furthermore, simulations on flexible sheets with two enzyme patches have revealed complex motility behaviors. The first patch produces substrates for the second, while the products from the second patch inhibit the enzymes in the first patch. The spatial separation between these two enzyme patches led to the time delay required for chemical oscillations in the system. The resulting fluid flow induced oscillatory mechanical deformation, displaying diverse motility behaviors. (188) These strategies utilizing ERNs to enable communication between spatially segregated bioreactors are crucial for designing systems with diverse life-like applications.
6. Cell-Free Synthesis
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Cell-free biosynthesis is a rapidly growing field in which complex biochemical pathways are assembled in vitro. The aim of this field is to better understand how such pathways function and how they can be manipulated. The reconstruction of these pathways in vitro also provides a route to the synthesis of valuable organic molecules using environmentally friendly processes and without the need for culturing organisms. Reaction networks carried out in cell-free environments can, in principle, be much better controlled by optimizing the pH, temperature, and enzyme loading. Furthermore, competition with other enzymatic pathways is excluded, and enzymes produced in various organisms can be combined into a single pathway. (190) In this section, we will focus on the biochemical pathways that consist of multiple enzymes. For the two-step enzyme cascades, we refer the reader to the reviews in refs (172) and (191−193) and specifically for enzymatic cofactor regeneration to the reviews in refs (194) and (195). Importantly, only cell-free synthesis of small molecules is covered in this review; cell-free protein synthesis has been the topic of several reviews. (196−198)
6.1. Sugars as Substrates
Bowie’s group demonstrated how complex enzymatic chemical reaction networks can be used to synthesize valuable molecules starting from simple substrates such as glucose or phosphoenolpyruvate (PEP) (Figure 8a). All reactions were performed using purified enzymes in batch conditions. One of the first complex enzymatic reaction pathways included isoprene synthesis, which was synthesized from PEP in a combined glycolysis and mevalonate pathway containing in total 12 enzymes. (199) ADP, NADPH, and coenzyme A (CoA) regeneration cycles were employed to reuse the cofactors and to avoid CoA build up, which was causing inhibition of the forward direction of the mevalonate pathway. After this, the same group showed how a 27-enzyme cell-free system in batch converts glucose into different monoterpenes using glycolysis and mevalonate. (200) The choice for the final enzyme in the pathway, a limonene synthase, pinene synthase, or limonene synthase-N345A mutant, determined which monoterpene (limonene, pinene, or sabinene) was produced. The whole pathway also included a molecular purge valve (Figure 8b). (201) Molecular purge valves maintain cofactor balance in the system without the need to perfectly match stoichiometric cofactor formation and carbon consumption. Once there is a NADPH cofactor buildup, the NADP+-dependent reductase enzyme is starved of oxidized cofactor and the pathway shuts down. In the meantime, the purge valve is turned on and NAD+-dependent reductase and NADH-specific oxidase are activated. In this case, it was used to control the balance of NAD(P)H/NAD(P)+ using three enzymes: glyceraldehyde-3-phosphate dehydrogenase (Gap), mutant glyceraldehyde-3-phosphate dehydrogenase (mGap), and NADH oxidase (NoxE). This cascade regenerates NADPH, purges the excess of NADH, and continues generating carbon building blocks for the glycolysis pathway. It was demonstrated that excluding at least one enzyme of the molecular purge valve reduced limonene production drastically, while a system without the NoxE enzyme did not convert any glucose to limonene. Researchers managed to produce all three terpenes starting from 500 mM glucose over 7 days at high titers.
Figure 8
Figure 8. (a) A diagram of low-cost substrate glucose being converted into pyruvate or acetyl-CoA as an intermediate and then depending on the selected pathway to isoprene, (199) monoterpenes, (200) PHB, (202) or cannabinoids (203) using purified enzymes (in orange) or to n-butanol, (205) mevalonate, (206) or limonene (208) using crude cell lysate (in yellow). (b) The purge valve concept, where the purge valve is “OFF” when there is low NADPH concentration and “ON” at high NADPH concentration. Adapted with permission from ref (201). Copyright 2014 Springer Nature.
Another example of synthetic pathways producing valuable organics includes the so-called PBG (pentose–bifido–glycolysis) cycle in which the final product is polyhydroxybutyrate (PHB), a bioplastic. (202) The whole cycle consists of 20 enzymes from three partial pathways: the pentose phosphate pathway, the bifidobacterium shunt, and the glycolysis pathway. This ERN also contained two purge valves to regulate NAD(P)H concentrations as well as a metabolite salvage pathway which allowed erythrose-4-phosphate to re-enter the cycle. Two new enzymes, a G6PDH and a 6-phosphogluconate dehydrogenase (Gnd) mutant, had to be engineered for the purge valve to favor NAD+ instead of NADP+, as their wild forms do. A batch reaction containing all enzymes was productive for up to 55 h, and only the last enzyme of the cycle─PHB synthase (PhaC)─had to be added again after each 10 h cycle. This was done because the enzyme was covalently linked to the growing end of the PHB product, which meant the enzyme would be removed every time during sampling of the bioplastic out of the reaction vessel to quantitatively measure produced PHB. Valliere et al. constructed an even more complex pathway consisting of 23 enzymes, which was used to synthesize a variety of prenylated molecules, including cannabinoids, from glucose. (203) In their work, a modified glycolysis module was developed which included a purge valve to balance NADPH concentration and carbon flux. This module was connected to acetyl coenzyme A (acetyl-CoA) and mevalonate modules to form geranyl-pyrophosphate (GPP). From there, different substrates and their complementary enzymes were added to yield various prenylated compounds. Performing this pathway using only purified enzymes is also advantageous as GPP is toxic to cells in medium concentrations. (204) The original system containing pyruvate dehydrogenase (PDH) had to be modified as it was found that PDH was inhibited by 1,6-dihydroxynaphthalene (1,6-DHN), which is the preferred substrate for wild-type prenyltransferase (NphB) enzyme. Therefore, a PDH bypass system consisting of two additional enzymes to convert pyruvate via acetyl phosphate to acetyl-CoA was introduced. After it was proved that various prenylated aromatic compounds can be generated using prenyl transferases, the authors focused on optimizing cannabinoid production. The results showed that the production rate of the cannabinoid precursor cannabigerolic acid (CBGA) using the primary system reached a not particularly high titer. However, a new mutant CBGA synthase (M23) was designed to produce CBGA using olivetolic acid (OA) without side products, which helped to reach a higher titer. In combination with in situ product removal using a flow system, a final CBGA titer was increased even more.
All of these examples demonstrated complex cell-free purified enzymatic reaction networks and possibilities for future industrial applications. Obviously, some important aspects of any industrial application include optimization, cost reduction, and yield increase. The authors proposed employing stable enzymes that could be used for longer periods of time in order to reduce costs, introducing cofactor (ATP/NAD(P)H) regeneration cycles to increase yields, (204) recycling enzymes, and introducing cheaper purification methods. (202) Another potential improvement could come from enzyme immobilization, which allows for easier purification and optimization at a cost of potentially reduced enzyme activity upon immobilization.
Instead of using purified enzymes, in some networks it would be desirable to construct ERNs in crude cell lysate. Karim et al. established a cell-free protein synthesis-driven metabolic engineering (CFPS-ME) framework for fast enzyme selection and pathway construction. (205) As a model system, they chose a CoA-dependent n-butanol pathway starting from glucose including 17 steps in vitro. For this, the authors combined cell-free metabolic engineering (CFME), where heterologous enzymes are selectively overexpressed, and cell-free protein synthesis (CFPS). First, it was shown that mixing five crude lysates each of them with different selectively overexpressed enzymes via CFME can activate the whole pathway and produce n-butanol. Next, CFPS was modularly integrated in CFME to speed up the biosynthesis. Hydroxybutyryl-CoA dehydrogenase from Clostridia beijerinckii (Hbd2) lysate was excluded from the mixture of overexpressed enzymes, and instead, the CFPS was performed for Hbd2. As the downstream enzymes could not be activated without their substrates, the pathway would stay inactive until Hbd2 was synthesized. Therefore, first, CFPS of Hbd2 was performed for 3 h, and glucose was added to initiate the n-butanol synthesis pathway. Later, a similar experiment was repeated by excluding every other one of the five n-butanol pathway enzymes from overexpressed enzyme lysate mix and synthesizing them using CFPS for 3 h. After adding glucose, NAD, and coenzyme-A (CoA) to initiate the CFPS–ME pathway reactions, all variants produced n-butanol. This study demonstrated how the CFPS–ME method can be used for fast enzyme screening for ERNs. The same group also demonstrated the synthesis of mevalonate from glucose using enzyme-enriched lysate mixtures (206) and investigated a pathway from mevalonate to limonene without any optimization. (207) This research laid the groundwork for a combined study involving a full pathway from glucose to limonene using enzyme-enriched Escherichia coli (E. coli) lysates requiring in total 20 metabolic steps. (208) To begin with, the endogenous metabolism in E. coli crude cell lysate already consisted of all of the glycolytic enzymes which transformed glucose to acetyl-CoA. This part was then connected to a single extract enriched in three enzymes─acetyl-CoA acetyltransferase (ACAT), hydroxymethylglutaryl-CoA synthase (HMGS), and hydroxymethylglutaryl-CoA reductase (HMGR)─converting acetyl-CoA to mevalonate. Last, it was coupled to a module of six extracts each enriched in a single enzyme using heterologous overexpression in vivo─mevalonate kinase (MK), phosphomevalonate kinase (PMK), pyrophosphomevalonate decarboxylase (PMD), isopentenyl pyrophosphate isomerase (IDI), geranyl diphosphate synthase (GPPS), and limonene synthase (LS). After additional experiments and optimization, the full pathway produced final molecule limonene.
Cell-free biosynthesis points out attractive possibilities of performing enzymatic synthesis and creating complex ERNs without any cell membrane constraints and avoiding cell toxicity. This growing field offers the opportunity of producing valuable compounds using sustainable biosynthesis. In the ethanol industry, commonly accepted thresholds required for industrial production are yields of 90%, productivity of 1 g/L/h, and titers of 40 g/L. (202) Some of the discussed network final product titers are 12.5 ± 0.3, 14.9 ± 0.6, and 15.9 ± 0.4 g/L and yields of 88.3 ± 4.6%, 103.9 ± 8.1%, and 94.5 ± 4.7% for limonene, pinene, and sabinene, respectively. (200) These final product values are close to the threshold values used in the ethanol industry, and it gives high hopes that cell-free biosynthesis will be applied for industrial purposes.
6.2. CO2 as Substrate
In addition to glucose, CO2 is also a versatile starting material for the sustainable synthesis of valuable compounds. Many inorganic and metallic catalysts, like SnO2, TiO2, Cu, and transition metal complexes, have been successfully developed as catalysts to reduce CO2. (209) These inorganic catalysts in general show low selectivity; hence, the products are mainly limited to C1 and C2 compounds, including methanol and ethanol. Nature has evolved several metabolic networks to reduce CO2 into higher carbon compounds. However, many of the key carboxylase enzymes that couple CO2 to organic substrates have low efficiency. (210−212) A new-to-nature carboxylase, glycolyl-CoA carboxylase, was recently developed and used in the successful in vitro implementation of the previously hypothesized tartronyl-CoA (TaCo) pathway, which had been proposed as a direct route for the assimilation of glycolate into central carbon metabolism and expected to outperform all naturally evolved glycolate assimilation routes. (213) In contrast, in vitro construction of artificial CO2 fixation ERNs by freely combining different enzymes from various biological sources is another promising approach. The Erb group assembled the so-called crotonyl–coenzyme A (CoA)/ethylmalonyl-CoA/hydroxybutyryl-CoA (CETCH) cycle with 17 enzymes from nine different organisms to convert CO2 into malate at a rate of 5 nmol of CO2 per minute per milligram of protein, providing a seventh synthetic alternative to the six naturally evolved CO2 fixation pathways (Figure 9a). (214) This is comparable to the CO2 fixation rate (1–3 nmol of CO2 per minute per milligram of protein) of the Calvin–Benson–Bassham (CBB) cycle, which fixes more than 90% of carbon in nature. (215) Moreover, this CETCH cycle could be solar powered through the incorporation of chloroplast extracts. (216) Subsequently, the output glyoxylate from the CETCH cycle can be applied to produce acetyl-CoA, the central precursor for many natural compounds. Hence, a series of value-added compounds, including monoterpenes, sesquiterpenes, polyketides, and 6-deoxyerythronolide B, has been successfully produced from CO2 in one pot based on the CETCH cycle. (217,218) In addition, a small ERN with four enzymes (pyruvate carboxylase–oxaloacetate acetylhydrolase–acetate-CoA ligase–pyruvate ferredoxin oxidoreductase), named the POAP cycle, was constructed to couple two CO2 into one oxalate at the expense of two ATP and one NAD(P)H, reaching a rate of 8 nmol/min/mg CO2-fixing enzymes (Figure 9b). (219) For a more detailed overview, the enzymatic conversion of CO2 has been discussed in a recent review on the topic. (220)
Figure 9
Figure 9. Synthetic CO2 fixation pathways. (a) CETCH cycle converting CO2 to malate. Adapted with permission from ref (214). Copyright 2016 The American Association for the Advancement of Science. (b) POAP cycle converting CO2 to oxalate. Adapted with permission from ref (219). Copyright 2022 American Chemical Society. (c) Chemoenzymatic ASAS pathway converting CO2 to starch. Adapted with permission from ref (221). Copyright 2021 The American Association for the Advancement of Science. (d) Chemoenzymatic pathway converting CO2 to bioplastic polyhydroxybutyrate (PHB). Adapted with permission from ref (223). Copyright 2023 Royal Society of Chemistry.
6.3. Other Substrates
In addition to using sugar and CO2 as substrates, progress has been made on fabricating ERNs to upgrade sustainable C1 and C2 stocks, like methanol, ethanol, and acetate, to compounds with longer chains and hence higher value. Cai et al. designed and constructed an 11-enzyme ERN to convert C1 methanol to polymeric starch (Figure 9c). (221) Zhou et al. designed several ERNs to convert methanol to ethylene glycol, glycolic acid, and d-erythrose. (222) In addition, the Bioplastic PHB has been produced from methanol and acetate (Figure 9d). (223,224) Furthermore, Liu et al. managed to use ethanol to produce acetyl-CoA and regenerate ATP using the isoprenol production pathway. (225)
7. Future Development
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The ERNs discussed so far exhibit a diverse range of functionalities and emergent properties. Yet, they remain relatively small based on several reactions occurring at once. However, as the sections on control over enzyme reactivity and the construction of elaborate reaction cascades for the synthesis of complex organic molecules have demonstrated, the field is now in a strong position to develop ERNs with much more advanced functionalities. In this section, we will discuss two such possible functions (without the ambition of providing complete overviews of those fields): synthetic cells and molecular computers.
7.1. Toward Building a Synthetic Cell from the Bottom Up
Building a synthetic cell from the bottom up could address the fundamental questions on how life emerged and evolved from nonliving components and at the same time generate a wide range of applications. Any functioning synthetic cell will inevitably require the construction of (minimal) metabolic ERNs for the continuous supply of building blocks and energy to support other cellular processes and also response to external environmental changes. (226) ATP fuels many cellular processes and also serves as a building block for gene transcription and translation. Lee et al. successfully designed and built a photosynthetic artificial organelle consisting of an ATP synthase and two photoconverters in a giant vesicle. This synthetic organelle was capable of harvesting light to produce ATP and to drive endergonic reactions, like pyruvate carboxylase-mediated carbon fixation and actin polymerization into filaments (Figure 10a). (227) Similarly, Berhanu et al. designed a light-harvesting artificial organelle, which was composed of two kinds of membrane proteins─bacteriorhodopsin and F-type ATP synthase. In this work, the organelle, reconstituted inside a GUV together with a cell-free protein synthesis system, generated ATP required in transcription and at the same time powered the synthesis of GTP and translation (Figure 10b). (228) Additionally, Bailoni et al. reconstituted the l-arginine breakdown pathway of three cytosolic enzymes to phosphorylate ADP into ATP to fuel the sustainable formation of phospholipid headgroups in synthetic cells. (229) Blanken et al. managed to encode seven phospholipid-producing enzymes in a synthetic minigenome, which were then expressed within cell-like liposomes. This de novo-constructed ERN can make use of fatty acyl coenzyme A and glycerol-3-phosphate as precursors to produce two phospholipids phosphatidylethanolamine (PE) and phosphatidylglycerol (PG) simultaneously (Figure 10c). The balance of PE and PG is transcriptionally regulated by the activity of specific genes together with a metabolic feedback mechanism. (230) In addition, Partipilo et al. constructed a small ERN with three enzymes in liposomes to control the redox status of NADH and NADPH cofactors. (231)
Figure 10
Figure 10. (a) Schematic of the artificial organelle harvesting light to produce ATP and to drive endergonic reactions. Adapted with permission from ref (227). Copyright 2018 Springer Nature. (b) Schematic of self-constituting protein synthesis in artificial photosynthetic cells. Adapted with permission from ref (228). Copyright 2019 licensed under CC 4.0 Springer Nature. (c) Schematic of the production of phospholipids PE and PG by de novo-synthesized enzymes. Adapted with permission from ref (230). Copyright 2020 licensed under CC 4.0 Springer Nature.
Living cells maintain physicochemical homeostasis (including pH, ionic strength, osmotic pressure) to enable the internal components to function near their optimum. This homeostasis state was successfully achieved inside a cell-like system by Pols et al., who co-reconstituted the arginine-breakdown pathway and an ionic strength-gated ATP-driven osmolyte transporter inside vesicles. The cell-like vesicles shrank under increased medium osmolality, causing the internal ionic strength and pH to increase, leading to the inactivation of enzymes. Once the internal ionic strength reached a critical value, the transporter was activated and glycine betaine was pumped in with the accompaniment of the influx of water into the vesicles. Hence, the volume of vesicles increased, and the internal ionic strength and pH decreased. As a result, homeostasis of the ATP/ADP ratio, internal ionic strength, and pH was achieved. (232) All of these examples clearly demonstrate how enzymatic modules are crucial for mimicking complex metabolic functions of living systems in the field of designing synthetic cells.
7.2. Information Processing and Computation
An emerging direction for the research on and application of ERNs is special-purpose information processing and computation. So far, the development of Boolean gates and logic circuits has seen a long tradition in enzymatic network research and has been extensively reviewed several times (examples are shown in Figure 11a). (233,234) However, the development of systems based on digital paradigms has been slowing down, as the principles of Boolean logic and von Neumann computer architectures are challenging to adapt in biochemical systems, which are intrinsically of a nonbinary and dynamic nature. Instead, the potential application of analog and neuromorphic computation principles in biochemistry is increasingly appreciated. (235) While these developments are still in their infancy, here we focus on recent progress in this direction.
Figure 11
Figure 11. (a) Schematic overview of commonly used ERN designs for two Boolean logic gates (AND and XOR). The computational output is based on an arbitrary threshold for the concentration of a product molecule. (b) A design for an ERN resulting in a multi-input perceptron, investigated in Pandi et al. (238) The design uses so-called transduction reactions to convert a range of different inputs into the same “transducer” substrate. Weights can be set by modifying enzyme concentrations. The final addition results in a roughly sigmoidal response. (c) Design for a chemical neuron, as investigated by Okumura et al. (239) A single neuron is constructed from specific DNA templates (curved lines) enabled by components of the polymerase–exonuclease–nickase (PEN) toolbox (shaded circle). (d) Design for a chemical multilayer perceptron. Individual neurons can be combined by replacing fluorescence-generating reporter strands by input strands for other neurons.
Rather than recreating digital infrastructure directly, recent work on small ERNs has aimed to establish modules and motifs capable of analogue computation. One example of this shows the development of small ERNs that are capable of arithmetic operations. (236) The key insight here is that specific enzymatic interactions, such as cascading pathways, inhibitors, and parallel product conversion, can be interpreted as arithmetic functions if operated in the right parameter regime. Similarly, specific forms of substrate competition between different enzymes can lead to nonlinear Boolean logic as the theoretical study by Genot and co-workers shows. (237) This work essentially presents network motifs that act like a nonlinear and nonadditive switch of the starting substrates. This type of research showcases the intrinsic computational power of ERNs, circumventing the requirements necessary for developing explicit digital circuitry.
Recently, this approach has been expanded to enzymatic systems capable of more generalized types of computation. Inspired by concepts from machine learning and neuromorphic computation, Pandi et al. created in vitro metabolic perceptrons. (238) They employed computational retrosynthesis tools to design enzymatic models that can act as transducers (converting different incoming metabolite signals into one substrate) and nonlinear actuators (to achieve sigmoidal responses) and when combined together can act as analogue adders (to perform a weighted sum of incoming signals, e.g., a perceptron). A schematic overview of this is shown in Figure 11b. These perceptrons are essentially based on ultrasensitivity network motifs to create nonlinear weighted adders (sigmoidal sums) of multiple metabolites but do so in a generalizable and extensible manner. Modifying the transducer reactions changes how the incoming signals are weighted, allowing the perceptron to be controlled and trained similar to an artificial neural network.
A different approach is investigated in the work of Okumura et al., where neural networks capable of nonlinear classification have been created by assembling various components of the polymerase–exonuclease–nickase (PEN) toolbox, as shown in Figure 11c. (239) These enzymes, respectively, polymerize, degrade, and cut DNA strands. Neurons are constructed by supplying specific DNA template strands that convert DNA (or RNA) inputs either into a “reporter” DNA strand or into an “antireporter” DNA strand. The reporter strand can be transformed into a fluorescent signal by another DNA template strand. The antireporter strand polymerizes with the reporter strand, inhibiting its output. Thus, modifying the concentration of reporter and antireporter templates impacts fluorescence, respectively, in a positive or negative way, establishing an effective weight on input conversion. These effective weights can be further modified by incorporating so-called “fake” weight templates, which competitively inhibit the antireporter–reporter interaction. The reporter signal is further amplified by an amplification template, resulting in an ultrasensitive (sigmoidal) response. Finally, the location of the ultrasensitive regime can be adjusted by inclusion of a “bias” template, which operates similarly to the antireporter strand but is not input dependent. Polymerase and nickase drive these DNA reactions, while exonuclease can be used to degrade the fluorescent output over time. The interaction of all enzymes and DNA template strands together establishes a perceptron where all weights and biases can be fine tuned by controlling the concentration of the reporter, antireporter, fake, and bias template strands. By exchanging the reporter templates for strands that can serve as input to another perceptron (Figure 11d), a multilayer neural network was created that could accurately classify input concentration regimes. Importantly, the computational power in this work originates from the highly heterogeneous substrates used in the three-enzyme ERN, in contrast to the previous example of a metabolic network where it originates from the design of the ERN itself.
The above examples show the significant progress that has been made in enzymatic information processing by directly exploiting similarities between enzymatic interactions and analogous principles instead of recreating small Boolean operations and assembling those in a circuit. By either rationally fine tuning the parameter regime in small existing ERNs, by computationally designing larger ERNs to convert many different substrates into the same products, or by intelligently choosing which substrates (and specifically which templates) to exploit using just a small ERN, impressive feats of computation can be achieved. However, further developments have been mostly theoretical up until now, including work showcasing different enzymatic network motifs capable of noise filtering, (240) pattern recognition and pulse counting, (241) and systems that can convert information into work and vice versa. (242) It remains to be seen if these proposals can be realized experimentally, but they highlight the potential for ERNs in future information processing tasks.
8. Conclusion
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We have presented this comprehensive review of the field of in vitro enzymatic reaction networks in the hope to inspire a broad group of researchers to participate in expanding the field. Key design principles, basic emergent properties, and the need for an integrated approach comprising experiments, computational approaches, and analytical methods have been highlighted. Several key challenges for future research were identified: there are currently no “blueprints” for the design of ERNs with functions that go beyond “simple” temporal behavior. For example: we know how to create oscillatory networks, but we cannot design networks that design with a specific frequency and amplitude. Most ERNs reported to date have relatively simple topologies. To significantly expand current designs, we need to establish new strategies for the reverse design of functional ERNs and establish high-throughput methods for the rapid evaluation of networks consisting of multiple feedback loops. Instead of current “trial-and-error” experiments to optimize network properties, iterative “design–build–test–learn” cycles need to be established, and we are hopeful that advances in machine learning and artificial intelligence could be leveraged to identify novel network topologies with hitherto “undesignable” properties. We note that many experimental designs (especially if they are aimed at constructing materials with life-like properties) can only be realized within a relatively narrow scope of kinetic values. Simulations can significantly accelerate discoveries of the most relevant experimental regime, but this will need to be coupled to methods to control or expand the kinetic parameters of enzymes. Although current networks are incomparable in complexity to ERNs found in extant life, our overview on possibilities to control enzymatic activity together with the many examples of feedback loops gives us confidence significantly more elaborate reaction networks will be within reach in the next decade. Indeed, the successful attempts to construct complex reaction cascades and cycles to synthesize complex organic molecules are testament to the potential of more complex ERNs.
We foresee many new developments in the construction of life-like materials, where properties such as homeostasis, sensing the environment, and motility are all carried out by coupled enzymatic reaction networks. The design of intelligent materials by incorporating ERNs, which are able to learn from their environment to adopt desired features, could open up innovative avenues in material science. In section 4, we have explored the influence of external stimuli to regulate ERN activity, serving as physical learning rules for enhanced task performance. Interestingly, these materials potentially can exhibit task adaptiveness through training with different stimuli. (243) Another important direction for designing life-like materials would be to incorporate some type of fuel regeneration and methods to maintain such materials out of equilibrium as many of the properties associated with living systems (motility, homeostasis, regeneration) require dissipative systems and continuous input of energy. In section 5, we have introduced different strategies like energy production, compartmentalization, and communication using ERNs, which facilitate crosstalk between multiscale processes to design truly autonomous systems. (244) Such materials could then find applications as delivery vehicles in medicine, in soft robotics, or as interfaces between living systems and electronic systems. In section 6, we demonstrated the progress in cell-free biosynthesis, showcasing the in vitro design of metabolic modules for unraveling complex metabolic machinery and producing value-added chemicals. Looking ahead, we anticipate the development of ever more complex ERNs for designing novel catalytic networks with promising industrial applications. Significantly, this can augment the design of novel synthetic cells as chemical factories. Finally, enzymes are key in processing information in biology, but the potential of synthetic ERNs to achieve similar capabilities remains largely unrealized. (245) Building on the multifarious uses of enzymes in complex (and not so complex) reaction networks, there is much potential for designing novel computing paradigms in which enzymes are used to process multimodal and multiplexed information at the molecular level.
Author Information
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- Wilhelm T. S. Huck - Institute for Molecules and Materials, Radboud University, Heyendaalseweg 135, 6525 AJ Nijmegen, The Netherlands;
https://orcid.org/0000-0003-4222-5411;
- Souvik Ghosh - Institute for Molecules and Materials, Radboud University, Heyendaalseweg 135, 6525 AJ Nijmegen, The Netherlands;https://orcid.org/0009-0007-3956-6588
- Mathieu G. Baltussen - Institute for Molecules and Materials, Radboud University, Heyendaalseweg 135, 6525 AJ Nijmegen, The Netherlands;https://orcid.org/0000-0001-7779-8899
- Tao Zhou - Institute for Molecules and Materials, Radboud University, Heyendaalseweg 135, 6525 AJ Nijmegen, The Netherlands;https://orcid.org/0000-0003-4520-9679
CRediT: Souvik Ghosh investigation, visualization, project administration, supervision, writing─original draft, writing─review and editing; Mathieu G. Baltussen investigation, visualization, writing─original draft, writing─review and editing; Nikita M. Ivanov investigation, visualization, writing─original draft, writing─review and editing; Rianne Haije investigation, visualization, writing─original draft, writing─review and editing; Miglė Jakštaitė investigation, visualization, writing─original draft, writing─review and editing; Tao Zhou investigation, visualization, writing─original draft, writing─review and editing; Wilhelm T. S. Huck conceptualization, investigation, visualization, funding acquisition, project administration, writing─original draft, supervision, writing─review and editing.
Biographies
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Souvik Ghosh
Souvik Ghosh received his B.Sc.–M.Sc. dual degree from IISER Kolkata, India (2021). For his Master’s research, he designed enzyme-powered nanomotors based on cross-β-amyloid nanotubes in the system chemistry lab at IISER Kolkata. In the same year, he started his Ph.D. work on the design and modulation of complex enzymatic reaction networks for information processing under the supervision of Wilhelm T. S. Huck at Radboud University. His research interests include flow chemistry, photochemistry, enzymatic reaction networks, enzyme immobilization, and reservoir computation.
Mathieu G. Baltussen
Mathieu G. Baltussen earned a double B.Sc. degree in Chemistry and Physics & Astronomy at Radboud University (2018) and a double M.Sc. honors degree in Nanomaterials Science and Experimental Physics at Utrecht University (2021). He is currently pursuing his Ph.D. degree under the supervision of Wilhelm T. S. Huck at Radboud University. His research interests focus on information processing and computation in (bio)chemical networks and developing techniques to use chemical reaction networks as computational systems.
Nikita M. Ivanov
Nikita M. Ivanov obtained his M.Sc. degree in Bioorganic Chemistry in 2019 from Lomonosov Moscow State University, Russia, with a diploma project on DNA aptamers to influenza hemagglutinin working with Alexei M. Kopylov. During these studies he also worked on the synthesis of perylene derivatives for nucleoside labeling and as antivirals in the laboratory of Vladimir A. Korshun at the Institute of Bioorganic Chemistry RAS, Moscow. In 2020, he started his Ph.D. work under supervision of Wilhelm T. S. Huck at Radboud University to focus on enzymatic reaction networks for molecular information processing and the physical chemistry of enzymes and photoinhibitors.
Rianne Haije
Rianne Haije received her B.Sc. and M.Sc. degrees in Chemistry from Radboud University. During this time, she focussed on different projects that included the development of an oscillating urease-based network, the synthesis of sialic acid-based inhibitors, and the development of tools to control enzymatic activity by using light. The goal of her Ph.D. work is to control dynamic reaction networks by using photoswitchable molecules in combination with light.
Miglė Jakštaitė
Miglė Jakštaitė obtained her B.Sc. degree in Chemistry from Vilnius University, Lithuania, in 2017. For her final thesis project, she investigated the synthesis of anionic molecular brushes containing glucuronate under the supervision of Tatjana Kavleiskaja. Later, she obtained her M.Sc. degree in Chemistry from Radboud University in 2019, where her main internship project focused on cell-free enzymatic reaction networks under the supervision of Wilhelm T. S. Huck. She rejoined the group of Wilhelm T. S. Huck to start her Ph.D. studies in 2019. Her research interest focuses on cell-free biosynthesis of valuable compounds in cascade reactions using immobilized enzymes.
Tao Zhou
Tao Zhou received his Ph.D. degree in Biochemistry at ETH Zurich, Switzerland, in 2020. During his Ph.D. work, he developed a biomimetic soft material-lipidic mesophases for enzymes in vitro to fulfill their full potentials in biocatalysis from room to cryogenic temperatures. Then, he conducted his postdoctoral research on complex enzymatic cascades at Radboud University in the laboratory of Wilhelm T. S. Huck. His research interests include biosynthesis, enzymatic reaction networks, enzyme immobilization, kinetic modeling, and flow chemistry.
Wilhelm T. S. Huck
Wilhelm T. S. Huck is a professor of Physical Organic Chemistry at Radboud University. After postdoctoral research at Harvard University, he took up a position in the Department of Chemistry at the University of Cambridge, where he became Director of the Melville Laboratory for Polymer Synthesis (2004) and Full Professor of Macromolecular Chemistry (2007). In 2010, he moved to Radboud University, where he developed a new line of research focusing on complex chemical systems. His group uses microfluidics, mathematical modeling, and, increasingly, AI and robotics to study chemical reaction networks.
Acknowledgments
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This work was funded by the European Research Council (ERC) under the European Union’s Horizon 2020 Research and Innovation Programme (ERC Adv. Grant Life-Inspired, Grant Agreement no. 833466), the European Union’s Horizon 2020 Research and Innovation Program (Grant Agreement No. 862081 (CLASSY)), and a Spinoza Grant of The Netherlands Organisation for Scientific Research (NWO).
| 1,6-DHN | 1,6-dihydroxynaphthalene |
| α-HL | α-hemolysin |
| ABTS | 2,2′-azinobis(3-ethylbenzothiazoline-6-sulfonic acid) |
| AcAc | acetylacetone |
| ACAT | acetyl-CoA acetyltransferase |
| acetyl-CoA | acetyl coenzyme A |
| AchE | acetylcholine esterase |
| ADH | alcohol dehydrogenase |
| ADP | adenosine 5/-diphosphate |
| AMG | amyloglucosidase |
| AMP | adenosine 5′-monophosphate |
| Ap | aminopeptidase |
| ATP | adenosine 5′-triphosphate |
| Cat | catalase |
| CBGA | cannabigerolic acid |
| CETCH | crotonyl-coenzyme A (CoA)/ethylmalonyl-CoA/hydroxybutyryl-CoA |
| CFPS-ME | cell-free protein synthesis-driven metabolic engineering |
| CFPS | cell-free protein synthesis |
| CFME | cell-free metabolic engineering |
| Cg | chymotrypsinogen |
| CoA | coenzyme A |
| COx | choline oxidase |
| Cr | chymotrypsin |
| CytC | cytochrome C |
| DASAs | donor–acceptor Stenhouse adducts |
| DNA | deoxyribonucleic acid |
| DS | dextransulfate |
| E. coli | Escherichia coli |
| Els | elastase |
| ERNs | enzymatic reaction networks |
| Est | esterase |
| FAD | flavin adenine dinucleotide |
| FADH2 | flavin adenine dinucleotide (hydroquinone form) |
| G6PDH | glucose-6-phosphate dehydrogenase |
| Gap | glyceraldehyde-3-phosphate dehydrogenase |
| Gnd | 6-phosphogluconate dehydrogenase |
| GOx | glucose oxidase |
| GPb | glycogen phosphorylase b |
| GPP | geranyl-pyrophosphate |
| GPPS | geranyl diphosphate synthase |
| GUVs | giant unilamellar vesicles |
| Hbd2 | hydroxybutyryl-CoA dehydrogenase |
| HMGR | hydroxymethylglutaryl-CoA reductase |
| HMGS | hydroxymethylglutaryl-CoA synthase |
| Hbd | hydroxybutyryl-CoA dehydrogenase |
| HK | hexokinase |
| HRP | horseradish peroxidase |
| IDI | isopentenyl pyrophosphate isomerase |
| LDH | l-lactate dehydrogenase |
| LO | l-lactate oxidase |
| LS | limonene synthase |
| M23 | mutant CBGA synthase |
| mGap | mutant glyceraldehyde-3-phosphate dehydrogenase |
| MK | mevalonate kinase |
| MM | Michaelis–Menten |
| MMP | matrix-metalloproteinases |
| NAD | nicotinamide adenine dinucleotide |
| NADP | nicotinamide adenine dinucleotide phosphate |
| NO | nitric oxide |
| NOxE | NADH oxidase |
| NphB | wild-type prenyltransferase |
| OA | olivetolic acid |
| PAAM | polyacrylamide |
| PBG | pentose–bifido–glycolysis |
| PDDA | poly(diallyldimethylammonium chloride) |
| PDH | pyruvate dehydrogenase |
| PE | phosphatidylethanolamine |
| PEG | polyethylene glycol |
| PEGMA | poly(ethylene glycol) methacrylate |
| PEN | polymerase–exonuclease–nickase |
| PEP | phosphoenolpyruvate |
| PG | phosphatidylglycerol |
| PGLA | poly(lactic-co-glycolic acid) |
| PhaC | PHB synthase |
| PHB | polyhydroxybutyrate |
| PKA | protein kinase A |
| PLA2 | phospholipase A2 |
| PMD | pyrophosphomevalonate decarboxylase |
| PMG | phosphoglucomutase |
| PMK | phosphomevalonate kinase |
| PO | peroxidase–oxidase |
| PP1 | protein phosphatase-1 |
| STI | soybean trypsin inhibitor |
| RLuc | renillaluciferase |
| RNA | ribonucleic acid |
| SOx | sarcosine oxidase |
| TaCo | tartronyl-CoA |
| Tg | trypsinogen |
| Tr | trypsin |
| UOx | urate oxidase |
| Ur | urease |
| UV | ultraviolet |
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- 8Kohyama, S.; Merino-Salomon, A.; Schwille, P. In vitro assembly, positioning and contraction of a division ring in minimal cells. Nat. Commun. 2022, 13 (1), 6098, DOI: 10.1038/s41467-022-33679-xGoogle Scholar8https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB38Xis1Oru7jO&md5=3c59deeb8978156ebc6202f7d269eb0cIn vitro assembly, positioning and contraction of a division ring in minimal cellsKohyama, Shunshi; Merino-Salomon, Adrian; Schwille, PetraNature Communications (2022), 13 (1), 6098CODEN: NCAOBW; ISSN:2041-1723. (Nature Portfolio)Constructing a minimal machinery for autonomous self-division of synthetic cells is a major goal of bottom-up synthetic biol. One paradigm has been the E. coli divisome, with the MinCDE protein system guiding assembly and positioning of a presumably contractile ring based on FtsZ and its membrane adaptor FtsA. Here, we demonstrate the full in vitro reconstitution of this machinery consisting of five proteins within lipid vesicles, allowing to observe the following sequence of events in real time: 1 Assembly of an isotropic filamentous FtsZ network, 2 its condensation into a ring-like structure, along with pole-to-pole mode selection of Min oscillations resulting in equatorial positioning, and 3 onset of ring constriction, deforming the vesicles from spherical shape. Besides demonstrating these essential features, we highlight the importance of decisive exptl. factors, such as macromol. crowding. Our results provide an exceptional showcase of the emergence of cell division in a minimal system, and may represent a step towards developing a synthetic cell.
- 9Fisher, A. K.; Freedman, B. G.; Bevan, D. R.; Senger, R. S. A review of metabolic and enzymatic engineering strategies for designing and optimizing performance of microbial cell factories. Comput. Struct. Biotechnol. J. 2014, 11 (18), 91– 99, DOI: 10.1016/j.csbj.2014.08.010Google Scholar9https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A280%3ADC%252BC2M3ms1Crsg%253D%253D&md5=a57acb850382c134d91c29404e683258A review of metabolic and enzymatic engineering strategies for designing and optimizing performance of microbial cell factoriesFisher Amanda K; Freedman Benjamin G; Senger Ryan S; Bevan David RComputational and structural biotechnology journal (2014), 11 (18), 91-9 ISSN:2001-0370.Microbial cell factories (MCFs) are of considerable interest to convert low value renewable substrates to biofuels and high value chemicals. This review highlights the progress of computational models for the rational design of an MCF to produce a target bio-commodity. In particular, the rational design of an MCF involves: (i) product selection, (ii) de novo biosynthetic pathway identification (i.e., rational, heterologous, or artificial), (iii) MCF chassis selection, (iv) enzyme engineering of promiscuity to enable the formation of new products, and (v) metabolic engineering to ensure optimal use of the pathway by the MCF host. Computational tools such as (i) de novo biosynthetic pathway builders, (ii) docking, (iii) molecular dynamics (MD) and steered MD (SMD), and (iv) genome-scale metabolic flux modeling all play critical roles in the rational design of an MCF. Genome-scale metabolic flux models are of considerable use to the design process since they can reveal metabolic capabilities of MCF hosts. These can be used for host selection as well as optimizing precursors and cofactors of artificial de novo biosynthetic pathways. In addition, recent advances in genome-scale modeling have enabled the derivation of metabolic engineering strategies, which can be implemented using the genomic tools reviewed here as well.
- 10Hirschi, S.; Ward, T. R.; Meier, W. P.; Muller, D. J.; Fotiadis, D. Synthetic biology: bottom-up assembly of molecular systems. Chem. Rev. 2022, 122 (21), 16294– 16328, DOI: 10.1021/acs.chemrev.2c00339Google Scholar10https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB38XisFWmtrjF&md5=598a8e43708beb84e525bd06c876310bSynthetic Biology: Bottom-Up Assembly of Molecular SystemsHirschi, Stephan; Ward, Thomas R.; Meier, Wolfgang P.; Muller, Daniel J.; Fotiadis, DimitriosChemical Reviews (Washington, DC, United States) (2022), 122 (21), 16294-16328CODEN: CHREAY; ISSN:0009-2665. (American Chemical Society)A review. The bottom-up assembly of biol. and chem. components opens exciting opportunities to engineer artificial vesicular systems for applications with previously unmet requirements. The modular combination of scaffolds and functional building blocks enables the engineering of complex systems with biomimetic or new-to-nature functionalities. Inspired by the compartmentalized organization of cells and organelles, lipid or polymer vesicles are widely used as model membrane systems to investigate the translocation of solutes and the transduction of signals by membrane proteins. The bottom-up assembly and functionalization of such artificial compartments enables full control over their compn. and can thus provide specifically optimized environments for synthetic biol. processes. This review aims to inspire future endeavors by providing a diverse toolbox of mol. modules, engineering methodologies, and different approaches to assemble artificial vesicular systems. Important tech. and practical aspects are addressed and selected applications are presented, highlighting particular achievements and limitations of the bottom-up approach. Complementing the cutting-edge technol. achievements, fundamental aspects are also discussed to cater to the inherently diverse background of the target audience, which results from the interdisciplinary nature of synthetic biol. The engineering of proteins as functional modules and the use of lipids and block copolymers as scaffold modules for the assembly of functionalized vesicular systems are explored in detail. Particular emphasis is placed on ensuring the controlled assembly of these components into increasingly complex vesicular systems. Finally, all descriptions are presented in the greater context of engineering valuable synthetic biol. systems for applications in biocatalysis, biosensing, bioremediation, or targeted drug delivery.
- 11Boguñá, M.; Bonamassa, I.; De Domenico, M.; Havlin, S.; Krioukov, D.; Serrano, M. Á. Network geometry. Nat. Rev. Phys. 2021, 3 (2), 114– 135, DOI: 10.1038/s42254-020-00264-4Google ScholarThere is no corresponding record for this reference.
- 12Ravasz, E.; Somera, A. L.; Mongru, D. A.; Oltvai, Z. N.; Barabási, A.-L. Hierarchical organization of modularity in metabolic networks. Science 2002, 297, 1551– 1555, DOI: 10.1126/science.1073374Google Scholar12https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD38XmslSjtLs%253D&md5=a7cf905a9f21a5fc614694635efa1f14Hierarchical organization of modularity in metabolic networksRavasz, E.; Somera, A. L.; Mongru, D. A.; Oltvai, Z. N.; Barabasi, A.-L.Science (Washington, DC, United States) (2002), 297 (5586), 1551-1555CODEN: SCIEAS; ISSN:0036-8075. (American Association for the Advancement of Science)Spatially or chem. isolated functional modules composed of several cellular components and carrying discrete functions are considered fundamental building blocks of cellular organization, but their presence in highly integrated biochem. networks lacks quant. support. Here, we show that the metabolic networks of 43 distinct organisms are organized into many small, highly connected topol. modules that combine in a hierarchical manner into larger, less cohesive units, with their no. and degree of clustering following a power law. Within Escherichia coli, the uncovered hierarchical modularity closely overlaps with known metabolic functions. The identified network architecture may be generic to system-level cellular organization.
- 13Barabási, A.-L.; Oltvai, Z. N. Network biology: understanding the cell’s functional organization. Nat. Rev. Genet. 2004, 5 (2), 101– 113, DOI: 10.1038/nrg1272Google Scholar13https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD2cXovV2mtg%253D%253D&md5=2ac9082dfaf88d56da1fdd75b9edddb4Network biology: Understanding the cell's functional organizationBarabasi, Albert-Laszlo; Oltvai, Zoltan N.Nature Reviews Genetics (2004), 5 (2), 101-113CODEN: NRGAAM; ISSN:1471-0056. (Nature Publishing Group)A key aim of postgenomic biomedical research is to systematically catalog all mols. and their interactions within a living cell. There is a clear need to understand how these mols. and the interactions between them det. the function of this enormously complex machinery, both in isolation and when surrounded by other cells. Rapid advances in network biol. indicate that cellular networks are governed by universal laws and offer a new conceptual framework that could potentially revolutionize our view of biol. and disease pathologies in the twenty-first century.
- 14Serrano, M. A.; Boguñá, M.; Sagués, F. Uncovering the hidden geometry behind metabolic networks. Mol. Biosyst. 2012, 8 (3), 843– 850, DOI: 10.1039/c2mb05306cGoogle Scholar14https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC38XitVSmtrk%253D&md5=7f0d358828c360215e2e49c1fc362131Uncovering the hidden geometry behind metabolic networksSerrano, M. Angeles; Boguna, Marian; Sagues, FrancescMolecular BioSystems (2012), 8 (3), 843-850CODEN: MBOIBW; ISSN:1742-2051. (Royal Society of Chemistry)Metab. is a fascinating cell machinery underlying life and disease and genome-scale reconstructions provide us with a captivating view of its complexity. However, deciphering the relationship between metabolic structure and function remains a major challenge. In particular, turning obsd. structural regularities into organizing principles underlying systemic functions is a crucial task that can be significantly addressed after endowing complex network representations of metab. with the notion of geometric distance. Here, we design a cartog. map of metabolic networks by embedding them into a simple geometry that provides a natural explanation for their obsd. network topol. and that codifies node proximity as a measure of hidden structural similarities. We assume a simple and general connectivity law that gives more probability of interaction to metabolite/reaction pairs which are closer in the hidden space. Remarkably, we find an astonishing congruency between the architecture of E. coli and human cell metabs. and the underlying geometry. In addn., the formalism unveils a backbone-like structure of connected biochem. pathways on the basis of a quant. cross-talk. Pathways thus acquire a new perspective which challenges their classical view as self-contained functional units.
- 15Kim, H.; Smith, H. B.; Mathis, C.; Raymond, J.; Walker, S. Universal scaling across biochemical networks on Earth. Sci. Adv. 2019, 5, eaau0149, DOI: 10.1126/sciadv.aau0149Google ScholarThere is no corresponding record for this reference.
- 16Gagler, D. C.; Karas, B.; Kempes, C. P.; Malloy, J.; Mierzejewski, V.; Goldman, A. D.; Kim, H.; Walker, S. I. Scaling laws in enzyme function reveal a new kind of biochemical universality. Proc. Natl. Acad. Sci. U. S. A. 2022, 119 (9), e2106655119, DOI: 10.1073/pnas.2106655119Google Scholar16https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB38Xns1Kltrg%253D&md5=d18d6cb44a63dc6882871166265aa65aScaling laws in enzyme function reveal a new kind of biochemical universalityGagler, Dylan C.; Karas, Bradley; Kempes, Christopher P.; Malloy, John; Mierzejewski, Veronica; Goldman, Aaron D.; Kim, Hyunju; Walker, Sara I.Proceedings of the National Academy of Sciences of the United States of America (2022), 119 (9), e2106655119CODEN: PNASA6; ISSN:0027-8424. (National Academy of Sciences)All life on Earth is unified by its use of a shared set of component chem. compds. and reactions, providing a detailed model for universal biochem. However, this notion of universality is specific to known biochem. and does not allow quant. predictions about examples not yet obsd. Here, we introduce a more generalizable concept of biochem. universality that is more akin to the kind of universality found in physics. Using annotated genomic datasets including an ensemble of 11,955 metagenomes, 1,282 archaea, 11,759 bacteria, and 200 eukaryotic taxa, we show how enzyme functions form universality classes with common scaling behavior in their relative abundances across the datasets. We verify that these scaling laws are not explained by the presence of compds., reactions, and enzyme functions shared across known examples of life. We demonstrate how these scaling laws can be used as a tool for inferring properties of ancient life by comparing their predictions with a consensus model for the last universal common ancestor (LUCA). We also illustrate how network analyses shed light on the functional principles underlying the obsd. scaling behaviors. Together, our results establish the existence of a new kind of biochem. universality, independent of the details of life on Earth's component chem., with implications for guiding our search for missing biochem. diversity on Earth or for biochemistries that might deviate from the exact chem. makeup of life as we know it, such as at the origins of life, in alien environments, or in the design of synthetic life.
- 17Guell, O.; Sagues, F.; Serrano, M. A. Essential plasticity and redundancy of metabolism unveiled by synthetic lethality analysis. PLoS Comput. Biol. 2014, 10 (5), e1003637, DOI: 10.1371/journal.pcbi.1003637Google Scholar17https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2cXhsVGkt7jP&md5=a89b8a58deb751d9506ef1432446fcddEssential plasticity and redundancy of metabolism unveiled by synthetic lethality analysisGuell, Oriol; Sagues, Francesc; Serrano, M. AngelesPLoS Computational Biology (2014), 10 (5), e1003637/1-e1003637/10, 10 pp.CODEN: PCBLBG; ISSN:1553-7358. (Public Library of Science)We unravel how functional plasticity and redundancy are essential mechanisms underlying the ability to survive of metabolic networks. We perform an exhaustive computational screening of synthetic lethal reaction pairs in Escherichia coli in a minimal medium and we find that synthetic lethal pairs divide in two different groups depending on whether the synthetic lethal interaction works as a backup or as a parallel use mechanism, the first corresponding to essential plasticity and the second to essential redundancy. In E. coli, the anal. of pathways entanglement through essential redundancy supports the view that synthetic lethality affects preferentially a single function or pathway. In contrast, essential plasticity, the dominant class, tends to be inter-pathway but strongly localized and unveils Cell Envelope Biosynthesis as an essential backup for Membrane Lipid Metab. When comparing E. coli and Mycoplasma pneumoniae, we find that the metabolic networks of the two organisms exhibit a large difference in the relative importance of plasticity and redundancy which is consistent with the conjecture that plasticity is a sophisticated mechanism that requires a complex organization. Finally, coessential reaction pairs are explored in different environmental conditions to uncover the interplay between the two mechanisms. We find that synthetic lethal interactions and their classification in plasticity and redundancy are basically insensitive to medium compn., and are highly conserved even when the environment is enriched with nonessential compds. or overconstrained to decrease max. biomass formation.
- 18Sambamoorthy, G.; Raman, K. Understanding the evolution of functional redundancy in metabolic networks. Bioinformatics 2018, 34 (17), i981– i987, DOI: 10.1093/bioinformatics/bty604Google Scholar18https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC1MXhtVamu77N&md5=dc84099605e8635841180083ca9daca9Understanding the evolution of functional redundancy in metabolic networksSambamoorthy, Gayathri; Raman, KarthikBioinformatics (2018), 34 (17), i981-i987CODEN: BOINFP; ISSN:1367-4811. (Oxford University Press)Metabolic networks have evolved to reduce the disruption of key metabolic pathways by the establishment of redundant genes/reactions. Synthetic lethals in metabolic networks provide a window to study these functional redundancies. While synthetic lethals have been previously studied in different organisms, there has been no study on how the synthetic lethals are shaped during adaptation/evolution. To understand the adaptive functional redundancies that exist in metabolic networks, we here explore a vast space of 'random' metabolic networks evolved on a glucose environment. We examine essential and synthetic lethal reactions in these random metabolic networks, evaluating over 39 billion phenotypes using an efficient algorithm previously developed in our lab, Fast-SL. We establish that nature tends to harbor higher levels of functional redundancies compared with random networks. We then examd. the propensity for different reactions to compensate for one another and show that certain key metabolic reactions that are necessary for growth in a particular growth medium show much higher redundancies, and can partner with hundreds of different reactions across the metabolic networks that we studied. We also observe that certain redundancies are unique to environments while some others are obsd. in all environments. Interestingly, we observe that even very diverse reactions, such as those belonging to distant pathways, show synthetic lethality, illustrating the distributed nature of robustness in metab. Our study paves the way for understanding the evolution of redundancy in metabolic networks, and sheds light on the varied compensation mechanisms that serve to enhance robustness.
- 19Alon, U. Network motifs: theory and experimental approaches. Nat. Rev. Genet. 2007, 8, 450– 461, DOI: 10.1038/nrg2102Google Scholar19https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD2sXlsVSktro%253D&md5=ad292568100bf626f7076dee816826ffNetwork motifs: theory and experimental approachesAlon, UriNature Reviews Genetics (2007), 8 (6), 450-461CODEN: NRGAAM; ISSN:1471-0056. (Nature Publishing Group)A review. Transcription regulation networks control the expression of genes. The transcription networks of well-studied microorganisms appear to be made up of a small set of recurring regulation patterns, called network motifs. The same network motifs have recently been found in diverse organisms from bacteria to humans, suggesting that they serve as basic building blocks of transcription networks. Here, the author reviews network motifs and their functions, with an emphasis on exptl. studies. Network motifs in other biol. networks are also mentioned, including signaling and neuronal networks.
- 20Goldbeter, A.; Koshland, D. E. An amplified sensitivity arising from covalent modification in biological systems. Proc. Natl. Acad. Sci. U. S. A. 1981, 78, 6840– 6844, DOI: 10.1073/pnas.78.11.6840Google Scholar20https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaL38XivVOi&md5=949ebb2ad47f4839b5e72c05abbc19f9An amplified sensitivity arising from covalent modification in biological systemsGoldbeter, Albert; Koshland, Daniel E., Jr.Proceedings of the National Academy of Sciences of the United States of America (1981), 78 (11), 6840-4CODEN: PNASA6; ISSN:0027-8424.The transient and steady-state behavior of a reversible covalent modification system is examd. When the modifying enzymes operate outside the region of 1st-order kinetics, small percentage changes in the concn. of the effector controlling either of the modifying enzymes can give much larger percentage changes in the amt. of modified protein. This amplification of the response to a stimulus can provide addnl. sensitivity in biol. control, equiv. to that of allosteric proteins with high Hill coeffs.
- 21Koshland, D. E., Jr.; Goldbeter, A.; Stock, J. B. Amplification and adaptation in regulatory and sensory systems. Science 1982, 217, 220– 225, DOI: 10.1126/science.7089556Google Scholar21https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaL38XkvVOisLs%253D&md5=e2492db5ae8999e0775f3bd576e1cc98Amplification and adaptation in regulatory and sensory systemsKoshland, Daniel E., Jr.; Goldbeter, Albert; Stock, Jeffry B.Science (Washington, DC, United States) (1982), 217 (4556), 220-5CODEN: SCIEAS; ISSN:0036-8075.A review with 31 refs. on how biol. systems respond to sensory inputs and changing metabolic conditions both by amplifying signals and by adapting to them.
- 22Barkai, N.; Leibler, S. Robustness in simple biochemical networks. Nature 1997, 387, 913– 917, DOI: 10.1038/43199Google Scholar22https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaK2sXkt1Kiur8%253D&md5=76427ddf0b596f4c18e024acb21182cfRobustness in simple biochemical networksBarkai, N.; Leibler, S.Nature (London) (1997), 387 (6636), 913-917CODEN: NATUAS; ISSN:0028-0836. (Macmillan Magazines)A review, with 21 refs. Cells use complex networks of interacting mol. components to transfer and process information. These "computational devices of living cells" are responsible for many important cellular processes, including cell-cycle regulation and signal transduction. Here we address the issue of the sensitivity of the networks to variations in their biochem. parameters. We propose a mechanism for robust adaptation in simple signal transduction networks. We show that this mechanism applies in particular to bacterial chemotaxis. This is demonstrated within a quant. model which explains, in a unified way, many aspects of chemotaxis, including proper responses to chem. gradients. The adaptation property is a consequence of the networks's connectivity and does not require the 'fine-tuning of parameters. We argue that the key properties of biochem. networks should be robust to ensure their proper functioning.
- 23Milo, R.; Shen-Orr, S.; Itzkovitz, S.; Kashtan, N.; Chklovskii, D.; Alon, U. Network motifs: simple building blocks of complex networks. Science. 2002, 298, 824– 827, DOI: 10.1126/science.298.5594.824Google Scholar23https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD38XotFSntb4%253D&md5=a83828b13c33bee8c7528922d91502b7Network Motifs: Simple Building Blocks of Complex NetworksMilo, R.; Shen-Orr, S.; Itzkovitz, S.; Kashtan, N.; Chklovskii, D.; Alon, U.Science (Washington, DC, United States) (2002), 298 (5594), 824-827CODEN: SCIEAS; ISSN:0036-8075. (American Association for the Advancement of Science)Complex networks are studied across many fields of science. To uncover their structural design principles, we defined "network motifs," patterns of interconnections occurring in complex networks at nos. that are significantly higher than those in randomized networks. We found such motifs in networks from biochem., neurobiol., ecol., and engineering. The motifs shared by ecol. food webs were distinct from the motifs shared by the genetic networks of Escherichia coli and Saccharomyces cerevisiae or from those found in the World Wide Web. Similar motifs were found in networks that perform information processing, even though they describe elements as different as biomols. within a cell and synaptic connections between neurons in Caenorhabditis elegans. Motifs may thus define universal classes of networks. This approach may uncover the basic building blocks of most networks.
- 24Tyson, J. J.; Novak, B. Functional motifs in biochemical reaction networks. Annu. Rev. Phys. Chem. 2010, 61, 219– 240, DOI: 10.1146/annurev.physchem.012809.103457Google Scholar24https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3cXmt1ajsL8%253D&md5=8b514d486aa599e24f0185cfface6067Functional motifs in biochemical reaction networksTyson, John J.; Novak, BelaAnnual Review of Physical Chemistry (2010), 61 (), 219-240CODEN: ARPLAP; ISSN:0066-426X. (Annual Reviews Inc.)A review. The signal-response characteristics of a living cell are detd. by complex networks of interacting genes, proteins, and metabolites. Understanding how cells respond to specific challenges, how these responses are contravened in diseased cells, and how to intervene pharmacol. in the decision-making processes of cells requires an accurate theory of the information-processing capabilities of macromol. regulatory networks. Adopting an engineer's approach to control systems, we ask whether realistic cellular control networks can be decompd. into simple regulatory motifs that carry out specific functions in a cell. We show that such functional motifs exist and review the exptl. evidence that they control cellular responses as expected.
- 25Novak, B.; Tyson, J. J. Design principles of biochemical oscillators. Nat. Rev. Mol. Cell Biol. 2008, 9 (12), 981– 991, DOI: 10.1038/nrm2530Google Scholar25https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD1cXhsVWhurnL&md5=2014e5749f715415e81b8afeec1b6008Design principles of biochemical oscillatorsNovak, Bela; Tyson, John J.Nature Reviews Molecular Cell Biology (2008), 9 (12), 981-991CODEN: NRMCBP; ISSN:1471-0072. (Nature Publishing Group)A review. Cellular rhythms are generated by complex interactions among genes, proteins and metabolites. They are used to control every aspect of cell physiol., from signaling, motility and development to growth, division and death. We consider specific examples of oscillatory processes and discuss four general requirements for biochem. oscillations: neg. feedback, time delay, sufficient nonlinearity of the reaction kinetics and proper balancing of the timescales of opposing chem. reactions. Pos. feedback is one mechanism to delay the neg.-feedback signal. Biol. oscillators can be classified according to the topol. of the pos.- and neg.-feedback loops in the underlying regulatory mechanism.
- 26Araujo, R. P.; Liotta, L. A. Universal structures for adaptation in biochemical reaction networks. Nat. Commun. 2023, 14 (1), 2251, DOI: 10.1038/s41467-023-38011-9Google Scholar26https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB3sXos1yqsrk%253D&md5=51df0502a1ff9903b4006ae2e353234eUniversal structures for adaptation in biochemical reaction networksAraujo, Robyn P.; Liotta, Lance A.Nature Communications (2023), 14 (1), 2251CODEN: NCAOBW; ISSN:2041-1723. (Nature Portfolio)At the mol. level, the evolution of life is driven by the generation and diversification of adaptation mechanisms. A universal description of adaptation-capable chem. reaction network (CRN) structures has remained elusive until now, since currently-known criteria for adaptation apply only to a tiny subset of possible CRNs. Here we identify the definitive structural requirements that characterize all adaptation-capable collections of interacting mols., however large or complex. We show that these network structures implement a form of integral control in which multiple independent integrals can collaborate to confer the capacity for adaptation on specific mols. Using an algebraic algorithm informed by these findings, we demonstrate the existence of embedded integrals in a variety of biol. important CRNs that have eluded previous methods, and for which adaptation has been obsd. exptl. This definitive picture of biol. adaptation at the level of intermol. interactions represents a blueprint for adaptation-capable signaling networks across all domains of life, and for the design of synthetic biosystems.
- 27Shellman, E. R.; Burant, C. F.; Schnell, S. Network motifs provide signatures that characterize metabolism. Mol. Biosyst. 2013, 9 (3), 352– 360, DOI: 10.1039/c2mb25346aGoogle Scholar27https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3sXitVequ70%253D&md5=d1d810be1fa3c4459ff0e74fdb3d2f5fNetwork motifs provide signatures that characterize metabolismShellman, Erin R.; Burant, Charles F.; Schnell, SantiagoMolecular BioSystems (2013), 9 (3), 352-360CODEN: MBOIBW; ISSN:1742-2051. (Royal Society of Chemistry)Motifs are repeating patterns that det. the local properties of networks. In this work, we characterized all 3-node motifs using enzyme commission nos. of the International Union of Biochem. and Mol. Biol. to show that motif abundance is related to biochem. function. Further, we present a comparative anal. of motif distributions in the metabolic networks of 21 species across six kingdoms of life. We found the distribution of motif abundances to be similar between species, but unique across cellular organelles. Finally, we show that motifs are able to capture inter-species differences in metabolic networks and that mol. differences between some biol. species are reflected by the distribution of motif abundances in metabolic networks.
- 28Beber, M. E.; Fretter, C.; Jain, S.; Sonnenschein, N.; Muller-Hannemann, M.; Hutt, M. T. Artefacts in statistical analyses of network motifs: general framework and application to metabolic networks. J. R. Soc. Interface 2012, 9 (77), 3426– 3435, DOI: 10.1098/rsif.2012.0490Google Scholar28https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A280%3ADC%252BC38fotl2rtg%253D%253D&md5=e778ca3b28d1d464a65eaaaf70e21e5bArtefacts in statistical analyses of network motifs: general framework and application to metabolic networksBeber Moritz Emanuel; Fretter Christoph; Jain Shubham; Sonnenschein Nikolaus; Muller-Hannemann Matthias; Hutt Marc-ThorstenJournal of the Royal Society, Interface (2012), 9 (77), 3426-35 ISSN:.Few-node subgraphs are the smallest collective units in a network that can be investigated. They are beyond the scale of individual nodes but more local than, for example, communities. When statistically over- or under-represented, they are called network motifs. Network motifs have been interpreted as building blocks that shape the dynamic behaviour of networks. It is this promise of potentially explaining emergent properties of complex systems with relatively simple structures that led to an interest in network motifs in an ever-growing number of studies and across disciplines. Here, we discuss artefacts in the analysis of network motifs arising from discrepancies between the network under investigation and the pool of random graphs serving as a null model. Our aim was to provide a clear and accessible catalogue of such incongruities and their effect on the motif signature. As a case study, we explore the metabolic network of Escherichia coli and show that only by excluding ever more artefacts from the motif signature a strong and plausible correlation with the essentiality profile of metabolic reactions emerges.
- 29Piephoff, D. E.; Wu, J.; Cao, J. Conformational nonequilibrium enzyme kinetics: generalized Michaelis-Menten equation. J. Phys. Chem. Lett. 2017, 8 (15), 3619– 3623, DOI: 10.1021/acs.jpclett.7b01210Google Scholar29https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2sXht1Sgu7rF&md5=9f5fa6ac5ff779cfe901d56b6dd7eaf0Conformational Nonequilibrium Enzyme Kinetics: Generalized Michaelis-Menten EquationPiephoff, D. Evan; Wu, Jianlan; Cao, JianshuJournal of Physical Chemistry Letters (2017), 8 (15), 3619-3623CODEN: JPCLCD; ISSN:1948-7185. (American Chemical Society)In a conformational nonequil. steady state (cNESS), enzyme turnover is modulated by the underlying conformational dynamics. On the basis of a discrete kinetic network model, we use the integrated probability flux balance method to derive the cNESS turnover rate for a conformation-modulated enzymic reaction. The traditional Michaelis-Menten (MM) rate equation is extended to a generalized form, which includes non-MM corrections induced by conformational population currents within combined cyclic kinetic loops. When conformational detailed balance is satisfied, the turnover rate reduces to the MM functional form, explaining its validity for many enzymic systems. For the first time, a one-to-one correspondence is established between non-MM terms and combined cyclic loops with unbalanced conformational currents. Cooperativity resulting from nonequil. conformational dynamics can be achieved in enzymic reactions, and we provide a novel, rigorous means of predicting and characterizing such behavior. Our generalized MM equation affords a systematic approach for exploring cNESS enzyme kinetics.
- 30Rohwer, J. M.; Hanekom, A. J.; Crous, C.; Snoep, J. L.; Hofmeyr, J. H. Evaluation of a simplified generic bi-substrate rate equation for computational systems biology. Syst. Biol. (Stevenage) 2006, 153 (5), 338– 41, DOI: 10.1049/ip-syb:20060026Google Scholar30https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A280%3ADC%252BD28rmtVyktQ%253D%253D&md5=a8759b27f01cd966cccbffc6dd2085a4Evaluation of a simplified generic bi-substrate rate equation for computational systems biologyRohwer J M; Hanekom A J; Crous C; Snoep J L; Hofmeyr J H SSystems biology (2006), 153 (5), 338-41 ISSN:1741-2471.The evaluation of a generic simplified bi-substrate enzyme kinetic equation, whose derivation is based on the assumption of equilibrium binding of substrates and products in random order, is described. This equation is much simpler than the mechanistic (ordered and ping-pong) models, in that it contains fewer parameters (that is, no K(i) values for the substrates and products). The generic equation fits data from both the ordered and the ping-pong models well over a wide range of substrate and product concentrations. In the cases where the fit is not perfect, an improved fit can be obtained by considering the rate equation for only a single set of product concentrations. Due to its relative simplicity in comparison to the mechanistic models, this equation will be useful for modelling bi-substrate reactions in computational systems biology.
- 31Transtrum, M. K.; Machta, B. B.; Brown, K. S.; Daniels, B. C.; Myers, C. R.; Sethna, J. P. Perspective: sloppiness and emergent theories in physics, biology, and beyond. J. Chem. Phys. 2015, 143 (1), 010901 DOI: 10.1063/1.4923066Google Scholar31https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2MXhtFSktr3N&md5=558fce9562787043593223a854d25541Perspective: Sloppiness and emergent theories in physics, biology, and beyondTranstrum, Mark K.; Machta, Benjamin B.; Brown, Kevin S.; Daniels, Bryan C.; Myers, Christopher R.; Sethna, James P.Journal of Chemical Physics (2015), 143 (1), 010901/1-010901/13CODEN: JCPSA6; ISSN:0021-9606. (American Institute of Physics)Large scale models of phys. phenomena demand the development of new statistical and computational tools in order to be effective. Many such models are "sloppy," i.e., exhibit behavior controlled by a relatively small no. of parameter combinations. We review an information theoretic framework for analyzing sloppy models. This formalism is based on the Fisher information matrix, which is interpreted as a Riemannian metric on a parameterized space of models. Distance in this space is a measure of how distinguishable two models are based on their predictions. Sloppy model manifolds are bounded with a hierarchy of widths and extrinsic curvatures. The manifold boundary approxn. can ext. the simple, hidden theory from complicated sloppy models. We attribute the success of simple effective models in physics as likewise emerging from complicated processes exhibiting a low effective dimensionality. We discuss the ramifications and consequences of sloppy models for biochem. and science more generally. We suggest that the reason our complex world is understandable is due to the same fundamental reason: simple theories of macroscopic behavior are hidden inside complicated microscopic processes. (c) 2015 American Institute of Physics.
- 32Gutenkunst, R. N.; Waterfall, J. J.; Casey, F. P.; Brown, K. S.; Myers, C. R.; Sethna, J. P. Universally sloppy parameter sensitivities in systems biology models. PLoS Comput. Biol. 2007, 3 (10), e189, DOI: 10.1371/journal.pcbi.0030189Google ScholarThere is no corresponding record for this reference.
- 33Transtrum, M. K.; Qiu, P. Model reduction by manifold boundaries. Phys. Rev. Lett. 2014, 113 (9), 098701 DOI: 10.1103/PhysRevLett.113.098701Google Scholar33https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2cXhslSht7nO&md5=00bb730d154a553d6ae135f505f0c54fModel reduction by manifold boundariesTranstrum, Mark K.; Qiu, PengPhysical Review Letters (2014), 113 (9), 098701/1-098701/6CODEN: PRLTAO; ISSN:0031-9007. (American Physical Society)Understanding the collective behavior of complex systems from their basic components is a difficult yet fundamental problem in science. Existing model redn. techniques are either applicable under limited circumstances or produce "black boxes" disconnected from the microscopic physics. We propose a new approach by translating the model redn. problem for an arbitrary statistical model into a geometric problem of constructing a low-dimensional, submanifold approxn. to a high-dimensional manifold. When models are overly complex, we use the observation that the model manifold is bounded with a hierarchy of widths and propose using the boundaries as submanifold approxns. We refer to this approach as the manifold boundary approxn. method. We apply this method to several models, including a sum of exponentials, a dynamical systems model of protein signaling, and a generalized Ising model. By focusing on parameters rather than phys. degrees of freedom, the approach unifies many other model redn. techniques, such as singular limits, equil. approxns., and the renormalization group, while expanding the domain of tractable models. The method produces a series of approxns. that decrease the complexity of the model and reveal how microscopic parameters are systematically "compressed" into a few macroscopic degrees of freedom, effectively building a bridge between the microscopic and the macroscopic descriptions.
- 34van de Schoot, R.; Depaoli, S.; King, R.; Kramer, B.; Märtens, K.; Tadesse, M. G.; Vannucci, M.; Gelman, A.; Veen, D.; Willemsen, J.; Yau, C. Bayesian statistics and modelling. Nat. Rev. Methods Primers 2021, 1, 16, DOI: 10.1038/s43586-020-00001-2Google Scholar34https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB38XjsVGgur0%253D&md5=fc3e76187d5fa0268f12409a1960dacfPublisher Correction: Bayesian statistics and modellingvan de Schoot, Rens; Depaoli, Sarah; King, Ruth; Kramer, Bianca; Maertens, Kaspar; Tadesse, Mahlet G.; Vannucci, Marina; Gelman, Andrew; Veen, Duco; Willemsen, Joukje; Yau, ChristopherNature Reviews Methods Primers (2021), 1 (1), 16CODEN: NRMPAT; ISSN:2662-8449. (Nature Portfolio)A Correction to this paper has been published: https://doi.org/10.1038/s43586-021-00017-2.
- 35Choi, B.; Rempala, G. A.; Kim, J. K. Beyond the Michaelis-Menten equation: accurate and efficient estimation of enzyme kinetic parameters. Sci. Rep. 2017, 7 (1), 17018, DOI: 10.1038/s41598-017-17072-zGoogle Scholar35https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A280%3ADC%252BC1M3otlWmug%253D%253D&md5=24801e3eb36b6bd649d17583ae5db895Beyond the Michaelis-Menten equation: Accurate and efficient estimation of enzyme kinetic parametersChoi Boseung; Rempala Grzegorz A; Kim Jae KyoungScientific reports (2017), 7 (1), 17018 ISSN:.Examining enzyme kinetics is critical for understanding cellular systems and for using enzymes in industry. The Michaelis-Menten equation has been widely used for over a century to estimate the enzyme kinetic parameters from reaction progress curves of substrates, which is known as the progress curve assay. However, this canonical approach works in limited conditions, such as when there is a large excess of substrate over enzyme. Even when this condition is satisfied, the identifiability of parameters is not always guaranteed, and often not verifiable in practice. To overcome such limitations of the canonical approach for the progress curve assay, here we propose a Bayesian approach based on an equation derived with the total quasi-steady-state approximation. In contrast to the canonical approach, estimates obtained with this proposed approach exhibit little bias for any combination of enzyme and substrate concentrations. Importantly, unlike the canonical approach, an optimal experiment to identify parameters with certainty can be easily designed without any prior information. Indeed, with this proposed design, the kinetic parameters of diverse enzymes with disparate catalytic efficiencies, such as chymotrypsin, fumarase, and urease, can be accurately and precisely estimated from a minimal amount of timecourse data. A publicly accessible computational package performing such accurate and efficient Bayesian inference for enzyme kinetics is provided.
- 36Linden, N. J.; Kramer, B.; Rangamani, P. Bayesian parameter estimation for dynamical models in systems biology. PLoS Comput. Biol. 2022, 18 (10), e1010651, DOI: 10.1371/journal.pcbi.1010651Google Scholar36https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB38XivVanu7bF&md5=06868bf56a1deae8d2eceeb29755e8b1Bayesian parameter estimation for dynamical models in systems biologyLinden, Nathaniel J.; Kramer, Boris; Rangamani, PadminiPLoS Computational Biology (2022), 18 (10), e1010651CODEN: PCBLBG; ISSN:1553-7358. (Public Library of Science)Dynamical systems modeling, particularly via systems of ordinary differential equations, has been used to effectively capture the temporal behavior of different biochem. components in signal transduction networks. Despite the recent advances in exptl. measurements, including sensor development and '-omics' studies that have helped populate protein-protein interaction networks in great detail, modeling in systems biol. lacks systematic methods to est. kinetic parameters and quantify assocd. uncertainties. This is because of multiple reasons, including sparse and noisy exptl. measurements, lack of detailed mol. mechanisms underlying the reactions, and missing biochem. interactions. Addnl., the inherent nonlinearities with respect to the states and parameters assocd. with the system of differential equations further compd. the challenges of parameter estn. In this study, we propose a comprehensive framework for Bayesian parameter estn. and complete quantification of the effects of uncertainties in the data and models. We apply these methods to a series of signaling models of increasing math. complexity. Systematic anal. of these dynamical systems showed that parameter estn. depends on data sparsity, noise level, and model structure, including the existence of multiple steady states. These results highlight how focused uncertainty quantification can enrich systems biol. modeling and enable addnl. quant. analyses for parameter estn.
- 37Baltussen, M. G.; van de Wiel, J.; Fernandez Regueiro, C. L.; Jakstaite, M.; Huck, W. T. S. A Bayesian Approach to Extracting kinetic information from artificial enzymatic networks. Anal. Chem. 2022, 94 (20), 7311– 7318, DOI: 10.1021/acs.analchem.2c00659Google Scholar37https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB38Xht1Ghu7fL&md5=68b8f5735f0b650622ff30064c2ea456A Bayesian approach to extracting kinetic information from artificial enzymatic networksBaltussen, Mathieu G.; van de Wiel, Jeroen; Fernandez Regueiro, Cristina Lia; Jakstaite, Migle; Huck, Wilhelm T. S.Analytical Chemistry (Washington, DC, United States) (2022), 94 (20), 7311-7318CODEN: ANCHAM; ISSN:0003-2700. (American Chemical Society)In order to create artificial enzymic networks capable of increasingly complex behavior, an improved methodol. in understanding and controlling the kinetics of these networks is needed. Here, we introduce a Bayesian anal. method allowing for the accurate inference of enzyme kinetic parameters and detn. of most likely reaction mechanisms, by combining data from different expts. and network topologies in a single probabilistic anal. framework. This Bayesian approach explicitly allows us to continuously improve our parameter ests. and behavior predictions by iteratively adding new data to our models, while automatically taking into account uncertainties introduced by the exptl. setups or the chem. processes in general. We demonstrate the potential of this approach by characterizing systems of enzymes compartmentalized in beads inside flow reactors. The methods we introduce here provide a new approach to the design of increasingly complex artificial enzymic networks, making the design of such networks more efficient, and robust against the accumulation of exptl. errors.
- 38Gábor, A.; Villaverde, A. F.; Banga, J. R. Parameter identifiability analysis and visualization in large-scale kinetic models of biosystems. BMC Syst. Biol. 2017, 11 (1), 54, DOI: 10.1186/s12918-017-0428-yGoogle Scholar38https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC1cXhtlSjurzP&md5=502fde6dca6c0ea22c784ac57f79f396Parameter identifiability analysis and visualization in large-scale kinetic models of biosystemsGabor, Attila; Villaverde, Alejandro F.; Banga, Julio R.BMC Systems Biology (2017), 11 (), 54/1-54/16CODEN: BSBMCC; ISSN:1752-0509. (BioMed Central Ltd.)Kinetic models of biochem. systems usually consist of ordinary differential equations that have many unknown parameters. Some of these parameters are often practically unidentifiable, i.e., their values cannot be uniquely detd. from the available data. Possible causes are lack of influence on the measured outputs, interdependence among parameters, and poor data quality. Uncorrelated parameters can be seen as the key tuning knobs of a predictive model. Therefore, before attempting to perform parameter estn. (model calibration) it is important to characterize the subset(s) of identifiable parameters and their interplay. Once this is achieved, it is still necessary to perform parameter estn., which poses addnl. challenges. We present a methodol. that (i) detects high-order relationships among parameters, and (ii) visualizes the results to facilitate further anal. We use a collinearity index to quantify the correlation between parameters in a group in a computationally efficient way. Then we apply integer optimization to find the largest groups of uncorrelated parameters. We also use the collinearity index to identify small groups of highly correlated parameters. The results files can be visualized using Cytoscape, showing the identifiable and non-identifiable groups of parameters together with the model structure in the same graph. Our contributions alleviate the difficulties that appear at different stages of the identifiability anal. and parameter estn. process. We show how to combine global optimization and regularization techniques for calibrating medium and large scale biol. models with moderate computation times. Then we evaluate the practical identifiability of the estd. parameters using the proposed methodol. The identifiability anal. techniques are implemented as a MATLAB toolbox called VisId, which is freely available as open source from GitHub. Our approach is geared towards scalability. It enables the practical identifiability anal. of dynamic models of large size, and accelerates their calibration. The visualization tool allows modellers to detect parts that are problematic and need refinement or reformulation, and provides experimentalists with information that can be helpful in the design of new expts.
- 39Villaverde, A. F.; Evans, N. D.; Chappell, M. J.; Banga, J. R. Input-dependent structural identifiability of nonlinear systems. IEEE Control Systems Letters 2019, 3 (2), 272– 277, DOI: 10.1109/LCSYS.2018.2868608Google ScholarThere is no corresponding record for this reference.
- 40van Sluijs, B.; Maas, R. J. M.; van der Linden, A. J.; de Greef, T. F. A.; Huck, W. T. S. A microfluidic optimal experimental design platform for forward design of cell-free genetic networks. Nat. Commun. 2022, 13 (1), 3626, DOI: 10.1038/s41467-022-31306-3Google Scholar40https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB38XhslWqurfF&md5=ededa61719057fc65d2e305c7ec2287aA microfluidic optimal experimental design platform for forward design of cell-free genetic networksvan Sluijs, Bob; Maas, Roel J. M.; van der Linden, Ardjan J.; de Greef, Tom F. A.; Huck, Wilhelm T. S.Nature Communications (2022), 13 (1), 3626CODEN: NCAOBW; ISSN:2041-1723. (Nature Portfolio)Abstr.: Cell-free protein synthesis has been widely used as a "breadboard" for design of synthetic genetic networks. However, due to a severe lack of modularity, forward engineering of genetic networks remains challenging. Here, we demonstrate how a combination of optimal exptl. design and microfluidics allows us to devise dynamic cell-free gene expression expts. providing max. information content for subsequent non-linear model identification. Importantly, we reveal that applying this methodol. to a library of genetic circuits, that share common elements, further increases the information content of the data resulting in higher accuracy of model parameters. To show modularity of model parameters, we design a pulse decoder and bistable switch, and predict their behavior both qual. and quant. Finally, we update the parameter database and indicate that network topol. affects parameter estn. accuracy. Utilizing our methodol. provides us with more accurate model parameters, a necessity for forward engineering of complex genetic networks.
- 41Hold, C.; Billerbeck, S.; Panke, S. Forward design of a complex enzyme cascade reaction. Nat. Commun. 2016, 7, 12971, DOI: 10.1038/ncomms12971Google Scholar41https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC28Xhs1Smu73E&md5=dddec507e4149c520cf922bc32e42344Forward design of a complex enzyme cascade reactionHold, Christoph; Billerbeck, Sonja; Panke, SvenNature Communications (2016), 7 (), 12971CODEN: NCAOBW; ISSN:2041-1723. (Nature Publishing Group)Enzymic reaction networks are unique in that one can operate a large no. of reactions under the same set of conditions concomitantly in one pot, but the nonlinear kinetics of the enzymes and the resulting system complexity have so far defeated rational design processes for the construction of such complex cascade reactions. Here we demonstrate the forward design of an in vitro 10-membered system using enzymes from highly regulated biol. processes such as glycolysis. For this, we adapt the characterization of the biochem. system to the needs of classical engineering systems theory: we combine online mass spectrometry and continuous system operation to apply std. system theory input functions and to use the detailed dynamic system responses to parameterize a model of sufficient quality for forward design. This allows the facile optimization of a 10-enzyme cascade reaction for fine chem. prodn. purposes.
- 42Wong, A. S. Y.; Huck, W. T. S. Grip on complexity in chemical reaction networks. Beilstein J. Org. Chem. 2017, 13, 1486– 1497, DOI: 10.3762/bjoc.13.147Google Scholar42https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2sXhs1eksbnI&md5=d7e6fbaf3b1e405476e35c2a85845c69Grip on complexity in chemical reaction networksWong, Albert S. Y.; Huck, Wilhelm T. S.Beilstein Journal of Organic Chemistry (2017), 13 (), 1486-1497CODEN: BJOCBH; ISSN:1860-5397. (Beilstein-Institut zur Foerderung der Chemischen Wissenschaften)A new discipline of "systems chem." is emerging, which aims to capture the complexity obsd. in natural systems within a synthetic chem. framework. Living systems rely on complex networks of chem. reactions to control the concn. of mols. in space and time. Despite the enormous complexity in biol. networks, it is possible to identify network motifs that lead to functional outputs such as bistability or oscillations. To truly understand how living systems function, we need a complete understanding of how chem. reaction networks (CRNs) create function. We propose the development of a bottom-up approach to design and construct CRNs where we can follow the influence of single chem. entities on the properties of the network as a whole. Ultimately, this approach should allow us to not only understand such complex networks but also to guide and control their behavior.
- 43Hanopolskyi, A. I.; Smaliak, V. A.; Novichkov, A. I.; Semenov, S. N. Autocatalysis: Kinetics, mechanisms and design. ChemSystemsChem. 2021, 3 (1), e2000026, DOI: 10.1002/syst.202000026Google Scholar43https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB3MXovFyltrw%253D&md5=374e03d4de2ae8956a15e2072fd7bd87Autocatalysis: Kinetics, Mechanisms and DesignHanopolskyi, Anton I.; Smaliak, Viktoryia A.; Novichkov, Alexander I.; Semenov, Sergey N.ChemSystemsChem (2021), 3 (1), e2000026CODEN: CSCHCD; ISSN:2570-4206. (Wiley-VCH Verlag GmbH & Co. KGaA)The importance of autocatalysis spans from practical applications such as in chem. amplified photoresists, to autocatalysis playing a fundamental role in evolution as well as a plausible key role in the origin of life. The phenomenon of autocatalysis is characterized by its kinetic signature rather than by its mechanistic aspects. The mols. that form autocatalytic systems and the mechanisms underlying autocatalytic reactions are very diverse. This chem. diversity, combined with the strong involvement of chem. kinetics, creates a formidable barrier for entrance to the field. Understanding these challenges, we wrote this Review with three main goals in mind: (i) To provide a basic introduction to the kinetics of autocatalytic systems and its relation to the role of autocatalysis in evolution, (ii) To provide a comprehensive overview, including tables, of synthetic chem. autocatalytic systems, and (iii) To provide an in-depth anal. of the concept of autocatalytic reaction networks, their design, and perspectives for their development.
- 44Bánsági, T.; Taylor, A. F. Exploitation of feedback in enzyme-catalysed reactions. Isr. J. Chem. 2018, 58 (6–7), 706– 713, DOI: 10.1002/ijch.201700141Google Scholar44https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC1cXmvVehsbo%253D&md5=61c49644fc12c9f8eeedc356273fb44aExploitation of Feedback in Enzyme-catalysed ReactionsBansagi, Tamas, Jr.; Taylor, Annette F.Israel Journal of Chemistry (2018), 58 (6-7), 706-713CODEN: ISJCAT; ISSN:0021-2148. (Wiley-VCH Verlag GmbH & Co. KGaA)A review. Some cellular systems, such as yeast, bacteria and slime mold, display dynamic behavior including switches and rhythms driven by feedback in enzyme-catalyzed reactions. The mechanisms of these processes have been well investigated and recent attention has turned to generating similar responses in synthetic biocatalytic systems, with a view to creating bioinspired analogs for applications. Here we discuss how feedback arises in the reaction mechanisms of some enzyme-catalyzed reactions in vitro, the behavior obtained and the emerging applications. These autocatalytic reactions may provide insights into behavior in cellular systems as well as new methods for drug delivery, sensing and repair that can be exploited in living systems.
- 45Plasson, R.; Brandenburg, A.; Jullien, L.; Bersini, H. Autocatalyses. J. Phys. Chem. A 2011, 115 (28), 8073– 8085, DOI: 10.1021/jp110079pGoogle Scholar45https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3MXnvFKitLs%253D&md5=e25b4c31569eb18c58e325585c868cbcAutocatalysesPlasson, Raphael; Brandenburg, Axel; Jullien, Ludovic; Bersini, HuguesJournal of Physical Chemistry A (2011), 115 (28), 8073-8085CODEN: JPCAFH; ISSN:1089-5639. (American Chemical Society)Autocatalysis is a fundamental concept, used in a wide range of domains. From the most general definition of autocatalysis, i.e., a process in which a chem. compd. is able to catalyze its own formation, several different systems can be described. We detail the different categories of autocatalyzes and compare them on the basis of their mechanistic, kinetic, and dynamic properties. It is shown how autocatalytic patterns can be generated by different systems of chem. reactions. With the notion of autocatalysis covering a large variety of mechanistic realizations with very similar behaviors, it is proposed that the key signature of autocatalysis is its kinetic pattern expressed in a math. form.
- 46Budroni, M. A.; Rossi, F.; Rongy, L. From transport phenomena to systems chemistry: chemohydrodynamic oscillations in A+B⃗C systems. ChemSystemsChem. 2022, 4 (1), e202100023, DOI: 10.1002/syst.202100023Google Scholar46https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB38Xot1aktA%253D%253D&md5=d37da696a0bfb395198c3468512bf6d7From Transport Phenomena to Systems Chemistry: Chemohydrodynamic Oscillations in A+B→C SystemsBudroni, Marcello A.; Rossi, Federico; Rongy, LaurenceChemSystemsChem (2022), 4 (1), e202100023CODEN: CSCHCD; ISSN:2570-4206. (Wiley-VCH Verlag GmbH & Co. KGaA)Can simple chem. drive the emergence of self-organised complex behaviors. Addressing this big-picture question crucially impacts the comprehension of fundamental mechanisms at the basis of stationary and dynamical spatio-temporal chem. patterns which represent an integral part of Origin of Life studies and morphogenesis. This is also of paramount importance in cutting-edge approaches for the design and control of bio-inspired self-organised functional materials as well as for understanding how complex biol. networks work. So far, spontaneous chem. self-organization has constituted the realm of Nonlinear Chem. Oscillations, waves, Turing structures have been typically obtained in systems characterised by a complex network of nonlinear reactions activated on appropriate relative timescales. Here we revisit the emergence of oscillatory dynamics in systems characterised by a kinetics as simple and general as a bimol. process, provided that it is actively coupled with transport phenomena, in the absence of any nonlinear or external kinetic feedback. We also present new numerical expts. to substantiate and clarify the minimal ingredients underlying these complex dynamics. The objective of this paper is to discuss chemo-hydrodynamics as a possible mechanism for activating self-organised structures and functional behaviors in contexts characterised by a minimal chem. like prebiotic conditions. In this view, we also highlight the necessity to include convective phenomena in the paradigm of Systems Chem.
- 47Menon, G.; Krishnan, J. Spatial localisation meets biomolecular networks. Nat. Commun. 2021, 12 (1), 5357, DOI: 10.1038/s41467-021-24760-yGoogle Scholar47https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB3MXitVSju7%252FI&md5=3d9a558aa56e2a8bac080aa27a85c011Spatial localisation meets biomolecular networksMenon, Govind; Krishnan, J.Nature Communications (2021), 12 (1), 5357CODEN: NCAOBW; ISSN:2041-1723. (Nature Research)Spatial organization through localisation/compartmentalisation of species is a ubiquitous but poorly understood feature of cellular biomol. networks. Current technologies in systems and synthetic biol. (spatial proteomics, imaging, synthetic compartmentalisation) necessitate a systematic approach to elucidating the interplay of networks and spatial organization. We develop a systems framework toward this end and focus on the effect of spatial localisation of network components revealing its multiple facets: (i) As a key distinct regulator of network behavior, and an enabler of new network capabilities (ii) As a potent new regulator of pattern formation and self-organization (iii) As an often hidden factor impacting inference of temporal networks from data (iv) As an engineering tool for rewiring networks and network/circuit design. These insights, transparently arising from the most basic considerations of networks and spatial organization, have broad relevance in natural and engineered biol. and in related areas such as cell-free systems, systems chem. and bionanotechnol.
- 48Sharma, C.; Maity, I.; Walther, A. pH-feedback systems to program autonomous self-assembly and material lifecycles. Chem. Commun. 2023, 59, 1125– 1144, DOI: 10.1039/D2CC06402BGoogle Scholar48https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB3sXpt1amsQ%253D%253D&md5=c81df9a957b093d794e0fb904a0ded10pH-feedback systems to program autonomous self-assembly and material lifecyclesSharma, Charu; Maity, Indrajit; Walther, AndreasChemical Communications (Cambridge, United Kingdom) (2023), 59 (9), 1125-1144CODEN: CHCOFS; ISSN:1359-7345. (Royal Society of Chemistry)A review. PH-responsive systems have gained importance for the development of smart materials and for biomedical applications because they can switch between different states by simple acid/base triggers. However, such equil. systems lack the autonomous behavior that is so ubiquitous in living systems that self-regulate out of equil. As a contribution to the emerging field of autonomous chem. systems, we have developed pH-feedback systems (pH-FS) based on the coupling of acid- and base-producing steps in chem. reaction networks. The resulting autonomous nonlinear pH curves can be coupled with a variety of pH-sensitive building blocks to program the lifecycles of the assocd. transient state at the level of self-assemblies and material systems. In this article, we discuss the different generations of such pH-feedback systems, the principles of their coupling to self-assemblies with lifecycles and highlight emerging concepts for the design of autonomous functional materials. The specificity, robustness, and flexible operation of such pH-FS can also be used to realize chemo-structural and chemo-mech. feedbacks that extend the behavior of such materials systems toward complex and functional life-like systems.
- 49Dúzs, B.; Molnár, I.; Lagzi, I.; Szalai, I. Reaction–diffusion dynamics of pH oscillators in oscillatory forced open spatial reactors. ACS Omega 2021, 6 (50), 34367– 34374, DOI: 10.1021/acsomega.1c04269Google Scholar49https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB3MXislSgu7rI&md5=a6cc93dab9a55f296a18a8c68cbf6ef8Reaction-Diffusion Dynamics of pH Oscillators in Oscillatory Forced Open Spatial ReactorsDuzs, Brigitta; Molnar, Istvan; Lagzi, Istvan; Szalai, IstvanACS Omega (2021), 6 (50), 34367-34374CODEN: ACSODF; ISSN:2470-1343. (American Chemical Society)Studying the effect of coupling and forcing of oscillators is a significant area of interest within nonlinear dynamics and has provided evidence of many interesting phenomena, such as synchronization, beating, oscillatory death, and phase resetting. Many studies have also reported along this line in reaction-diffusion systems, which are preferably explored exptl. by using open reactors. These reactors consist of one or two homogeneous (well-stirred) tanks, which provide the boundary conditions for a spatially distributed part. The spatiotemporal dynamics of this configuration in the presence of temporal oscillations in the homogeneous part has not been systematically investigated. This paper aims to explore numerically the effect of time-periodic boundary conditions on the dynamics of open reactors provided by autonomous and forced oscillations in the well-stirred part. A simple model of pH oscillators can produce various phenomena under these conditions, for example, superposition and modulation of spatiotemporal oscillations and forced bursting. The autonomous oscillatory boundary conditions can be generated by the same kinetic instabilities that result in spatiotemporal oscillations in the spatially distributed part. The forced oscillations are induced by sinusoidal modulation on the inflow concn. of the activator in the tank. The simulations confirmed that this type of forcing is more effective when the modulation period is longer than the residence time of the well-stirred part. The use of time-periodic boundary conditions may open a new perspective in the control and design of spatiotemporal phenomena in open one-side-fed and two-side-fed reactors.
- 50Duzs, B.; Lagzi, I.; Szalai, I. Functional rhythmic chemical systems governed by pH-driven kinetic feedback. ChemSystemsChem 2023, 5, e202200032, DOI: 10.1002/syst.202200032Google Scholar50https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB38XjtFymsLfE&md5=ceef1e19f91a085dcb74f8bcea070d73Functional Rhythmic Chemical Systems Governed by pH-Driven Kinetic FeedbackDuzs, Brigitta; Lagzi, Istvan; Szalai, IstvanChemSystemsChem (2023), 5 (2), e202200032CODEN: CSCHCD; ISSN:2570-4206. (Wiley-VCH Verlag GmbH & Co. KGaA)Hydrogen ion autocatalytic reactions, esp. in combination with an appropriate neg. feedback process, show a wide range of dynamical phenomena, like clock behavior, bistability, oscillations, waves, and stationary patterns. The temporal or spatial variation of pH caused by these reactions is often significant enough to control the actual state (geometry, conformation, reactivity) or drive the mech. motion of coupled pH-sensitive physico-chem. systems. These autonomous operating systems provide nonlinear chem.'s most reliable applications, where the hydrogen ion autocatalytic reactions act as engines. This review briefly summarizes the nonlinear dynamics of these reactions and the different approaches developed to properly couple the pH-sensitive units (e. g., pH-sensitive equil., gels, mol. machines, colloids). We also emphasize the feedback of the coupled processes on the dynamics of the hydrogen ion autocatalytic reactions since the way of coupling is a crit. operational issue.
- 51Bubanja, I. N.; Bánsági, T.; Taylor, A. F. Kinetics of the urea–urease clock reaction with urease immobilized in hydrogel beads. Reac. Kinet. Mech. Catal. 2018, 123 (1), 177– 185, DOI: 10.1007/s11144-017-1296-6Google ScholarThere is no corresponding record for this reference.
- 52Miele, Y.; Jones, S. J.; Rossi, F.; Beales, P. A.; Taylor, A. F. Collective behavior of urease pH clocks in nano- and microvesicles controlled by fast ammonia transport. J. Phys. Chem. Lett. 2022, 13 (8), 1979– 1984, DOI: 10.1021/acs.jpclett.2c00069Google Scholar52https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB38Xktl2qurY%253D&md5=14a36537aa46cd55bd13745860a4bc8dCollective Behavior of Urease pH Clocks in Nano- and Microvesicles Controlled by Fast Ammonia TransportMiele, Ylenia; Jones, Stephen J.; Rossi, Federico; Beales, Paul A.; Taylor, Annette F.Journal of Physical Chemistry Letters (2022), 13 (8), 1979-1984CODEN: JPCLCD; ISSN:1948-7185. (American Chemical Society)The transmission of chem. signals via an extracellular soln. plays a vital role in collective behavior in cellular biol. systems and may be exploited in applications of lipid vesicles such as drug delivery. Here, we investigated chem. communication in synthetic micro- and nanovesicles contg. urease in a soln. of urea and acid. We combined expts. with simulations to demonstrate that the fast transport of ammonia to the external soln. governs the pH-time profile and synchronizes the timing of the pH clock reaction in a heterogeneous population of vesicles. This study shows how the rate of prodn. and emission of a small basic product controls pH changes in active vesicles with a distribution of sizes and enzyme amts., which may be useful in bioreactor or healthcare applications.
- 53Markovic, V. M.; Bánsági, T., Jr.; McKenzie, D.; Mai, A.; Pojman, J. A.; Taylor, A. F. Influence of reaction-induced convection on quorum sensing in enzyme-loaded agarose beads. Chaos 2019, 29 (3), 033130 DOI: 10.1063/1.5089295Google Scholar53https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC1MXlvVaht7Y%253D&md5=253ed3102986bffa416647741cc8542bInfluence of reaction-induced convection on quorum sensing in enzyme-loaded agarose beadsMarkovic, Vladimir M.; Bansagi, Tamas; McKenzie, Dennel; Mai, Anthony; Pojman, John A.; Taylor, Annette F.Chaos (2019), 29 (3), 033130/1-033130/8CODEN: CHAOEH; ISSN:1054-1500. (American Institute of Physics)In theory, groups of enzyme-loaded particles producing an acid or base may show complex behavior including dynamical quorum sensing, the appearance of synchronized oscillations above a crit. no. or d. of particles. Here, expts. were performed with the enzyme urease loaded into mm-sized agarose beads and placed in a soln. of urea, resulting in an increase in pH. This behavior was found to be dependent upon the no. of beads present in the array; however, reaction-induced convection occurred and plumes of high pH developed that extended to the walls of the reactor. The convection resulted in the motion of the mm-sized particles and conversion of the soln. to high pH. Simulations in a simple model of the beads demonstrated the suppression of dynamical quorum sensing in the presence of flow. (c) 2019 American Institute of Physics.
- 54Muzika, F.; RuZicka, M.; Schreiberova, L.; Schreiber, I. Oscillations of pH in the urea-urease system in a membrane reactor. Phys. Chem. Chem. Phys. 2019, 21 (17), 8619– 8622, DOI: 10.1039/C9CP00630CGoogle Scholar54https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC1MXmtlyksbY%253D&md5=a78ec2e5982160a8ec8ca2a83a397ab1Oscillations of pH in the urea-urease system in a membrane reactorMuzika, Frantisek; Ruzicka, Matej; Schreiberova, Lenka; Schreiber, IgorPhysical Chemistry Chemical Physics (2019), 21 (17), 8619-8622CODEN: PPCPFQ; ISSN:1463-9076. (Royal Society of Chemistry)Herein, we present direct exptl. evidence of pH oscillatory dynamics in the urea-urease enzymic reaction conducted in a continuous reactor-membrane-reservoir system. Our results are consistent with earlier model predictions requiring differential transport of H+ and substrate. We report oscillations with periods in hundreds of seconds and the amplitude of ∼0.1 pH units.
- 55Muzika, F.; Bánsági, T., Jr.; Schreiber, I.; Schreiberova, L.; Taylor, A. F. A bistable switch in pH in urease-loaded alginate beads. Chem. Commun. 2014, 50 (76), 11107– 11109, DOI: 10.1039/C4CC03936JGoogle Scholar55https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2cXht1ylsLfM&md5=ab6e71b5ceabd086472dc0da60a86b16A bistable switch in pH in urease-loaded alginate beadsMuzika, F.; Bansagi, T.; Schreiber, I.; Schreiberova, L.; Taylor, A. F.Chemical Communications (Cambridge, United Kingdom) (2014), 50 (76), 11107-11109CODEN: CHCOFS; ISSN:1359-7345. (Royal Society of Chemistry)A bistable switch from a low pH (unreacted "off") state to a high pH (reacted "on") state was obtained in enzyme-loaded gel beads in response to supra-threshold substrate concns.
- 56Hu, G.; Pojman, J. A.; Scott, S. K.; Wrobel, M. M.; Taylor, A. F. Base-catalyzed feedback in the urea-urease reaction. J. Phys. Chem. B 2010, 114 (44), 14059– 14063, DOI: 10.1021/jp106532dGoogle Scholar56https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3cXht12lt7nI&md5=03f423f0c3cf6e8af398a2b90feaf934Base-Catalyzed Feedback in the Urea-Urease ReactionHu, Gang; Pojman, John A.; Scott, Stephen K.; Wrobel, Magdelena M.; Taylor, Annette F.Journal of Physical Chemistry B (2010), 114 (44), 14059-14063CODEN: JPCBFK; ISSN:1520-6106. (American Chemical Society)The bell-shaped rate-pH curve coupled to prodn. of base in the urea-urease reaction was utilized to give feedback-driven behavior: an acid-to-base pH clock (a kinetic switch), bistability and hysteresis between an acid/base state when the initial pH was adjusted by a strong acid, and aperiodic pH oscillations when the initial pH was adjusted by a weak acid in an open reactor. A simple model of the reaction reproduced most of the exptl. results and provided insight into the role of self-buffering in the dynamics. This reaction suggests new possibilities in the development of biocompatible feedback to couple to pH-sensitive processes for bioinspired applications in medicine, engineering, or materials science.
- 57Straube, A. V.; Winkelmann, S.; Schütte, C.; Höfling, F. Stochastic pH oscillations in a model of the urea-urease reaction confined to lipid vesicles. J. Phys. Chem. Lett. 2021, 12 (40), 9888– 9893, DOI: 10.1021/acs.jpclett.1c03016Google Scholar57https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB3MXitFKgs7vL&md5=302f4240f6ef6671cea1a311d812818bStochastic pH Oscillations in a Model of the Urea-Urease Reaction Confined to Lipid VesiclesStraube, Arthur V.; Winkelmann, Stefanie; Schuette, Christof; Hoefling, FelixJournal of Physical Chemistry Letters (2021), 12 (40), 9888-9893CODEN: JPCLCD; ISSN:1948-7185. (American Chemical Society)The urea-urease clock reaction is a pH switch from acid to basic that can turn into a pH oscillator if it occurs inside a suitable open reactor. The authors numerically study the confinement of the reaction to lipid vesicles, which permit the exchange with an external reservoir by differential transport, enabling the recovery of the pH level and yielding a const. supply of urea mols. For microscopically small vesicles, the discreteness of the no. of mols. requires a stochastic treatment of the reaction dynamics. The authors' anal. shows that intrinsic noise induces a significant statistical variation of the oscillation period, which increases as the vesicles become smaller. The mean period, however, is remarkably robust for vesicle sizes down to ∼200 nm, but the periodicity of the rhythm is gradually destroyed for smaller vesicles. The obsd. oscillations are explained as a canard-like limit cycle that differs from the wide class of conventional feedback oscillators.
- 58Bánsági, T.; Taylor, A. F. Switches induced by quorum sensing in a model of enzyme-loaded microparticles. J. R. Soc. Interface. 2018, 15, 20170945, DOI: 10.1098/rsif.2017.0945Google Scholar58https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC1cXitFyksrjM&md5=7879b8b536206bf09b4f3be0510234a9Switches induced by quorum sensing in a model of enzyme-loaded microparticlesBansagi, Tamas, Jr.; Taylor, Annette F.Journal of the Royal Society, Interface (2018), 15 (140), 20170945/1-20170945/10CODEN: JRSICU; ISSN:1742-5662. (Royal Society)Quorum sensing refers to the ability of bacteria and other single-celled organisms to respond to changes in cell d. or no. with population- wide changes in behavior. Here, simulations were performed to investigate quorum sensing in groups of diffusively coupled enzyme microparticles using a well-characterized autocatalytic reaction which raises the pH of the medium: hydrolysis of urea by urease. The enzyme urease is found in both plants and microorganisms, and has been widely exploited in engineering processes. We demonstrate how increases in group size can be used to achieve a sigmoidal switch in pH at high enzyme loading, oscillations in pH at intermediate enzyme loading and a bistable, hysteretic switch at low enzyme loading. Thus, quorum sensing can be exploited to obtain different types of response in the same system, depending on the enzyme concn. The implications for microorganisms in colonies are discussed, and the results could help in the design of synthetic quorum sensing for biotechnol. applications such as drug delivery.
- 59Heinen, L.; Heuser, T.; Steinschulte, A.; Walther, A. Antagonistic enzymes in a biocatalytic pH feedback system program autonomous DNA hydrogel life cycles. Nano Lett. 2017, 17 (8), 4989– 4995, DOI: 10.1021/acs.nanolett.7b02165Google Scholar59https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2sXhtVOntr7J&md5=7e4447431de1ec8deba85a2979c81bfaAntagonistic Enzymes in a Biocatalytic pH Feedback System Program Autonomous DNA Hydrogel Life CyclesHeinen, Laura; Heuser, Thomas; Steinschulte, Alexander; Walther, AndreasNano Letters (2017), 17 (8), 4989-4995CODEN: NALEFD; ISSN:1530-6984. (American Chemical Society)Enzymes regulate complex functions and active behavior in natural systems and have shown increasing prospect for developing self-regulating soft matter systems. Striving for advanced autonomous hydrogel materials with fully programmable, self-regulated life cycles, we combine two enzymes with an antagonistic pH-modulating effect in a feedback-controlled biocatalytic reaction network (BRN) and couple it to pH-responsive DNA hydrogels to realize hydrogel systems with distinct preprogrammable lag times and lifetimes in closed systems. The BRN enables precise and orthogonal internal temporal control of the "ON" and "OFF" switching times of the temporary gel state by modulation of programmable, non-linear pH changes. The timescales are tunable by variation of the enzyme concns. and addnl. buffer substances. The resulting material system operates in full autonomy after injection of the chem. fuels driving the BRN. The concept may open new applications inherent to DNA hydrogels, for instance, autonomous shape memory behavior for soft robotics. We further foresee general applicability to achieve autonomous life cycles in other pH switchable systems.
- 60Fan, X.; Walther, A. Autonomous transient pH flips shaped by layered compartmentalization of antagonistic enzymatic reactions. Angew. Chem., Int. Ed. 2021, 60, 3619– 3624, DOI: 10.1002/anie.202009542Google Scholar60https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB3cXis1agtbnO&md5=83cc1ce50746ba7ef36516d4d684b92bAutonomous transient pH flips shaped by layered compartmentalization of antagonistic enzymatic reactionsFan, Xinlong; Walther, AndreasAngewandte Chemie, International Edition (2021), 60 (7), 3619-3624CODEN: ACIEF5; ISSN:1433-7851. (Wiley-VCH Verlag GmbH & Co. KGaA)Transient signaling orchestrates complex spatiotemporal behavior in living organisms via (bio)chem. reaction networks (CRNs). Compartmentalization of signal processing is an important aspect for controlling such networks. However, artificial CRNs mostly focus on homogeneous solns. to program autonomous self-assembling systems, which limits their accessible behavior and tuneability. Here, we introduce layered compartments housing antagonistic pH-modulating enzymes and demonstrate that transient pH signals in a supernatant soln. can be programmed based on spatial delays. This overcomes limitations of activity mismatches of antagonistic enzymes in soln. and allows to flexibly program acidic and alk. pH lifecycles beyond the possibilities of homogeneous solns. Lag time, lifetime, and the pH min. and maxima can be precisely programmed by adjusting spatial and kinetic conditions. We integrate these spatially controlled pH flips with switchable peptides, furnishing time-programmed self-assemblies and hydrogel material system.
- 61Wang, X.; Moreno, S.; Boye, S.; Wen, P.; Zhang, K.; Formanek, P.; Lederer, A.; Voit, B.; Appelhans, D. Feedback-induced and oscillating pH regulation of a binary enzyme–polymersomes. System. Chem. Mater. 2021, 33 (17), 6692– 6700, DOI: 10.1021/acs.chemmater.1c00897Google ScholarThere is no corresponding record for this reference.
- 62Dúzs, B.; Lagzi, I.; Szalai, I. Functional rhythmic chemical systems governed by pH-Driven kinetic feedback. ChemSystemsChem 2023, 5 (2), e202200032, DOI: 10.1002/syst.202200032Google Scholar62https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB38XjtFymsLfE&md5=ceef1e19f91a085dcb74f8bcea070d73Functional Rhythmic Chemical Systems Governed by pH-Driven Kinetic FeedbackDuzs, Brigitta; Lagzi, Istvan; Szalai, IstvanChemSystemsChem (2023), 5 (2), e202200032CODEN: CSCHCD; ISSN:2570-4206. (Wiley-VCH Verlag GmbH & Co. KGaA)Hydrogen ion autocatalytic reactions, esp. in combination with an appropriate neg. feedback process, show a wide range of dynamical phenomena, like clock behavior, bistability, oscillations, waves, and stationary patterns. The temporal or spatial variation of pH caused by these reactions is often significant enough to control the actual state (geometry, conformation, reactivity) or drive the mech. motion of coupled pH-sensitive physico-chem. systems. These autonomous operating systems provide nonlinear chem.'s most reliable applications, where the hydrogen ion autocatalytic reactions act as engines. This review briefly summarizes the nonlinear dynamics of these reactions and the different approaches developed to properly couple the pH-sensitive units (e. g., pH-sensitive equil., gels, mol. machines, colloids). We also emphasize the feedback of the coupled processes on the dynamics of the hydrogen ion autocatalytic reactions since the way of coupling is a crit. operational issue.
- 63Jee, E.; Bánsági, T., Jr.; Taylor, A. F.; Pojman, J. A. Temporal control of gelation and polymerization fronts driven by an autocatalytic enzyme reaction. Angew. Chem., Int. Ed. 2016, 55 (6), 2127– 2131, DOI: 10.1002/anie.201510604Google Scholar63https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC28XktlSmsA%253D%253D&md5=2ed1fdf6d04860d0dbf9745a6bb289dbTemporal Control of Gelation and Polymerization Fronts Driven by an Autocatalytic Enzyme ReactionJee, Elizabeth; Bansagi, Tamas Jr.; Taylor, Annette F.; Pojman, John A.Angewandte Chemie, International Edition (2016), 55 (6), 2127-2131CODEN: ACIEF5; ISSN:1433-7851. (Wiley-VCH Verlag GmbH & Co. KGaA)Chem. systems that remain kinetically dormant until activated have numerous applications in materials science. Herein we present a method for the control of gelation that exploits an inbuilt switch: the increase in pH after an induction period in the urease-catalyzed hydrolysis of urea was used to trigger the base-catalyzed Michael addn. of a water-sol. trithiol to a polyethylene glycol diacrylate. The time to gelation (minutes to hours) was either preset through the initial concns. or the reaction was initiated locally by a base, thus resulting in polymn. fronts that converted the mixt. from a liq. into a gel (ca. 0.1 mm min-1). The rate of hydrolytic degrdn. of the hydrogel depended on the initial concns., thus resulting in a gel lifetime of hours to months. In this way, temporal programming of gelation was possible under mild conditions by using the output of an autocatalytic enzyme reaction to drive both the polymn. and subsequent degrdn. of a hydrogel.
- 64Mai, A. Q.; Bánsági, T., Jr.; Taylor, A. F.; Pojman, J. A., Sr. Reaction-diffusion hydrogels from urease enzyme particles for patterned coatings. Commun. Chem. 2021, 4 (1), 101, DOI: 10.1038/s42004-021-00538-7Google Scholar64https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB3MXitF2gur3I&md5=af0f5c0f3dcbe53e166935d27ac84dcdReaction-diffusion hydrogels from urease enzyme particles for patterned coatingsMai, Anthony Q.; Bansagi Jr., Tamas; Taylor, Annette F.; Pojman Sr., John A.Communications Chemistry (2021), 4 (1), 101CODEN: CCOHCT; ISSN:2399-3669. (Nature Research)Abstr.: The reaction and diffusion of small mols. is used to initiate the formation of protective polymeric layers, or biofilms, that attach cells to surfaces. Here, inspired by biofilm formation, we present a general method for the growth of hydrogels from urease enzyme-particles by combining prodn. of ammonia with a pH-regulated polymn. reaction in soln. We show through expts. and simulations how the propagating basic front and thiol-acrylate polymn. were continuously maintained by the localized urease reaction in the presence of urea, resulting in hydrogel layers around the enzyme particles at surfaces, interfaces or in motion. The hydrogels adhere the enzyme-particles to surfaces and have a tunable growth rate of the order of 10 μm min-1 that depends on the size and spatial distribution of particles. This approach can be exploited to create enzyme-hydrogels or chem. patterned coatings for applications in biocatalytic flow reactors.
- 65Maity, I.; Sharma, C.; Lossada, F.; Walther, A. Feedback and communication in active hydrogel spheres with pH fronts: facile approaches to grow soft hydrogel structures. Angew. Chem., Int. Ed. 2021, 60 (41), 22537– 22546, DOI: 10.1002/anie.202109735Google Scholar65https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB3MXhvFGmsbvO&md5=fc6e8605c1188dfbdcd3e9571568898bFeedback and Communication in Active Hydrogel Spheres with pH Fronts: Facile Approaches to Grow Soft Hydrogel StructuresMaity, Indrajit; Sharma, Charu; Lossada, Francisco; Walther, AndreasAngewandte Chemie, International Edition (2021), 60 (41), 22537-22546CODEN: ACIEF5; ISSN:1433-7851. (Wiley-VCH Verlag GmbH & Co. KGaA)Compartmentalized reaction networks regulating signal processing, communication and pattern formation are central to living systems. Towards achieving life-like materials, the authors compartmentalized urea-urease and more complex urea-urease/ester-esterase pH-feedback reaction networks into hydrogel spheres and study how fuel-driven pH fronts can be sent out from these spheres and regulated by internal reaction networks. Membrane characteristics are installed by covering urease spheres with responsive hydrogel shells. The authors then encapsulate the two networks (urea-urease and ester-esterase) sep. into different hydrogel spheres to devise communication, pattern formation and attraction. Moreover, these pH fronts and patterns can be used for self-growing hydrogels, and for developing complex geometries from non-injectable hydrogels without 3D printing tools. This study opens possibilities for compartmentalized feedback reactions and their use in next generation materials fabrication.
- 66Rauner, N.; Buenger, L.; Schuller, S.; Tiller, J. C. Post-polymerization of urease-induced calcified, polymer hydrogels. Macromol. Rapid Commun. 2015, 36 (2), 224– 230, DOI: 10.1002/marc.201400426Google Scholar66https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2MXosFyksg%253D%253D&md5=f7c7d33c8fffcdc22bc61188ce70bbc8Post-Polymerization of Urease-Induced Calcified, Polymer HydrogelsRauner, Nicolas; Buenger, Lea; Schuller, Stefanie; Tiller, Joerg C.Macromolecular Rapid Communications (2015), 36 (2), 224-230CODEN: MRCOE3; ISSN:1022-1336. (Wiley-VCH Verlag GmbH & Co. KGaA)Urease-induced calcification is an innovative method to artificially produce highly filled CaCO3-based composite materials by intrinsic mineralization of hydrogels. The mech. properties of these hybrid materials based on poly(2-hydroxyethylacrylate) cross-linked by triethylene glycol dimethacrylate are poor. Increasing the degree of calcification to up to 94 wt% improves the Young's moduli (YM) of the materials from some 40 MPa to more than 300 MPa. The introduction of calcium carbonate affine groups to the hydrogel matrix by copolymg. acrylic acid and [2-(methacryloyloxy) ethyl]trimethylammonium chloride, resp., does not increase the stiffness of the composites. A Young's modulus of more than 1 GPa is achieved by post-polymn. (PP) of the calcified hydrogels, which proves that the size of the contact area between the matrix and calcium carbonate crystals is the most crucial parameter for controlling the stiffness of hybrid materials. Switching from low Tg to high Tg hydrogel matrixes (based on poly(N,N-di-Me acrylamide)) results in a YM of up to 3.5 GPa after PP.
- 67Semenov, S. N.; Wong, A. S.; van der Made, R. M.; Postma, S. G.; Groen, J.; van Roekel, H. W.; de Greef, T. F.; Huck, W. T. Rational design of functional and tunable oscillating enzymatic networks. Nat. Chem. 2015, 7 (2), 160– 165, DOI: 10.1038/nchem.2142Google Scholar67https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2MXmtFSgsQ%253D%253D&md5=e5010fdd61b6a845eb70077cfebb232fRational design of functional and tunable oscillating enzymatic networksSemenov, Sergey N.; Wong, Albert S. Y.; van der Made, R. Martijn; Postma, Sjoerd G. J.; Groen, Joost; van Roekel, Hendrik W. H.; de Greef, Tom F. A.; Huck, Wilhelm T. S.Nature Chemistry (2015), 7 (2), 160-165CODEN: NCAHBB; ISSN:1755-4330. (Nature Publishing Group)Life is sustained by complex systems operating far from equil. and consisting of a multitude of enzymic reaction networks. The operating principles of biol.'s regulatory networks are known, but the in vitro assembly of out-of-equil. enzymic reaction networks proved challenging, limiting the development of synthetic systems showing autonomous behavior. Here, the authors present a strategy for the rational design of programmable functional reaction networks that exhibit dynamic behavior. A network built around autoactivation and delayed neg. feedback of the enzyme trypsin is capable of producing sustained oscillating concns. of active trypsin for over 65 h. Other functions, such as amplification, analog-to-digital conversion and periodic control over equil. systems, were obtained by linking multiple network modules in microfluidic flow reactors. The methodol. developed here provides a general framework to construct dissipative, tunable and robust (bio)chem. reaction networks.
- 68Maguire, O. R.; Wong, A. S. Y.; Westerdiep, J. H.; Huck, W. T. S. Early warning signals in chemical reaction networks. Chem. Commun. 2020, 56 (26), 3725– 3728, DOI: 10.1039/D0CC01010CGoogle Scholar68https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB3cXjvFKiu7c%253D&md5=5bef9631196a02efb799d83b92408a71Early warning signals in chemical reaction networksMaguire, Oliver R.; Wong, Albert S. Y.; Westerdiep, Jan Harm; Huck, Wilhelm T. S.Chemical Communications (Cambridge, United Kingdom) (2020), 56 (26), 3725-3728CODEN: CHCOFS; ISSN:1359-7345. (Royal Society of Chemistry)Complex systems such as ecosystems, the climate and stock markets produce emergent behavior which is capable of undergoing dramatic change when pushed beyond a tipping point. Such complex systems display Early Warning Signals in their behavior when they are close to a tipping point. Here we show that a complex chem. reaction network can also display early warning signals when it is in close proximity to the boundary between oscillatory and steady state concn. behaviors. We identify early warning signals using both an active sensing method, based on the recovery time of an oscillatory response after a perturbation in temp., and a passive sensing method, based upon a change in the shape of the oscillations. The presence of the early warning signals indicates that complex, dissipative chem. networks can intrinsically sense their proximity to a boundary between behaviors.
- 69Maguire, O. R.; Wong, A. S. Y.; Baltussen, M. G.; van Duppen, P.; Pogodaev, A. A.; Huck, W. T. S. Dynamic environments as a tool to preserve desired output in a chemical reaction network. Chem. Eur. J. 2020, 26, 1676– 1682, DOI: 10.1002/chem.201904725Google Scholar69https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB3cXht1Khu7o%253D&md5=eb697b3888e013a653c608bf30c55677Dynamic environments as tool to preserve desired output in chemical reaction networkMaguire, Oliver R.; Wong, Albert S. Y.; Baltussen, Mathieu G.; van Duppen, Peer; Pogodaev, Aleksandr A.; Huck, Wilhelm T. S.Chemistry - A European Journal (2020), 26 (7), 1676-1682CODEN: CEUJED; ISSN:0947-6539. (Wiley-VCH Verlag GmbH & Co. KGaA)Current efforts to design functional mol. systems have overlooked the importance of coupling out-of-equil. behavior with changes in the environment. Here, the authors use an oscillating reaction network and demonstrate that the application of environmental forcing, in the form of periodic changes in temp. and in the inflow of the concn. of one of the network components, removes the dependency of the periodicity of this network on temp. or flow rates and enforces a stable periodicity across a wide range of conditions. Coupling a system to a dynamic environment can thus be used as a simple tool to regulate the output of a network. In addn., the authors show that coupling can also induce an increase in behavioral complexity to include quasi-periodic oscillations.
- 70Helwig, B.; van Sluijs, B.; Pogodaev, A. A.; Postma, S. G. J.; Huck, W. T. S. Bottom-up construction of an adaptive enzymatic reaction network. Angew. Chem., Int. Ed. 2018, 57 (43), 14065– 14069, DOI: 10.1002/anie.201806944Google Scholar70https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC1cXhvVejs7%252FI&md5=376c5ca98743d8247e0116a3309693e1Bottom-Up Construction of an Adaptive Enzymatic Reaction NetworkHelwig, Britta; van Sluijs, Bob; Pogodaev, Aleksandr A.; Postma, Sjoerd G. J.; Huck, Wilhelm T. S.Angewandte Chemie, International Edition (2018), 57 (43), 14065-14069CODEN: ACIEF5; ISSN:1433-7851. (Wiley-VCH Verlag GmbH & Co. KGaA)The reprodn. of emergent behaviors in nature using reaction networks is an important objective in synthetic biol. and systems chem. Herein, the first exptl. realization of an enzymic reaction network capable of an adaptive response is reported. The design is based on the dual activity of trypsin (Tr), which activates chymotrypsin (Cr) while at the same time generating a fluorescent output from a fluorogenic substrate. Once activated, chymotrypsin counteracts the trypsin output by competing for the fluorogenic substrate and producing a non-fluorescent output. It is demonstrated that this network produces a transient fluorescent output under out-of-equil. conditions while the input signal persists. Importantly, in agreement with math. simulations, we show that optimization of the pulse-like response is an inherent trade-off between max. amplitude and lowest residual fluorescence.
- 71Semenov, S. N.; Markvoort, A. J.; de Greef, T. F.; Huck, W. T. Threshold sensing through a synthetic enzymatic reaction-diffusion network. Angew. Chem., Int. Ed. 2014, 53 (31), 8066– 8069, DOI: 10.1002/anie.201402327Google Scholar71https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2cXlsFyisrw%253D&md5=57191864d1fcaa1a82b52db1977c29d4Threshold Sensing Through a Synthetic Enzymatic Reaction-Diffusion NetworkSemenov, Sergey N.; Markvoort, Albert J.; de Greef, Tom F. A.; Huck, Wilhelm T. S.Angewandte Chemie, International Edition (2014), 53 (31), 8066-8069CODEN: ACIEF5; ISSN:1433-7851. (Wiley-VCH Verlag GmbH & Co. KGaA)A wet stamping method to precisely control concns. of enzymes and inhibitors in place and time inside layered gels is reported. By combining enzymic reactions such as autocatalysis and inhibition with spatial delivery of components through soft lithog. techniques, a biochem. reaction network capable of recognizing the spatial distribution of an enzyme was constructed. The exptl. method can be used to assess fundamental principles of spatiotemporal order formation in chem. reaction networks.
- 72Kriukov, D. V.; Koyuncu, A. H.; Wong, A. S. Y. History dependence in a chemical reaction network enables dynamic switching. Small 2022, 18 (16), 2107523, DOI: 10.1002/smll.202107523Google Scholar72https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB38Xms1Omu7c%253D&md5=fc91fbb1e0b1af424562d6dda6e51c76History Dependence in a Chemical Reaction Network Enables Dynamic SwitchingKriukov, Dmitrii V.; Koyuncu, A. Hazal; Wong, Albert S. Y.Small (2022), 18 (16), 2107523CODEN: SMALBC; ISSN:1613-6810. (Wiley-VCH Verlag GmbH & Co. KGaA)This work describes an enzymic autocatalytic network capable of dynamic switching under out-of-equil. conditions. The network, wherein a mol. fuel (trypsinogen) and an inhibitor (soybean trypsin inhibitor) compete for a catalyst (trypsin), is kept from reaching equil. using a continuous flow stirred tank reactor. A so-called 'linear inhibition sweep' is developed (i.e., a mol. analog of linear sweep voltammetry) to intentionally perturb the competition between autocatalysis and inhibition, and used to demonstrate that a simple mol. system, comprising only three components, is already capable of a variety of essential neuromorphic behaviors (hysteresis, synchronization, resonance, and adaptation). This research provides the first steps in the development of a strategy that uses the principles in systems chem. to transform chem. reaction networks into platforms capable of neural network computing.
- 73Pogodaev, A. A.; Fernández Regueiro, C. L.; Jakštaitė, M.; Hollander, M. J.; Huck, W. T. S. Modular design of small enzymatic reaction networks based on reversible and cleavable inhibitors. Angew. Chem., Int. Ed. 2019, 58 (41), 14539– 14543, DOI: 10.1002/anie.201907995Google Scholar73https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC1MXhslWkurnM&md5=8e800d63a72c0a71f00f1ecf02c54b04Modular Design of Small Enzymatic Reaction Networks Based on Reversible and Cleavable InhibitorsPogodaev, Aleksandr A.; Fernandez Regueiro, Cristina Lia; Jakstaite, Migle; Hollander, Marijn J.; Huck, Wilhelm T. S.Angewandte Chemie, International Edition (2019), 58 (41), 14539-14543CODEN: ACIEF5; ISSN:1433-7851. (Wiley-VCH Verlag GmbH & Co. KGaA)Systems chem. aims to mimic the functional behavior of living systems by constructing chem. reaction networks with well-defined dynamic properties. Enzymes can play a key role in such networks, but there is currently no general and scalable route to the design and construction of enzymic reaction networks. Here, we introduce reversible, cleavable peptide inhibitors that can link proteolytic enzymic activity into simple network motifs. As a proof-of-principle, we show auto-activation topologies producing sigmoidal responses in enzymic activity, explore cross-talk in minimal systems, design a simple enzymic cascade, and introduce non-inhibiting phosphorylated peptides that can be activated using a phosphatase.
- 74Yamazaki, I.; Yokota, K.; Nakajima, R. Oscillatory oxidations of reduced pyridine nucleotide by peroxidase. Biochem. Biophys. Res. Commun. 1965, 21 (6), 582– 6, DOI: 10.1016/0006-291X(65)90525-5Google Scholar74https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaF28XjslehtA%253D%253D&md5=944b16272e50aff3e58d1c39ae519aebOscillatory oxidations of reduced pyridine nucleotide by peroxidaseYamazaki, I.; Yokota, K.; Nakajima, R.Biochemical and Biophysical Research Communications (1965), 21 (6), 582-6CODEN: BBRCA9; ISSN:0006-291X.Observation of periodic reactions catalyzed by peroxidase in the presence of reduced pyridine nucleotide under aerobic conditions is described. Addn. of glucose-6-phosphate dehydrogenase to a soln. contg. NADP, Mn2+, glucose 6-phosphate, and peroxidase results in a transient redn. of NADP, followed by a few cyclic responses of oxidn. and redn. until all of the O is consumed. A rapid oxidn. of NADP suddenly occurs at a certain reaction time which corresponds to the termination of Compound III accumulation. Addn. of NADH to an aerobic soln. of peroxidase results in a rapid appearance of Compound III. The rate of concomitant consumption of O overcomes that of the O supply and the O concentration of the soln. decreases, followed by the decompn. of Compound III. The formation of Compound III is mainly due to the reaction of peroxidase with the perhydroxyl radical which causes the chain reaction of the NADH oxidn. The sudden decay of Compound III in the presence of low O concns. is likely due to the redn. of Compound III by intermediates, such as the NAD radical and ferrous peroxidase.
- 75Folke Olsen, L. Complex dynamics in an unexplored simple model of the peroxidase-oxidase reaction. Chaos 2023, 33 (2), 023102 DOI: 10.1063/5.0129095Google Scholar75https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB3sXisF2ru7Y%253D&md5=881e99d3a1fba1340d8b1276a9220ea5Complex dynamics in an unexplored simple model of the peroxidase-oxidase reactionFolke Olsen, LarsChaos (2023), 33 (2), 023102CODEN: CHAOEH; ISSN:1054-1500. (American Institute of Physics)A previously overlooked version of the so-called Olsen model of the peroxidase-oxidase reaction has been studied numerically using 2D isospike stability and max. Lyapunov exponent diagrams and reveals a rich variety of dynamic behaviors not obsd. before. The model has a complex bifurcation structure involving mixed-mode and bursting oscillations as well as quasiperiodic and chaotic dynamics. In addn., multiple periodic and non-periodic attractors coexist for the same parameters. For some parameter values, the model also reveals formation of mosaic patterns of complex dynamic states. The complex dynamic behaviors exhibited by this model are compared to those of another version of the same model, which has been studied in more detail. The two models show similarities, but also notable differences between them, e.g., the organization of mixed-mode oscillations in parameter space and the relative abundance of quasiperiodic and chaotic oscillations. In both models, domains with chaotic dynamics contain apparently disorganized subdomains of periodic attractors with dinoflagellate-like structures, while the domains with mainly quasiperiodic behavior contain subdomains with periodic attractors organized as regular filamentous structures. These periodic attractors seem to be organized according to Stern-Brocot arithmetics. Finally, it appears that toroidal (quasiperiodic) attractors develop into first wrinkled and then fractal tori before they break down to chaotic attractors. (c) 2023 American Institute of Physics.
- 76Olsen, L. F.; Lunding, A. Chaos in the peroxidase-oxidase oscillator. Chaos 2021, 31 (1), 013119 DOI: 10.1063/5.0022251Google Scholar76https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB3MXhtFGnsb%252FP&md5=040d7f99e066ac615c9bb5dc635c0993Chaos in the peroxidase-oxidase oscillatorOlsen, Lars F.; Lunding, AnitaChaos (2021), 31 (1), 013119CODEN: CHAOEH; ISSN:1054-1500. (American Institute of Physics)The peroxidase-oxidase (PO) reaction involves the oxidn. of reduced NAD by mol. oxygen. When both reactants are supplied continuously to a reaction mixt. contg. the enzyme and a phenolic compd., the reaction will exhibit oscillatory behavior. In fact, the reaction exhibits a zoo of dynamical behaviors ranging from simple periodic oscillations to period-doubled and mixed mode oscillations to quasiperiodicity and chaos. The routes to chaos involve period-doubling, period-adding, and torus bifurcations. The dynamic behaviors in the exptl. system can be simulated by detailed semiquant. models. Previous models of the reaction have omitted the phenolic compd. from the reaction scheme. In the current paper, we present new exptl. results with the oscillating PO reaction that add to our understanding of its rich dynamics, and we describe a new variant of a previous model, which includes the chem. of the phenol in the reaction mechanism. This new model can simulate most of the exptl. behaviors of the exptl. system including the new observations presented here. For example, the model reproduces the two main routes to chaos obsd. in expts.: (i) a period-doubling scenario, which takes place at low pH, and a period-adding scenario involving mixed mode oscillations (MMOs), which occurs at high pH. Our simulations suggest alternative explanations for the pH-sensitivity of the dynamics. We show that the MMO domains are sepd. by narrow parameter regions of chaotic behavior or quasiperiodicity. These regions start as tongues of secondary quasiperiodicity and develop into strange attractors through torus breakdown. (c) 2021 American Institute of Physics.
- 77Scheeline, A.; Olson, D. L.; Williksen, E. P.; Horras, G. A.; Klein, M. L.; Larter, R. The peroxidase–oxidase oscillator and its constituent chemistries. Chem. Rev. 1997, 97 (3), 739– 756, DOI: 10.1021/cr960081aGoogle Scholar77https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaK2sXisF2murg%253D&md5=ee8592a17b3b80df9865655a040f4e7bThe Peroxidase-Oxidase Oscillator and Its Constituent ChemistriesScheeline, Alexander; Olson, Dean L.; Williksen, Erik P.; Horras, Gregg A.; Klein, Margaret L.; Larter, RaimaChemical Reviews (Washington, D. C.) (1997), 97 (3), 739-756CODEN: CHREAY; ISSN:0009-2665. (American Chemical Society)A review with 165 refs. on the chem. and biochem. of peroxidase concg. on aspect relevant to periodic and chaotic oscillations.
- 78Zhang, Y.; Tsitkov, S.; Hess, H. Complex dynamics in a two-enzyme reaction network with substrate competition. Nat. Catal. 2018, 1 (4), 276– 281, DOI: 10.1038/s41929-018-0053-1Google ScholarThere is no corresponding record for this reference.
- 79Gyevi-Nagy, L.; Lantos, E.; Gehér-Herczegh, T.; Tóth, A.; Bagyinka, C.; Horváth, D. Reaction fronts of the autocatalytic hydrogenase reaction. J. Chem. Phys. 2018, 148 (16), 165103, DOI: 10.1063/1.5022359Google Scholar79https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC1cXosl2htL4%253D&md5=5b7e7b1a5325910a0eb4ea79b2627f91Reaction fronts of the autocatalytic hydrogenase reactionGyevi-Nagy, Laszlo; Lantos, Emese; Geher-Herczegh, Tunde; Toth, Agota; Bagyinka, Csaba; Horvath, DezsoJournal of Chemical Physics (2018), 148 (16), 165103/1-165103/8CODEN: JCPSA6; ISSN:0021-9606. (American Institute of Physics)We have built a model to describe the hydrogenase catalyzed, autocatalytic, reversible hydrogen oxidn. reaction where one of the enzyme forms is the autocatalyst. The model not only reproduces the exptl. obsd. front properties, but also explains the found hydrogen ion dependence. Furthermore, by linear stability anal., two different front types are found in good agreement with the expts. (c) 2018 American Institute of Physics.
- 80Bagyinka, C.; Pankotai-Bodó, G.; Branca, R. M. M.; Debreczeny, M. Oscillating hydrogenase reaction. Int. J. Hydrogen Energy 2014, 39 (32), 18551– 18555, DOI: 10.1016/j.ijhydene.2014.02.015Google Scholar80https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2cXjsVWhsLg%253D&md5=79655c55851cbb0e6f5d40627e5c7abaOscillating hydrogenase reactionBagyinka, Csaba; Pankotai-Bodo, Gabriella; Branca, Rui M. M.; Debreczeny, MonikaInternational Journal of Hydrogen Energy (2014), 39 (32), 18551-18555CODEN: IJHEDX; ISSN:0360-3199. (Elsevier Ltd.)The hydrogenase-catalyzed oxidn. of H2 includes an autocatalytic step in the reaction cycle. The reaction also exhibits different pH dependence in the H2 oxidn. and in the proton redn. directions. This is not only due to the pH titrn. of the amino acid side-chains as protons are also either the substrates or the products of the reaction. Utilizing the autocatalytic nature of the hydrogenase reaction and the multiple roles of protons therein, together with appropriate limitation of the substrate (gaseous H2) supply, oscillations can be induced in the system. The reaction oscillates both in space and in time, and can last for days with decreasing frequency until reaching chem. equil. Of all biol. oscillating systems described so far, this one is the simplest in that it has the fewest biol. components.
- 81Claaßen, C.; Gerlach, T.; Rother, D. Stimulus-responsive regulation of enzyme activity for one-step and multi-step syntheses. Adv. Synth. Catal. 2019, 361 (11), 2387– 2401, DOI: 10.1002/adsc.201900169Google Scholar81https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC1MXntleru74%253D&md5=aa201db447575541f52c94d6d9f6c3d7Stimulus-Responsive Regulation of Enzyme Activity for One-Step and Multi-Step SynthesesClaassen, Christiane; Gerlach, Tim; Rother, DoerteAdvanced Synthesis & Catalysis (2019), 361 (11), 2387-2401CODEN: ASCAF7; ISSN:1615-4150. (Wiley-VCH Verlag GmbH & Co. KGaA)A review. Multi-step biocatalytic reactions have gained increasing importance in recent years because the combination of different enzymes enables the synthesis of a broad variety of industrially relevant products. However, the more enzymes combined, the more crucial it is to avoid cross-reactivity in these cascade reactions and thus achieve high product yields and high purities. The selective control of enzyme activity, i.e., remote on-/off-switching of enzymes, might be a suitable tool to avoid the formation of unwanted byproducts in multi-enzyme reactions. This review compiles a range of methods that are known to modulate enzyme activity in a stimulus-responsive manner. It focuses predominantly on in vitro systems and is subdivided into reversible and irreversible enzyme activity control. Furthermore, a discussion section provides indications as to which factors should be considered when designing and choosing activity control systems for biocatalysis. Finally, an outlook is given regarding the future prospects of the field.
- 82Chen, Z.; Zhao, Y.; Liu, Y. Advanced strategies of enzyme activity regulation for biomedical applications. ChemBioChem 2022, 23 (21), e202200358, DOI: 10.1002/cbic.202200358Google Scholar82https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB38XitV2ksb3O&md5=a6e13b3b38222804c88636870c7dd7ecAdvanced Strategies of Enzyme Activity Regulation for Biomedical ApplicationsChen, Zihan; Zhao, Yu; Liu, YangChemBioChem (2022), 23 (21), e202200358CODEN: CBCHFX; ISSN:1439-4227. (Wiley-VCH Verlag GmbH & Co. KGaA)A review. Enzymes are important macromol. biocatalysts that accelerate chem. and biochem. reactions in living organisms. Most human diseases are related to alterations in enzyme activity. Moreover, enzymes are potential therapeutic tools for treating different diseases, such as cancer, infections, and cardiovascular and cerebrovascular diseases. Precise remote enzyme activity regulation provides new opportunities to combat diseases. This review summarizes recent advances in the field of enzyme activity regulation, including reversible and irreversible regulation. It also discusses the mechanisms and approaches for on-demand control of these activities. Furthermore, a range of stimulus-responsive inhibitors, polymers, and nanoparticles for regulating enzyme activity and their prospective biomedical applications are summarized. Finally, the current challenges and future perspectives on enzyme activity regulation are discussed.
- 83Hoorens, M. W. H.; Szymanski, W. Reversible, spatial and temporal control over protein activity using light. Trends Biochem. Sci. 2018, 43 (8), 567– 575, DOI: 10.1016/j.tibs.2018.05.004Google Scholar83https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC1cXhtFSru7vI&md5=6802043365a055b6a166db2f493fb391Reversible, Spatial and Temporal Control over Protein Activity Using LightHoorens, Mark W. H.; Szymanski, WiktorTrends in Biochemical Sciences (2018), 43 (8), 567-575CODEN: TBSCDB; ISSN:0968-0004. (Elsevier Ltd.)A review. In biomedical sciences, the function of a protein of interest is investigated by altering its net activity and assessing the consequences for the cell or organism. To change the activity of a protein, a wide variety of chem. and genetic tools have been developed. The drawback of most of these tools is that they do not allow for reversible, spatial and temporal control. Here, we describe selected developments in photopharmacol. that aim at establishing such control over protein activity through bioactive mols. with photo-controlled potency. We also discuss why such control is desired and what challenges still need to be overcome for photopharmacol. to reach its maturity as a chem. biol. research tool.
- 84Kneuttinger, A. C. A guide to designing photocontrol in proteins: methods, strategies and applications. Biol. Chem. 2022, 403 (5–6), 573– 613, DOI: 10.1515/hsz-2021-0417Google Scholar84https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB38XhtVCmtLzF&md5=f11065ba65174acc0ee78139a82f764dA guide to designing photocontrol in proteins: methods, strategies and applicationsKneuttinger, Andrea C.Biological Chemistry (2022), 403 (5-6), 573-613CODEN: BICHF3; ISSN:1431-6730. (Walter de Gruyter GmbH)A review. Light is essential for various biochem. processes in all domains of life. In its presence certain proteins inside a cell are excited, which either stimulates or inhibits subsequent cellular processes. The artificial photocontrol of specifically proteins is of growing interest for the investigation of scientific questions on the organismal, cellular and mol. level as well as for the development of medicinal drugs or biocatalytic tools. For the targeted design of photocontrol in proteins, three major methods have been developed over the last decades, which employ either chem. engineering of small-mol. photosensitive effectors (photopharmacol.), incorporation of photoactive non-canonical amino acids by genetic code expansion (photoxenoprotein engineering), or fusion with photoreactive biol. modules (hybrid protein optogenetics). This review compares the different methods as well as their strategies and current applications for the light-regulation of proteins and provides background information useful for the implementation of each technique.
- 85Volarić, J.; Szymanski, W.; Simeth, N. A.; Feringa, B. L. Molecular photoswitches in aqueous environments. Chem. Soc. Rev. 2021, 50 (22), 12377– 12449, DOI: 10.1039/D0CS00547AGoogle Scholar85https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB3MXitFCgtbvK&md5=4fd8e15de13066519d17b50d2fe7fde0Molecular photoswitches in aqueous environmentsVolaric, Jana; Szymanski, Wiktor; Simeth, Nadja A.; Feringa, Ben L.Chemical Society Reviews (2021), 50 (22), 12377-12449CODEN: CSRVBR; ISSN:0306-0012. (Royal Society of Chemistry)A review. Mol. photoswitches enable dynamic control of processes with high spatiotemporal precision, using light as external stimulus, and hence are ideal tools for different research areas spanning from chem. biol. to smart materials. Photoswitches are typically org. mols. that feature extended arom. systems to make them responsive to (visible) light. However, this renders them inherently lipophilic, while water-soly. is of crucial importance to apply photoswitchable org. mols. in biol. systems, like in the rapidly emerging field of photopharmacol. Several strategies for solubilizing org. mols. in water are known, but there are not yet clear rules for applying them to photoswitchable mols. Importantly, rendering photoswitches water-sol. has a serious impact on both their photophys. and biol. properties, which must be taken into consideration when designing new systems. Altogether, these aspects pose considerable challenges for successfully applying mol. photoswitches in aq. systems, and in particular in biol. relevant media. In this , we focus on fully water-sol. photoswitches, such as those used in biol. environments, in both in vitro and in vivo studies. We discuss the design principles and prospects for water-sol. photoswitches to inspire and enable their future applications.
- 86Pogodaev, A. A.; Lap, T. T.; Huck, W. T. S. The dynamics of an oscillating enzymatic reaction network is crucially determined by side reactions. ChemSystemsChem 2021, 3, e2000033, DOI: 10.1002/syst.202000033Google Scholar86https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB3MXovFylsbk%253D&md5=242d0992326356525f85f0960ece3499The Dynamics of an Oscillating Enzymatic Reaction Network is Crucially Determined by Side ReactionsPogodaev, Aleksandr A.; Lap, Tijs T.; Huck, Wilhelm T. S.ChemSystemsChem (2021), 3 (1), e2000033CODEN: CSCHCD; ISSN:2570-4206. (Wiley-VCH Verlag GmbH & Co. KGaA)Synthetic complex chem. systems are often subject to perturbations in reaction conditions. To ensure robust functioning of these systems in real-world applications, a better understanding is required of how resilience to perturbations could be included in the design of these systems. In order to develop such an understanding we need a deeper insight into how chem. systems respond to perturbations. Here, we study the effect of spiking concns. in an oscillating enzymic reaction network. We identify that a different magnitude of a perturbation triggers two distinctive responses: obtaining sustained oscillations and causing the loss of the amplitude. We rationalise our findings based on non-linear dynamics and identify that non-essential side reactions crucially tailor the obsd. behavior.
- 87Crespi, S.; Simeth, N. A.; König, B. Heteroaryl azo dyes as molecular photoswitches. Nat. Rev. Chem. 2019, 3, 133– 146, DOI: 10.1038/s41570-019-0074-6Google Scholar87https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC1MXnsFCltL0%253D&md5=b8fd12702cc1d5c5ad102de3b6ce6378Heteroaryl azo dyes as molecular photoswitchesCrespi, Stefano; Simeth, Nadja A.; Koenig, BurkhardNature Reviews Chemistry (2019), 3 (3), 133-146CODEN: NRCAF7; ISSN:2397-3358. (Nature Research)A review. We have known of azobenzene for over 150 years, the past 80 of which have seen the study and application of its photochromism. Azobenzene derivs. are now considered archetypical mol. switches, and their stability and reliability make them amenable to many fields of modern chem., materials science, biol. and photopharmacol. When developing a photoswitch for a given application, a common approach is to tune the properties of an azobenzene. It is also possible to instead use heteroaryl azo dyes - motifs that are less popular even though their diversity offers distinct features. Despite the first discoveries of switching behavior in heteroaryl azos and azobenzenes being coincident, the former have only recently begun to attract attention. This Review describes how the versatile and multifaceted characteristics of these scaffolds make them serious alternatives to azobenzene derivs. in mol. photoactuation. Heteroaryl azo photoswitches arguably deserve more consideration, and our survey of these systems includes challenges to their successful deployment.
- 88Teders, M.; Pogodaev, A. A.; Bojanov, G.; Huck, W. T. S. Reversible photoswitchable inhibitors generate ultrasensitivity in out-of-equilibrium enzymatic reactions. J. Am. Chem. Soc. 2021, 143 (15), 5709– 5716, DOI: 10.1021/jacs.0c12956Google Scholar88https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB3MXosVWntLo%253D&md5=4aa47d83f4357ed58cd6585db025bb59Reversible photoswitchable inhibitors generate ultrasensitivity in out-of-equil. enzymic reactionsTeders, Michael; Pogodaev, Aleksandr A.; Bojanov, Glenn; Huck, Wilhelm T. S.Journal of the American Chemical Society (2021), 143 (15), 5709-5716CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)Ultrasensitivity is a ubiquitous emergent property of biochem. reaction networks. The design and construction of synthetic reaction networks exhibiting ultrasensitivity has been challenging, but would greatly expand the potential properties of life-like materials. Herein, we exploit a general and modular strategy to reversibly regulate the activity of enzymes using light and show how ultrasensitivity arises in simple out-of-equil. enzymic systems upon incorporation of reversible photoswitchable inhibitors (PIs). Utilizing a chromophore/warhead strategy, PIs of the protease α-chymotrypsin were synthesized, which led to the discovery of inhibitors with large differences in inhibition consts. (Ki) for the different photoisomers. A microfluidic flow setup was used to study enzymic reactions under out-of-equil. conditions by continuous addn. and removal of reagents. Upon irradn. of the continuously stirred tank reactor with different light pulse sequences, i.e., varying the pulse duration or frequency of UV and blue light irradn., reversible switching between photoisomers resulted in ultrasensitive responses in enzymic activity as well as frequency filtering of input signals. This general and modular strategy enables reversible and tunable control over the kinetic rates of individual enzyme-catalyzed reactions and makes a programmable linkage of enzymes to a wide range of network topologies feasible.
- 89Teders, M.; Murray, N. R.; Huck, W. T. S. Reversible photoswitchable inhibitors enable wavelength-selective regulation of out-of-equilibrium bi-enzymatic systems. ChemSystemsChem 2021, 3 (6), e2100020, DOI: 10.1002/syst.202100020Google Scholar89https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB3MXisFSjsrrF&md5=2266f480fd59765360dab98b60932eb3Reversible Photoswitchable Inhibitors Enable Wavelength-Selective Regulation of Out-of-Equilibrium Bi-enzymatic SystemsTeders, Michael; Murray, Nicholas R.; Huck, Wilhelm T. S.ChemSystemsChem (2021), 3 (6), e2100020CODEN: CSCHCD; ISSN:2570-4206. (Wiley-VCH Verlag GmbH & Co. KGaA)The construction of synthetic enzymic reaction networks can provide new insights into the design principles of living systems. However, the programmable connection of enzymes into a wide range of network topologies has been challenging due to the lack of a general strategy enabling a reversible activity regulation of individual network enzymes. Here, we exploit a general and modular strategy based on the external regulation of enzymes using light and photoswitchable inhibitors (PIs) that enables the bottom-up construction and control of enzymic systems studied under out-of-equil. conditions. Upon synthesis and incorporation of potent photoswitchable trypsin inhibitors (Tr-PIs), the output of several functional enzymic systems could be photoregulated using 390/460 nm light as a trigger signal. In addn., the wavelength-selective control over the activity of two enzymes within a functional bi-enzymic system was achieved using a suitable combination of two PIs.
- 90Li, K.; Liu, M. D.; Huang, Q. X.; Liu, C. J.; Zhang, X. Z. Nanoplatforms with donor-acceptor Stenhouse adduct molecular switch for enzymatic reactions remotely controlled with near-infrared light. Sci. China Mater. 2023, 66, 375– 384, DOI: 10.1007/s40843-022-2107-0Google Scholar90https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB38XhvVGkur3L&md5=b5f8248b37bd48da19c39abc5f802662Nanoplatforms with donor-acceptor Stenhouse adduct molecular switch for enzymatic reactions remotely controlled with near-infrared lightLi, Ke; Liu, Miao-Deng; Huang, Qian-Xiao; Liu, Chuan-Jun; Zhang, Xian-ZhengScience China Materials (2023), 66 (1), 375-384CODEN: SCMCDB; ISSN:2095-8226. (Science China Press)Enzymes have been widely used in biomedical applications owing to their excellent biocatalysis functions. However, the precise control of enzymic reactions remains a challenge in vivo. Here, a nanoplatform functionalized with upconversion nanoparticles (UCNPs) as the core and a donor-acceptor Stenhouse adduct as a mol. switch (MS) was designed for near-IR (NIR)-controlled enzymic reactions. Under NIR irradn., the UCNPs could emit bright green light to isomerize the MS and then transform the permeability of the MS-gated polymer layer, allowing small mols. to penetrate the polymer layer. Consequently, the contact between the enzyme and substrate could be regulated to control the enzymic reaction remotely. As the photoisomerism of MS is reversible and the enzyme activity is not changed, the enzyme reactor based on this nanoplatform is also reversible, which can achieve precise temporal and spatial control of the enzyme reaction. Glucose oxidase (GOX), lactate oxidase (LOX), urate oxidase (UOX), and luciferase were used to assess the controllability of the enzymic reactions. Both the substrates in and out of the nanoplatform were satisfactorily controlled, indicating that the gated nanoplatform equipped with valve-like characteristics could realize remotely controlled enzymic reactions upon NIR irradn.
- 91Hindley, J. W.; Elani, Y.; McGilvery, C. M.; Ali, S.; Bevan, C. L.; Law, R. V.; Ces, O. Light-triggered enzymatic reactions in nested vesicle reactors. Nat. Commun. 2018, 9 (1), 1093, DOI: 10.1038/s41467-018-03491-7Google Scholar91https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A280%3ADC%252BC1MnhslKisg%253D%253D&md5=96a3060c2d2721132e189f186947f5c3Light-triggered enzymatic reactions in nested vesicle reactorsHindley James W; Elani Yuval; Law Robert V; Ces Oscar; Hindley James W; Elani Yuval; Law Robert V; Ces Oscar; McGilvery Catriona M; Ali Simak; Bevan Charlotte LNature communications (2018), 9 (1), 1093 ISSN:.Cell-sized vesicles have tremendous potential both as miniaturised pL reaction vessels and in bottom-up synthetic biology as chassis for artificial cells. In both these areas the introduction of light-responsive modules affords increased functionality, for example, to initiate enzymatic reactions in the vesicle interior with spatiotemporal control. Here we report a system composed of nested vesicles where the inner compartments act as phototransducers, responding to ultraviolet irradiation through diacetylene polymerisation-induced pore formation to initiate enzymatic reactions. The controlled release and hydrolysis of a fluorogenic β-galactosidase substrate in the external compartment is demonstrated, where the rate of reaction can be modulated by varying ultraviolet exposure time. Such cell-like nested microreactor structures could be utilised in fields from biocatalysis through to drug delivery.
- 92Babii, O.; Afonin, S.; Diel, C.; Huhn, M.; Dommermuth, J.; Schober, T.; Koniev, S.; Hrebonkin, A.; Nesterov-Mueller, A.; Komarov, I. V. Diarylethene-based photoswitchable inhibitors of serine proteases. Angew. Chem., Int. Ed. 2021, 60 (40), 21789– 21794, DOI: 10.1002/anie.202108847Google Scholar92https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB3MXhvFShu7zP&md5=204d0959bc8b05f71e2b016406e4554cDiarylethene-Based Photoswitchable Inhibitors of Serine ProteasesBabii, Oleg; Afonin, Sergii; Diel, Christian; Huhn, Marcel; Dommermuth, Jennifer; Schober, Tim; Koniev, Serhii; Hrebonkin, Andrii; Nesterov-Mueller, Alexander; Komarov, Igor V.; Ulrich, Anne S.Angewandte Chemie, International Edition (2021), 60 (40), 21789-21794CODEN: ACIEF5; ISSN:1433-7851. (Wiley-VCH Verlag GmbH & Co. KGaA)A bicyclic peptide scaffold was chem. adapted to generate diarylethene-based photoswitchable inhibitors of serine protease Bos taurus trypsin 1 (T1). Starting from a prototype mol.-sunflower trypsin inhibitor-1 (SFTI-1)-the authors obtained light-controllable inhibitors of T1 with Ki in the low nanomolar range, whose activity could be modulated over 20-fold by irradn. The inhibitory potency as well as resistance to proteolytic degrdn. were systematically studied on a series of 17 SFTI-1 analogs. The hydrogen bond network that stabilizes the structure of inhibitors and possibly the enzyme-inhibitor binding dynamics were affected by isomerization of the photoswitch. The feasibility of manipulating enzyme activity in time and space was demonstrated by controlled digestion of gelatin-based hydrogel and an antimicrobial peptide BP100-RW. Finally, the authors' design principles of diarylethene photoswitches apply also for the development of other serine protease inhibitors.
- 93Rifaie-Graham, O.; Yeow, J.; Najer, A.; Wang, R.; Sun, R.; Zhou, K.; Dell, T. N.; Adrianus, C.; Thanapongpibul, C.; Chami, M. Photoswitchable gating of non-equilibrium enzymatic feedback in chemically communicating polymersome nanoreactors. Nat. Chem. 2023, 15 (1), 110– 118, DOI: 10.1038/s41557-022-01062-4Google Scholar93https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB38XivVajs7bL&md5=b01721d704940a001212e55306e28618Photoswitchable gating of non-equilibrium enzymatic feedback in chemically communicating polymersome nanoreactorsRifaie-Graham, Omar; Yeow, Jonathan; Najer, Adrian; Wang, Richard; Sun, Rujie; Zhou, Kun; Dell, Tristan N.; Adrianus, Christopher; Thanapongpibul, Chalaisorn; Chami, Mohamed; Mann, Stephen; de Alaniz, Javier Read; Stevens, Molly M.Nature Chemistry (2023), 15 (1), 110-118CODEN: NCAHBB; ISSN:1755-4330. (Nature Portfolio)The circadian rhythm generates out-of-equil. metabolite oscillations that are controlled by feedback loops under light/dark cycles. Here we describe a non-equil. nanosystem comprising a binary population of enzyme-contg. polymersomes capable of light-gated chem. communication, controllable feedback and coupling to macroscopic oscillations. The populations consist of esterase-contg. polymersomes functionalized with photo-responsive donor-acceptor Stenhouse adducts (DASA) and light-insensitive semipermeable urease-loaded polymersomes. The DASA-polymersome membrane becomes permeable under green light, switching on esterase activity and decreasing the pH, which in turn initiates the prodn. of alkali in the urease-contg. population. A pH-sensitive pigment that absorbs green light when protonated provides a neg. feedback loop for deactivating the DASA-polymersomes. Simultaneously, increased alkali prodn. deprotonates the pigment, reactivating esterase activity by opening the membrane gate. We utilize light-mediated fluctuations of pH to perform non-equil. communication between the nanoreactors and use the feedback loops to induce work as chemomech. swelling/deswelling oscillations in a crosslinked hydrogel. We envision possible applications in artificial organelles, protocells and soft robotics.
- 94Bisswanger, H. Enzyme assays. Perspectives in Science 2014, 1 (1–6), 41– 55, DOI: 10.1016/j.pisc.2014.02.005Google ScholarThere is no corresponding record for this reference.
- 95Zhang, Y.; Wang, Q.; Hess, H. Increasing enzyme cascade throughput by pH-engineering the microenvironment of individual enzymes. ACS Catal. 2017, 7 (3), 2047– 2051, DOI: 10.1021/acscatal.6b03431Google Scholar95https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2sXisVOkurk%253D&md5=50fb5a9647fbe533d55dff99c707712fIncreasing enzyme cascade throughput by pH-engineering the microenvironment of individual enzymesZhang, Yifei; Wang, Qin; Hess, HenryACS Catalysis (2017), 7 (3), 2047-2051CODEN: ACCACS; ISSN:2155-5435. (American Chemical Society)The throughput of enzyme cascade reactions could be increased if the environmental conditions could be optimized for each enzyme in the cascade individually. Here, we describe the engineering of the microenvironment of a hemoprotein, cytochrome c (cyt c), which is active under acidic conditions, with respect to pH, so that it operates optimally together with a the enzyme, D-amino acid oxidase, which is active under alk. conditions. Conjugation of cyt c with a neg. charged polyelectrolyte [poly(methacrylic acid)] lowered the local pH at the cyt c active site and enabled a 10-fold enhancement in cascade throughput.
- 96Fan, X.; Walther, A. pH feedback lifecycles programmed by enzymatic logic gates using common foods as fuels. Angew. Chem., Int. Ed. 2021, 60, 11398, DOI: 10.1002/anie.202017003Google Scholar96https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB3MXotlGktrs%253D&md5=eec6c760a9a40322bed51e3b5b458040pH Feedback Lifecycles Programmed by Enzymatic Logic Gates Using Common Foods as FuelsFan, Xinlong; Walther, AndreasAngewandte Chemie, International Edition (2021), 60 (20), 11398-11405CODEN: ACIEF5; ISSN:1433-7851. (Wiley-VCH Verlag GmbH & Co. KGaA)Artificial temporal signaling systems, which mimic living out-of-equil. conditions, have made large progress. However, systems programmed by enzymic reaction networks in multicomponent and unknown environments, and using biocompatible components remain a challenge. Herein, we demonstrate an approach to program temporal pH signals by enzymic logic gates. They are realized by an enzymic disaccharide-to-monosaccharide-to-sugar acid reaction cascade catalyzed by two metabolic chains: invertase-glucose oxidase and β-galactosidase-glucose oxidase, resp. Lifetimes of the transient pH signal can be programmed from less than 15 min to more than 1 day. We study enzymic kinetics of the reaction cascades and reveal the underlying regulatory mechanisms. Operating with all-food grade chems. and coupling to self-regulating hydrogel, our system is quite robust to work in a complicated medium with unknown components and in a biocompatible fashion.
- 97Che, H.; Cao, S.; van Hest, J. C. M. Feedback-induced temporal control of ″breathing″ polymersomes to create self-adaptive nanoreactors. J. Am. Chem. Soc. 2018, 140 (16), 5356– 5359, DOI: 10.1021/jacs.8b02387Google Scholar97https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC1cXntVCmurk%253D&md5=178dddacb91d1c063f0d6104e216d2e6Feedback-Induced Temporal Control of "Breathing" Polymersomes To Create Self-Adaptive NanoreactorsChe, Hailong; Cao, Shoupeng; van Hest, Jan C. M.Journal of the American Chemical Society (2018), 140 (16), 5356-5359CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)Here the authors present the development of self-regulated "breathing" polymersome nanoreactors that show temporally programmable biocatalysis induced by a chem. fuel. pH-sensitive polymersomes loaded with horseradish peroxidase (HRP) and urease were developed. Addn. of an acidic urea soln. ("fuel") endowed the polymersomes with a transient size increase and permeability enhancement, driving a temporal "ON" state of the HRP enzymic catalysis; subsequent depletion of fuel led to shrinking of the polymersomes, resulting in the catalytic "OFF" state. Moreover, the nonequil. nanoreactors could be reinitiated several cycles as long as fuel was supplied. This feedback-induced temporal control of catalytic activity in polymersome nanoreactors provides a platform for functional nonequil. systems as well as for artificial organelles with precisely controlled adaptivity.
- 98Wells, P. K.; Smutok, O.; Melman, A.; Katz, E. Switchable biocatalytic reactions controlled by interfacial pH changes produced by orthogonal biocatalytic processes. ACS Appl. Mater. Interfaces 2021, 13 (29), 33830– 33839, DOI: 10.1021/acsami.1c07393Google Scholar98https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB3MXhsFCltrvK&md5=35c6c8a040f735ffddb70ceb0fe21816Switchable Biocatalytic Reactions Controlled by Interfacial pH Changes Produced by Orthogonal Biocatalytic ProcessesWells, Paulina K.; Smutok, Oleh; Melman, Artem; Katz, EvgenyACS Applied Materials & Interfaces (2021), 13 (29), 33830-33839CODEN: AAMICK; ISSN:1944-8244. (American Chemical Society)Enzymes immobilized on a nano-structured surface were used to switch the activity of one enzyme by a local pH change produced by another enzyme. Immobilized amyloglucosidase (AMG) and trypsin were studied as examples of the pH-dependent switchable "target enzymes." The reactions catalyzed by co-immobilized urease or esterase were increasing or decreasing the local pH, resp., thus operating as "actuator enzymes." Both kinds of the enzymes, producing local pH changes and changing biocatalytic activity with the pH variation, were orthogonal in terms of the biocatalytic reactions; however, their operation was coupled with the local pH produced near the surface with the immobilized enzymes. The "target enzymes" (AMG and trypsin) were changed reversibly between the active and inactive states by applying input signals (urea or ester, substrates for the urease or esterase operating as the "actuator enzymes") and washing them out with a new portion of the background soln. The developed approach can potentially lead to switchable operation of several enzymes, while some of them are inhibited when the others are activated upon receiving external signals processed by the "actuator enzymes." More complex systems with branched biocatalytic cascades can be controlled by orthogonal biocatalytic reactions activating selected pathways and changing the final output.
- 99Wang, C.; Fischer, A.; Ehrlich, A.; Nahmias, Y.; Willner, I. Biocatalytic reversible control of the stiffness of DNA-modified responsive hydrogels: applications in shape-memory, self-healing and autonomous controlled release of insulin. Chem. Sci. 2020, 11 (17), 4516– 4524, DOI: 10.1039/D0SC01319FGoogle Scholar99https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB3cXmvFOitrc%253D&md5=8da648a257fc758d89335776e8164d59Biocatalytic reversible control of the stiffness of DNA-modified responsive hydrogels: applications in shape-memory, self-healing and autonomous controlled release of insulinWang, Chen; Fischer, Amit; Ehrlich, Avner; Nahmias, Yaakov; Willner, ItamarChemical Science (2020), 11 (17), 4516-4524CODEN: CSHCCN; ISSN:2041-6520. (Royal Society of Chemistry)The enzymes glucose oxidase (GOx), acetylcholine esterase (AchE) and urease that drive biocatalytic transformations to alter pH, are integrated into pH-responsive DNA-based hydrogels. A two-enzyme-loaded hydrogel composed of GOx/urease or AchE/urease and a three-enzyme-loaded hydrogel composed of GOx/AchE/urease are presented. The biocatalytic transformations within the hydrogels lead to the dictated reconfiguration of nucleic acid bridges and the switchable control over the stiffness of the resp. hydrogels. The switchable stiffness features are used to develop biocatalytically guided shape-memory and self-healing matrixes. In addn., loading of GOx/insulin in a pH-responsive DNA-based hydrogel yields a glucose-triggered matrix for the controlled release of insulin, acting as an artificial pancreas. The release of insulin is controlled by the concns. of glucose, hence, the biocatalytic insulin-loaded hydrogel provides an interesting sense-and-treat carrier for controlling diabetes.
- 100Zhang, Y.; Nie, N.; Wang, H.; Tong, Z.; Xing, H.; Zhang, Y. Smart enzyme catalysts capable of self-separation by sensing the reaction extent. Biosens. Bioelectron. 2023, 239, 115585, DOI: 10.1016/j.bios.2023.115585Google Scholar100https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB3sXhslWns73P&md5=3e0c482c8735cb453cfa2580577caf5eSmart enzyme catalysts capable of self-separation by sensing the reaction extentZhang, Yinchen; Nie, Ning; Wang, Haoran; Tong, Ziyi; Xing, Hao; Zhang, YifeiBiosensors & Bioelectronics (2023), 239 (), 115585CODEN: BBIOE4; ISSN:0956-5663. (Elsevier B.V.)A smart biocatalyst should dissolve homogeneously for catalysis and recover spontaneously at the end of the reaction. In this study, we present a strategy for prepg. self-pptg. enzyme catalysts by exploiting reaction-induced pH decreases, which connect the reaction extent to the catalyst aggregation state. Using poly(methacrylic acid)-functionalized gold nanoparticles as carriers, we construct smart catalysts with three model systems, including the glucose oxidase (GOx)-catalase (CAT) cascade, the alc. dehydrogenase (ADH)-glucose dehydrogenase (GDH) cascade, and a combination of two lipases. All smart catalysts can self-sep. with a nearly 100% recovery efficiency when a certain conversion threshold is reached. The threshold can be adjusted depending on the reaction demand and buffer capacity. By monitoring the optical signals caused by the dissoln./pptn. of smart catalysts, we propose a prototypic automation system that may enable unsupervised batch/fed-batch bioprocessing.
- 101Jain, M.; Ravoo, B. J. Fuel-driven and enzyme-regulated redox-responsive supramolecular hydrogels. Angew. Chem., Int. Ed. 2021, 60 (38), 21062– 21068, DOI: 10.1002/anie.202107917Google Scholar101https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB3MXhsl2ltb7F&md5=644dd8f13ce610d6bbbdd4018b3e5a5cFuel-Driven and Enzyme-Regulated Redox-Responsive Supramolecular HydrogelsJain, Mehak; Ravoo, Bart JanAngewandte Chemie, International Edition (2021), 60 (38), 21062-21068CODEN: ACIEF5; ISSN:1433-7851. (Wiley-VCH Verlag GmbH & Co. KGaA)Chem. reaction networks (CRN) embedded in hydrogels can transform responsive materials into complex self-regulating materials that generate feedback to counter the effect of external stimuli. This study presents hydrogels contg. the β-cyclodextrin (CD) and ferrocene (Fc) host-guest pair as supramol. crosslinks where redox-responsive behavior is driven by the enzyme-fuel couples horse radish peroxidase (HRP)-H2O2 and glucose oxidase (GOx)-D-glucose. The hydrogel can be tuned from a responsive to a self-regulating supramol. system by varying the concn. of added redn. fuel D-glucose. The onset of self-regulating behavior is due to formation of oxidn. fuel in the hydrogel by a cofactor intermediate GOx[FADH2]. UV/Vis spectroscopy, rheol., and kinetic modeling were employed to understand the emergence of out-of-equil. behavior and reveal the programmable neg. feedback response of the hydrogel, including the adaptation of its elastic modulus and its potential as a glucose sensor.
- 102Eixelsberger, T.; Nidetzky, B. Enzymatic redox cascade for one-pot synthesis of uridine 5′-diphosphate xylose from uridine 5′-diphosphate glucose. Adv. Synth. Catal. 2014, 356 (17), 3575– 3584, DOI: 10.1002/adsc.201400766Google Scholar102https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2cXhvVKlsLvO&md5=fcd9da668f65eddf73f1bc5bb9886e73Enzymatic Redox Cascade for One-Pot Synthesis of Uridine 5'-Diphosphate Xylose from Uridine 5'-Diphosphate GlucoseEixelsberger, Thomas; Nidetzky, BerndAdvanced Synthesis & Catalysis (2014), 356 (17), 3575-3584CODEN: ASCAF7; ISSN:1615-4150. (Wiley-VCH Verlag GmbH & Co. KGaA)Synthetic ways towards UDP (UDP)-xylose are scarce and not well established, although this compd. plays an important role in the glycobiol. of various organisms and cell types. We show here how UDP-glucose 6-dehydrogenase (hUGDH) and UDP-xylose synthase 1 (hUXS) from Homo sapiens can be used for the efficient prodn. of pure UDP-α-xylose from UDP-glucose. In a mimic of the natural biosynthetic route, UDP-glucose is converted to UDP-glucuronic acid by hUGDH, followed by subsequent formation of UDP-xylose by hUXS. The NAD (NAD+) required in the hUGDH reaction is continuously regenerated in a three-step chemo-enzymic cascade. In the first step, reduced NAD+ (NADH) is recycled by xylose reductase from Candida tenuis via redn. of 9,10-phenanthrenequinone (PQ). Radical chem. re-oxidn. of this mediator in the second step reduces mol. oxygen to hydrogen peroxide (H2O2) that is cleaved by bovine liver catalase in the last step. A comprehensive anal. of the coupled chemo-enzymic reactions revealed pronounced inhibition of hUGDH by NADH and UDP-xylose as well as an adequate oxygen supply for PQ re-oxidn. as major bottlenecks of effective performance of the overall multi-step reaction system. Net oxidn. of UDP-glucose to UDP-xylose by hydrogen peroxide (H2O2) could thus be achieved when using an in situ oxygen supply through periodic external feed of H2O2 during the reaction. Engineering of the interrelated reaction parameters finally enabled prodn. of 19.5 mM (10.5 g L-1) UDP-α-xylose. After two-step chromatog. purifn. the compd. was obtained in high purity (>98%) and good overall yield (46%). The results provide a strong case for application of multi-step redox cascades in the synthesis of nucleotide sugar products.
- 103Kuk, S. K.; Singh, R. K.; Nam, D. H.; Singh, R.; Lee, J. K.; Park, C. B. Photoelectrochemical reduction of carbon dioxide to methanol through a highly efficient enzyme cascade. Angew. Chem., Int. Ed. 2017, 56 (14), 3827– 3832, DOI: 10.1002/anie.201611379Google Scholar103https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2sXhs1ems7o%253D&md5=db856189622518381382e2fd5d0b43b6Photoelectrochemical Reduction of Carbon Dioxide to Methanol through a Highly Efficient Enzyme CascadeKuk, Su Keun; Singh, Raushan K.; Nam, Dong Heon; Singh, Ranjitha; Lee, Jung-Kul; Park, Chan BeumAngewandte Chemie, International Edition (2017), 56 (14), 3827-3832CODEN: ACIEF5; ISSN:1433-7851. (Wiley-VCH Verlag GmbH & Co. KGaA)Natural photosynthesis is an effective route for the clean and sustainable conversion of CO2 into high-energy chems. Inspired by the natural process, a tandem photoelectrochem. (PEC) cell with an integrated enzyme-cascade (TPIEC) system was designed, which transfers photogenerated electrons to a multienzyme cascade for the biocatalyzed redn. of CO2 to methanol. A hematite photoanode and a bismuth ferrite photocathode were applied to fabricate the iron oxide based tandem PEC cell for visible-light-assisted regeneration of the nicotinamide cofactor (NADH). The cell utilized water as an electron donor and spontaneously regenerated NADH. To complete the TPIEC system, a superior three-dehydrogenase cascade system was employed in the cathodic part of the PEC cell. Under applied bias, the TPIEC system achieved a high methanol conversion output of 220 μm h-1, 1280 μmol g-1 h-1 using readily available solar energy and water.
- 104Mallawarachchi, S.; Gejji, V.; Sierra, L. S.; Wang, H.; Fernando, S. Electrical field reversibly modulates enzyme kinetics of hexokinase entrapped in an electro-responsive hydrogel. ACS Appl. Bio Mater. 2019, 2 (12), 5676– 5686, DOI: 10.1021/acsabm.9b00748Google Scholar104https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC1MXitVOht7rO&md5=349857855cebc50da63f91fdc2e2f765Electrical Field Reversibly Modulates Enzyme Kinetics of Hexokinase Entrapped in an Electro-Responsive HydrogelMallawarachchi, Samavath; Gejji, Varun; Sierra, Laura Soto; Wang, Haoqi; Fernando, SandunACS Applied Bio Materials (2019), 2 (12), 5676-5686CODEN: AABMCB; ISSN:2576-6422. (American Chemical Society)In this paper, the potential use of electro-responsive poly(acrylic acid) (PAA) gels as reversible enzyme activity regulators is analyzed. This was evaluated by measuring the glucose conversion by hexokinase embedded PAA hydrogels under external elec. stimuli. Hexokinase phys. entrapped within PAA gels showed a significant increase in activity under an elec. stimulus as compared to in the absence of a stimulus. Kinetic studies revealed that the change in reaction rate could be attributed to the change of Vmax under a stimulus, while Km was unaffected by the stimulus, which suggested that the increase in reaction rate under an elec. stimulus was due to increased accessibility of the active site. Optimum stimuli-responsive behavior that resulted in max. conversion under a stimulus and min. conversion in the absence of a stimulus was obtained at 5.5 pH and 30 °C. The significant difference between the pH optima for the entrapped enzyme and the pure enzyme can be attributed to the acidic nature of the polymeric matrix. Higher cross-linker concns. resulted in a redn. of both enzyme release and glucose conversion, and a reasonable trade-off between conversion and release could be obtained at 5% cross-linker concn. Application of a stepwise elec. stimulus revealed that the entrapped enzymes could sustain responsive properties over multiple cycles of elec. switching. Entrapped hexokinase also showed much better reusability compared to pure hexokinase, a combined result of higher enzyme retention and increased stability. No significant impact of the polymer on the interaction between enzyme and glucose was obsd. Thus, this system enables electro-responsive modulation of enzyme activity without any redn. in enzyme activity. The studies revealed that conjugation of electro-responsive polymers to enzymes has the potential to reversibly modulate enzymic reactions via the application of external elec. stimuli, which is promising for bioprocessing and enzymic sepn. applications.
- 105Morello, G.; Megarity, C. F.; Armstrong, F. A. The power of electrified nanoconfinement for energising, controlling and observing long enzyme cascades. Nat. Commun. 2021, 12 (1), 340, DOI: 10.1038/s41467-020-20403-wGoogle Scholar105https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB3MXhsVelsrk%253D&md5=144f16dcc5a3103a0ecddb9dabb20714The power of electrified nanoconfinement for energising, controlling and observing long enzyme cascadesMorello, Giorgio; Megarity, Clare F.; Armstrong, Fraser A.Nature Communications (2021), 12 (1), 340CODEN: NCAOBW; ISSN:2041-1723. (Nature Research)Abstr.: Multistep enzyme-catalyzed cascade reactions are highly efficient in nature due to the confinement and concn. of the enzymes within nanocompartments. In this way, rates are exceptionally high, and loss of intermediates minimised. Similarly, extended enzyme cascades trapped and crowded within the nanoconfined environment of a porous conducting metal oxide electrode material form the basis of a powerful way to study and exploit myriad complex biocatalytic reactions and pathways. One of the confined enzymes, ferredoxin-NADP+ reductase, serves as a transducer, rapidly and reversibly recycling nicotinamide cofactors electrochem. for immediate delivery to the next enzyme along the chain, thereby making it possible to energize, control and observe extended cascade reactions driven in either direction depending on the electrode potential that is applied. Here we show as proof of concept the synthesis of aspartic acid from pyruvic acid or its reverse oxidative decarboxylation/deamination, involving five nanoconfined enzymes.
- 106Milton, R. D.; Cai, R.; Abdellaoui, S.; Leech, D.; De Lacey, A. L.; Pita, M.; Minteer, S. D. Bioelectrochemical Haber-Bosch process: an ammonia-producing H2/N2 fuel cell. Angew. Chem., Int. Ed. 2017, 56 (10), 2680– 2683, DOI: 10.1002/anie.201612500Google Scholar106https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2sXitVKnurg%253D&md5=fe1ceeac4a36827692ba7997994321c3Bioelectrochemical Haber-Bosch Process: An Ammonia-Producing H2/N2 Fuel CellMilton, Ross D.; Cai, Rong; Abdellaoui, Sofiene; Leech, Donal; De Lacey, Antonio L.; Pita, Marcos; Minteer, Shelley D.Angewandte Chemie, International Edition (2017), 56 (10), 2680-2683CODEN: ACIEF5; ISSN:1433-7851. (Wiley-VCH Verlag GmbH & Co. KGaA)Nitrogenases are the only enzymes known to reduce mol. nitrogen (N2) to ammonia (NH3). By using Me viologen (N,N'-dimethyl-4,4'-bipyridinium) to shuttle electrons to nitrogenase, N2 redn. to NH3 can be mediated at an electrode surface. The coupling of this nitrogenase cathode with a bioanode that utilizes the enzyme hydrogenase to oxidize mol. hydrogen (H2) results in an enzymic fuel cell (EFC) that is able to produce NH3 from H2 and N2 while simultaneously producing an elec. current. To demonstrate this, a charge of 60 mC was passed across H2 /N2 EFCs, which resulted in the formation of 286 nmol NH3 mg-1 MoFe protein, corresponding to a Faradaic efficiency of 26.4 %.
- 107Cao, Y.; Wang, Y. Temperature-mediated regulation of enzymatic activity. ChemCatChem. 2016, 8 (17), 2740– 2747, DOI: 10.1002/cctc.201600406Google Scholar107https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC28XhtFersb7I&md5=3fa509ec15c4fdab3c62abebd2bc6bf7Temperature-Mediated Regulation of Enzymatic ActivityCao, Yuanyuan; Wang, YapeiChemCatChem (2016), 8 (17), 2740-2747CODEN: CHEMK3; ISSN:1867-3880. (Wiley-VCH Verlag GmbH & Co. KGaA)A review. Controllable nanobiocatalysis using enzymes has evolved to play an important role in the specific and efficient prepn. of bioproducts. Temp. change is one promising tool owing to the dependence of enzyme catalysis on temp. Some examples of regulation of enzymic activity through direct or indirect heating methods with the assistance of thermoresponsive or thermal harvesting materials are summarized in this concept article. Several fascinating applications including remote control of enzyme and/or substrate release, on-off enzyme reactions triggered by temp. variation to sep. products from enzymes and remote acceleration of enzyme reactions even at room temp. have been highlighted. All this provides a new perspective for establishing on-demand enzyme catalysis to further their applications in biol. engineering, chem. industry, and scientific research fields.
- 108Zhang, S.; Wang, C.; Chang, H.; Zhang, Q.; Cheng, Y. Off-on switching of enzyme activity by near-infrared light-induced photothermal phase transition of nanohybrids. Sci. Adv. 2019, 5, eaaw4252, DOI: 10.1126/sciadv.aaw4252Google ScholarThere is no corresponding record for this reference.
- 109Gobbo, P.; Patil, A. J.; Li, M.; Harniman, R.; Briscoe, W. H.; Mann, S. Programmed assembly of synthetic protocells into thermoresponsive prototissues. Nat. Mater. 2018, 17 (12), 1145– 1153, DOI: 10.1038/s41563-018-0183-5Google Scholar109https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC1cXhvV2rurfM&md5=f18bafe4760311f97789519a09e1fcf6Programmed assembly of synthetic protocells into thermoresponsive prototissuesGobbo, Pierangelo; Patil, Avinash J.; Li, Mei; Harniman, Robert; Briscoe, Wuge H.; Mann, StephenNature Materials (2018), 17 (12), 1145-1153CODEN: NMAACR; ISSN:1476-1122. (Nature Research)Although several new types of synthetic cell-like entities are now available, their structural integration into spatially interlinked prototissues that communicate and display coordinated functions remains a considerable challenge. Here the authors describe the programmed assembly of synthetic prototissue constructs based on the bio-orthogonal adhesion of a spatially confined binary community of protein-polymer protocells, termed proteinosomes. The thermoresponsive properties of the interlinked proteinosomes were used collectively to generate prototissue spheroids capable of reversible contractions that can be enzymically modulated and exploited for mechanochem. transduction. Overall, the authors' methodol. opens up a route to the fabrication of artificial tissue-like materials capable of collective behaviors, and addresses important emerging challenges in bottom-up synthetic biol. and bioinspired engineering.
- 110Szekeres, K.; Bollella, P.; Kim, Y.; Minko, S.; Melman, A.; Katz, E. Magneto-controlled enzyme activity with locally produced pH changes. J. Phys. Chem. Lett. 2021, 12 (10), 2523– 2527, DOI: 10.1021/acs.jpclett.1c00036Google Scholar110https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB3MXlslWgtro%253D&md5=a0a2d91316b29753fa48826093def8b6Magneto-Controlled Enzyme Activity with Locally Produced pH ChangesSzekeres, Krisztina; Bollella, Paolo; Kim, Yongwook; Minko, Sergiy; Melman, Artem; Katz, EvgenyJournal of Physical Chemistry Letters (2021), 12 (10), 2523-2527CODEN: JPCLCD; ISSN:1948-7185. (American Chemical Society)Biocatalytic activity of amyloglucosidase (AMG), immobilized on superparamagnetic nanoparticles, is dynamically and reversibly activated or inhibited by applying a magnetic field. The magnetic field triggers aggregation/deaggregation of magnetic particles that are also functionalized with urease or esterase enzymes. These enzymes produce a local pH change in the vicinity of the particles changing the AMG activity.
- 111Dhasaiyan, P.; Ghosh, T.; Lee, H. G.; Lee, Y.; Hwang, I.; Mukhopadhyay, R. D.; Park, K. M.; Shin, S.; Kang, I. S.; Kim, K. Cascade reaction networks within audible sound induced transient domains in a solution. Nat. Commun. 2022, 13 (1), 2372, DOI: 10.1038/s41467-022-30124-xGoogle Scholar111https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB38XhtFyisrbJ&md5=f9d49f81b13de09e13f615b0b5d3f163Cascade reaction networks within audible sound induced transient domains in a solutionDhasaiyan, Prabhu; Ghosh, Tanwistha; Lee, Hong-Guen; Lee, Yeonsang; Hwang, Ilha; Mukhopadhyay, Rahul Dev; Park, Kyeng Min; Shin, Seungwon; Kang, In Seok; Kim, KimoonNature Communications (2022), 13 (1), 2372CODEN: NCAOBW; ISSN:2041-1723. (Nature Portfolio)Spatiotemporal control of chem. cascade reactions within compartmentalized domains is one of the difficult challenges to achieve. To implement such control, scientists have been working on the development of various artificial compartmentalized systems such as liposomes, vesicles, polymersomes, etc. Although a considerable amt. of progress has been made in this direction, one still needs to develop alternative strategies for controlling cascade reaction networks within spatiotemporally controlled domains in a soln., which remains a non-trivial issue. Herein, we present the utilization of audible sound induced liq. vibrations for the generation of transient domains in an aq. medium, which can be used for the control of cascade chem. reactions in a spatiotemporal fashion. This approach gives us access to highly reproducible spatiotemporal chem. gradients and patterns, in situ growth and aggregation of gold nanoparticles at predetd. locations or domains formed in a soln. Our strategy also gives us access to nanoparticle patterned hydrogels and their applications for region specific cell growth.
- 112Küchler, A.; Yoshimoto, M.; Luginbühl, S.; Mavelli, F.; Walde, P. Enzymatic reactions in confined environments. Nat. Nanotechnol. 2016, 11 (5), 409– 420, DOI: 10.1038/nnano.2016.54Google Scholar112https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC28XnsVWruro%253D&md5=f476bcda9da7f4fccdb11cb2cd11ccd2Enzymatic reactions in confined environmentsKuchler, Andreas; Yoshimoto, Makoto; Luginbuhl, Sandra; Mavelli, Fabio; Walde, PeterNature Nanotechnology (2016), 11 (5), 409-420CODEN: NNAABX; ISSN:1748-3387. (Nature Publishing Group)A review. Within each biol. cell, surface- and vol.-confined enzymes control a highly complex network of chem. reactions. These reactions are efficient, timely, and spatially defined. Efforts to transfer such appealing features to in vitro systems have led to several successful examples of chem. reactions catalyzed by isolated and immobilized enzymes. In most cases, these enzymes are either bound or adsorbed to an insol. support, phys. trapped in a macromol. network, or encapsulated within compartments. Advanced applications of enzymic cascade reactions with immobilized enzymes include enzymic fuel cells and enzymic nanoreactors, both for in vitro and possible in vivo applications. Here, the authors discuss some of the general principles of enzymic reactions confined on surfaces, at interfaces, and inside small vols. The authors also highlight the similarities and differences between the in vivo and in vitro cases and attempt to critically evaluate some of the necessary future steps to improve the fundamental understanding of these systems.
- 113Hwang, E. T.; Lee, S. Multienzymatic Cascade Reactions Enzyme complex by immobilization. ACS Catal. 2019, 9 (5), 4402– 4425, DOI: 10.1021/acscatal.8b04921Google Scholar113https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC1MXmvVemsL4%253D&md5=57580b50c6cd302510726c71da408601Multienzymatic cascade reactions via enzyme complex by immobilizationHwang, Ee Taek; Lee, SeonbyulACS Catalysis (2019), 9 (5), 4402-4425CODEN: ACCACS; ISSN:2155-5435. (American Chemical Society)A review. Multienzymic cascade reactions are a most important technol. to succeed in industrial process development, such as synthesis of pharmaceutical, cosmetic, and nutritional compds. Different strategies to construct multienzyme structures have been widely reported. Enzymes complexes are designed by three types of routes: (i) fusion proteins, (ii) enzyme scaffolds, or (iii) immobilization. As a result, enzyme complexes can enhance cascade enzymic activity through substrate channeling. In particular, recent advances in materials science have led to syntheses of various materials applicable for enzyme immobilization. This review discusses different cases for assembling multienzyme complexes via random co-immobilization, compartmentalization, and positional co-immobilization. The advantages of using immobilized multienzymes include not only improved cascade enzymic activity via substrate channeling but also enhanced enzyme stability and ease of recovery for reuse. In this review, we also consider the latest studies of different model enzyme reactions immobilized on various support materials, as multienzyme systems allow for economical product synthesis through bioprocesses.
- 114Ji, Q.; Wang, B.; Tan, J.; Zhu, L.; Li, L. Immobilized multienzymatic systems for catalysis of cascade reactions. Process Biochemistry 2016, 51 (9), 1193– 1203, DOI: 10.1016/j.procbio.2016.06.004Google Scholar114https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC28XhtVOntLjE&md5=769953a90f9288de812d6159383ea053Immobilized multienzymatic systems for catalysis of cascade reactionsJi, Qingzhi; Wang, Bochu; Tan, Jun; Zhu, Liancai; Li, LiuyingProcess Biochemistry (Oxford, United Kingdom) (2016), 51 (9), 1193-1203CODEN: PBCHE5; ISSN:1359-5113. (Elsevier Ltd.)A review. Cascade reactions catalyzed by immobilized multienzymic systems are an emerging technol., making catalytic strategies more sophisticated and effective. Thus, immobilized multienzymic systems that exploit the chemo-, regio-, and stereoselectivity of biocatalysts have been developed. Recently, a variety of bioinspired immobilized multienzymic systems was fabricated. In this review, immobilized multienzymic systems were divided into three types: stepwise immobilized enzymes, mixed immobilized enzymes, or co-immobilized enzymes. General considerations such as coupling pH, coupling temp., proportion of each enzyme, and cascade bottleneck anal. for the enzymic cascade reaction were presented. For greater insight, we analyzed the effects of substrate channeling, mass transport limitation, synergistic mechanisms, and promotion of cofactor regeneration on immobilized multienzymic systems. Some recent novel examples of immobilized multienzymic systems were summarized. The aim of this review is to promote and broaden the application of immobilized multienzymic systems in biocatalysis and other related fields.
- 115Kazenwadel, F.; Franzreb, M.; Rapp, B. E. Synthetic enzyme supercomplexes: co-immobilization of enzyme cascades. Anal. Methods 2015, 7 (10), 4030– 4037, DOI: 10.1039/C5AY00453EGoogle Scholar115https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2MXmvFartbY%253D&md5=d1e8c020ccd7c3ef41f9b4a42a5916faSynthetic enzyme supercomplexes: co-immobilization of enzyme cascadesKazenwadel, F.; Franzreb, M.; Rapp, B. E.Analytical Methods (2015), 7 (10), 4030-4037CODEN: AMNEGX; ISSN:1759-9679. (Royal Society of Chemistry)A review. A sustainable alternative to traditional chem. synthesis is the use of enzymes as biocatalysts. Using enzymes, different advantages such as mild reaction conditions and high turnover rates are combined. However, the approach of using sol. enzymes suffers from the fact that enzymes have to be sepd. from the product post-synthesis and can be inactivated by this process. Therefore, enzymes are often immobilized to solid carriers to enable easy sepn. from the product as well as stabilization of the enzyme structure. In order to mimic the metabolic pathways of living cells and thus to create more complex bioproducts in a cell-free manner, a series of consecutive reactions can be realized by applying whole enzyme cascades. As enzymes from different host organisms can be combined, this offers enormous opportunities for creating advanced metabolic pathways that do not occur in nature. When immobilizing these enzyme cascades in a co-localized pattern a further advantage emerges: as the product of the previous enzyme is directly transferred to its co-immobilized subsequent catalyst, the overall performance of the cascade can be enhanced. Furthermore when enzymes are in close proximity to each other, the generation of byproducts is reduced and obstructive effects like product inhibition and unfavorable kinetics can be disabled. This review provides an overview of the current state of the art in the application of enzyme cascades in immobilized forms. Furthermore it focuses on different immobilization techniques for structured immobilizates and the use of enzyme cascade in specially designed (microfluidic) reactor devices.
- 116Xu, K.; Chen, X.; Zheng, R.; Zheng, Y. Immobilization of multi-enzymes on support materials for efficient biocatalysis. Front. Bioeng. Biotechnol. 2020, 8, 660, DOI: 10.3389/fbioe.2020.00660Google Scholar116https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A280%3ADC%252BB38jlsFGmsw%253D%253D&md5=9429722fa4f60a98dc174b0dc75690e9Immobilization of Multi-Enzymes on Support Materials for Efficient BiocatalysisXu Kongliang; Chen Xuexiao; Zheng Renchao; Zheng Yuguo; Xu Kongliang; Chen Xuexiao; Zheng Renchao; Zheng YuguoFrontiers in bioengineering and biotechnology (2020), 8 (), 660 ISSN:2296-4185.Multi-enzyme biocatalysis is an important technology to produce many valuable chemicals in the industry. Different strategies for the construction of multi-enzyme systems have been reported. In particular, immobilization of multi-enzymes on the support materials has been proved to be one of the most efficient approaches, which can increase the enzymatic activity via substrate channeling and improve the stability and reusability of enzymes. A general overview of the characteristics of support materials and their corresponding attachment techniques used for multi-enzyme immobilization will be provided here. This review will focus on the materials-based techniques for multi-enzyme immobilization, which aims to present the recent advances and future prospects in the area of multi-enzyme biocatalysis based on support immobilization.
- 117Bié, J.; Sepodes, B.; Fernandes, P. C. B.; Ribeiro, M. H. L. Enzyme immobilization and co-immobilization: main framework, advances and some applications. Processes 2022, 10 (3), 494, DOI: 10.3390/pr10030494Google Scholar117https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB38XhtVSltr%252FL&md5=3138ce7588d05413bd16d4ea4514ac36Enzyme Immobilization and Co-Immobilization: Main Framework, Advances and Some ApplicationsBie, Joaquim; Sepodes, Bruno; Fernandes, Pedro C. B.; Ribeiro, Maria H. L.Processes (2022), 10 (3), 494CODEN: PROCCO; ISSN:2227-9717. (MDPI AG)Enzymes are outstanding (bio)catalysts, not solely on account of their ability to increase reaction rates by up to several orders of magnitude but also for the high degree of substrate specificity, regiospecificity and stereospecificity. The use and development of enzymes as robust biocatalysts is one of the main challenges in biotechnol. However, despite the high specificities and turnover of enzymes, there are also drawbacks. At the industrial level, these drawbacks are typically overcome by resorting to immobilized enzymes to enhance stability. Immobilization of biocatalysts allows their reuse, increases stability, facilitates process control, eases product recovery, and enhances product yield and quality. This is esp. important for expensive enzymes, for those obtained in low fermn. yield and with relatively low activity. This review provides an integrated perspective on (multi)enzyme immobilization that abridges a crit. evaluation of immobilization methods and carriers, biocatalyst metrics, impact of key carrier features on biocatalyst performance, trends towards miniaturization and detailed illustrative examples that are representative of biocatalytic applications promoting sustainability.
- 118Benitez-Mateos, A. I.; Roura Padrosa, D.; Paradisi, F. Multistep enzyme cascades as a route towards green and sustainable pharmaceutical syntheses. Nat. Chem. 2022, 14 (5), 489– 499, DOI: 10.1038/s41557-022-00931-2Google Scholar118https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB38Xht1Wis7rE&md5=926da86ead0775bd6729686f9f1afc4bMultistep enzyme cascades as a route towards green and sustainable pharmaceutical synthesesBenitez-Mateos, Ana I.; Roura Padrosa, David; Paradisi, FrancescaNature Chemistry (2022), 14 (5), 489-499CODEN: NCAHBB; ISSN:1755-4330. (Nature Portfolio)A review. Enzyme cascades are a powerful technol. to develop environmentally friendly and cost-effective synthetic processes to manuf. drugs, as they couple different biotransformations in sequential reactions to synthesize the product. These biocatalytic tools can address two key parameters for the pharmaceutical industry: an improved selectivity of synthetic reactions and a redn. of potential hazards by using biocompatible catalysts, which can be produced from sustainable sources, which are biodegradable and, generally, non-toxic. Here we outline a broad variety of enzyme cascades used either in vivo (whole cells) or in vitro (purified enzymes) to specifically target pharmaceutically relevant mols., from simple building blocks to complex drugs. We also discuss the advantages and requirements of multistep enzyme cascades and their combination with chem. catalysts through a series of reported examples. Finally, we examine the efficiency of enzyme cascades and how they can be further improved by enzyme engineering, process intensification in flow reactors and/or enzyme immobilization to meet all the industrial requirements.
- 119Liang, J.; Liang, K. Multi-enzyme cascade reactions in metal-organic frameworks. Chem. Rec. 2020, 20 (10), 1100– 1116, DOI: 10.1002/tcr.202000067Google Scholar119https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB3cXhsVClt7fF&md5=c3527de6b56da9e62ac78172ce1c6057Multi-enzyme Cascade Reactions in Metal-Organic FrameworksLiang, Jieying; Liang, KangChemical Record (2020), 20 (10), 1100-1116CODEN: CRHEAK; ISSN:1528-0691. (Wiley-VCH Verlag GmbH & Co. KGaA)Multi-enzyme cascade reactions are indispensable in biotechnol. and many industrial (bio)chem. processes. However, most natural enzymes have poor stability and reusability, and tend to inactivate in toxic media or high temp., which significantly limit their broader applications. Metal-org. frameworks (MOFs) are promising candidates for enzymes immobilization to produce nanocomposite structures that not only could shield the enzymes from harsh environments, but also facilitate selective diffusion of substrates and intermediates to the reactive site via their tailorable and ordered pore network. Multi-enzyme cascade reactions in MOFs have recently attracted considerable attention. This Personal Account discusses the different strategies for multi-enzyme-MOF interfaces and their cutting-edge applications from biosensing and catalytic nanomedicine to artificial/hybrid cells. At last, we provide a crit. evaluation and future prospects to outline future research directions.
- 120Tsitkov, S.; Hess, H. Design principles for a compartmentalized enzyme cascade reaction. ACS Catal. 2019, 9, 2432– 2439, DOI: 10.1021/acscatal.8b04419Google Scholar120https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC1MXitlChtLs%253D&md5=9e6ee51aa63f5a4311db731647ce276aDesign principles for a compartmentalized enzyme cascade reactionTsitkov, Stanislav; Hess, HenryACS Catalysis (2019), 9 (3), 2432-2439CODEN: ACCACS; ISSN:2155-5435. (American Chemical Society)Compartmentalization of enzyme cascade reactions can both create a safe space for volatile reaction intermediates, where they are protected from cellular degrdn. mechanisms, and a quarantine for toxic intermediates, where they are prevented from impeding cellular function. The quant. understanding of biol. compartments and the design of bioengineered compartments and synthetic cells would be facilitated by a general and concise model. Most existing models for studying compartmentalized cascades focus on a specific biol. system and are highly detailed. In this study, we develop a simple model for a compartmentalized two-enzyme cascade reaction and analyze it in the well-mixed, steady-state regime. An immediate and intuitive result is that the fundamental parameter governing compartment cascade throughput is the resistance to diffusion of substrate and intermediate mols. across the compartment boundary. We then use this model to develop a design process for a compartmentalized cascade, where intermediate loss is minimized while maintaining a desired product outflux. Finally, the model also reveals that there is a crit. threshold at which compartmentalization provides benefits over the free-soln. reaction. Our model not only provides many insights into the design of compartmentalized cascade reactions, but also captures the essential physics of the problem, as it can replicate the results of more complex models.
- 121Oh, W.; Jeong, D.; Park, J.-W. An Artificial compartmentalized biocatalytic cascade system constructed with enzyme-caged reticulate nanoporous membranes. Adv. Mater. Interfaces 2023, 10, 2300185, DOI: 10.1002/admi.202300185Google Scholar121https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB3sXhtVClt7jI&md5=61941208ae34d0a151e41830a1332838An Artificial Compartmentalized Biocatalytic Cascade System Constructed With Enzyme-Caged Reticulate Nanoporous MembranesOh, Wangsuk; Jeong, Dawoon; Park, Ji-WoongAdvanced Materials Interfaces (2023), 10 (17), 2300185CODEN: AMIDD2; ISSN:2196-7350. (Wiley-VCH Verlag GmbH & Co. KGaA)Compartmentalization is a ubiquitous feature of life, with a membrane interface that regulates mol. transport and exhibits bioactivities. An artificial enzyme-integrated membrane cascade system can mimic such interactive properties across different length scales for in vitro application. Here, it is shown that a reticulated nanoporous framework membrane with nano-caged enzymes presents the first modular macroscale platform for the compartmentalized biocatalytic system interfaced with an external medium. Catalase (CAT)-integrated polyurea membrane scaffold is prepd. by a simple pressurization procedure for a model cyclic cascade of glucose oxidase/catalase (GOx/CAT). The bicontinuous nanoporous membrane readily constructs a compartmentalized system interfacing the glucose/GOx soln. and the external environment and mediates glucose oxidn. by a cascade reaction under anaerobic or aerobic conditions. Furthermore, the membrane compartment exhibits multiple bioactive capabilities and barrier properties, including hydrogen peroxide detoxification, in situ oxygen generation, and protease exclusion. This work suggests a new strategy toward a robust modular biocatalytic membranous interface to mediate biochem. reaction cascades occurring in a compartment interfaced to an external environment, promising for potential applications in biohybrid devices embedded with tissues and cells.
- 122Diamanti, E.; Andrés-Sanz, D.; Orrego, A. H.; Carregal-Romero, S.; López-Gallego, F. Surpassing substrate–enzyme competition by compartmentalization. ACS Catal. 2023, 13 (17), 11441– 11454, DOI: 10.1021/acscatal.3c01965Google Scholar122https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB3sXhs1ygtb%252FO&md5=ac45f276ec4ceb2c664ef5c9988e8327Surpassing Substrate-Enzyme Competition by CompartmentalizationDiamanti, Eleftheria; Andres-Sanz, Daniel; Orrego, Alejandro H.; Carregal-Romero, Susana; Lopez-Gallego, FernandoACS Catalysis (2023), 13 (17), 11441-11454CODEN: ACCACS; ISSN:2155-5435. (American Chemical Society)Enzyme compartmentalization is one of the main strategies exploited by nature to create phys. sepd. chem. environments that allow simultaneous enzyme reactions within the cell metabolic networks. However, designing nanostructured architectures that mimic cellular compartments remains a challenge when two competing enzymes must work simultaneously over the same substrate. Herein, we develop a method to fabricate soft hybrids that phys. sep. two oxidoreductases that compete for NADH with greatly different kinetics. The less competitive enzyme is encapsulated into polymeric capsules capable of recruiting NADH, which are then assembled on porous agarose microbeads where the most competitive enzyme is immobilized. As a result, this functional hybrid enables the simultaneous action of two competing enzymes in the same reaction media, which would otherwise be impossible in a non-compartmentalized system. We demonstrate that substrate recruitment is a powerful approach to building up enzymic reaction networks with complex dynamics. Moreover, single-particle anal. under operando conditions reveals the impact of enzyme spatial organization on the overall performance of these soft hybrids, underlining the importance of understanding the functional variability within compartmentalized systems. Finally, integrating this compartmentalized system into a model cell-free biosynthetic cascade, we transform vinyl acetate into (S)-β-hydroxybutyrate with a 2 times higher titer than the non-compartmentalized free system. The proposed strategy can be generalized to produce compartmentalized cell-free biosynthetic pathways and multienzyme cascades where enzyme competition is an issue.
- 123Aumiller, W. M., Jr.; Uchida, M.; Douglas, T. Protein cage assembly across multiple length scales. Chem. Soc. Rev. 2018, 47, 3433– 3469, DOI: 10.1039/C7CS00818JGoogle Scholar123https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC1cXjs1OjsL0%253D&md5=da2a83077dd878cab68e8334e78c974cProtein cage assembly across multiple length scalesAumiller, William M., Jr.; Uchida, Masaki; Douglas, TrevorChemical Society Reviews (2018), 47 (10), 3433-3469CODEN: CSRVBR; ISSN:0306-0012. (Royal Society of Chemistry)Within the materials science community, proteins with cage-like architectures are being developed as versatile nanoscale platforms for use in protein nanotechnol. Much effort has been focused on the functionalization of protein cages with biol. and non-biol. moieties to bring about new properties of not only individual protein cages, but collective bulk-scale assemblies of protein cages. In this review, we report on the current understanding of protein cage assembly, both of the cages themselves from individual subunits, and the assembly of the individual protein cages into higher order structures. We start by discussing the key properties of natural protein cages (for example: size, shape and structure) followed by a review of some of the mechanisms of protein cage assembly and the factors that influence it. We then explore the current approaches for functionalizing protein cages, on the interior or exterior surfaces of the capsids. Lastly, we explore the emerging area of higher order assemblies created from individual protein cages and their potential for new and exciting collective properties.
- 124Jaekel, A.; Stegemann, P.; Saccà, B. Manipulating enzymes properties with DNA nanostructures. Molecules 2019, 24 (20), 3694, DOI: 10.3390/molecules24203694Google Scholar124https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC1MXit1yisb3P&md5=d3a16abadf59421439624c5f4f6360c4Manipulating enzymes properties with DNA nanostructuresJaekel, Andreas; Stegemann, Pierre; Sacca, BarbaraMolecules (2019), 24 (20), 3694CODEN: MOLEFW; ISSN:1420-3049. (MDPI AG)A review. Nucleic acids and proteins are two major classes of biopolymers in living systems. Whereas nucleic acids are characterized by robust mol. recognition properties, essential for the reliable storage and transmission of the genetic information, the variability of structures displayed by proteins and their adaptability to the environment make them ideal functional materials. One of the major goals of DNA nanotechnol.-and indeed its initial motivation-is to bridge these two worlds in a rational fashion. Combining the predictable base-pairing rule of DNA with chem. conjugation strategies and modern protein engineering methods has enabled the realization of complex DNA-protein architectures with programmable structural features and intriguing functionalities. In this review, we will focus on a special class of biohybrid structures, characterized by one or many enzyme mols. linked to a DNA scaffold with nanometer-scale precision. After an initial survey of the most important methods for coupling DNA oligomers to proteins, we will report the strategies adopted until now for organizing these conjugates in a predictable spatial arrangement. The major focus of this review will be on the consequences of such manipulations on the binding and kinetic properties of single enzymes and enzyme complexes: an interesting aspect of artificial DNA-enzyme hybrids, often reported in the literature, however, not yet entirely understood and whose full comprehension may open the way to new opportunities in protein science.
- 125Jiao, Y.; Shang, Y.; Li, N.; Ding, B. DNA-based enzymatic systems and their applications. iScience 2022, 25, 104018, DOI: 10.1016/j.isci.2022.104018Google Scholar125https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB38XhsVOmsbbE&md5=4b88367fa3b27d7f14575f2345c4c014DNA-based enzymatic systems and their applicationsJiao, Yunfei; Shang, Yingxu; Li, Na; Ding, BaoquaniScience (2022), 25 (4), 104018CODEN: ISCICE; ISSN:2589-0042. (Elsevier B.V.)A review. DNA strands with unique secondary structures can catalyze various chem. reactions and mimic natural enzymes with the assistance of cofactors, which have attracted much research attention. At the same time, the emerging DNA nanotechnol. provides an efficient platform to organize functional components of the enzymic systems and regulate their catalytic performances. In this review, we summarize the recent progress of DNA-based enzymic systems. First, DNAzymes (Dzs) are introduced, and their versatile utilities are summarized. Then, G-quadruplex/hemin (G4/hemin) Dzs with unique oxidase/peroxidase-mimicking activities and representative examples where these Dzs served as biosensors are explicitly elaborated. Next, the DNA-based enzymic cascade systems fabricated by the structural DNA nanotechnol. are depicted. In addn., the applications of catalytic DNA nanostructures in biosensing and biomedicine are included. At last, the challenges and the perspectives of the DNA-based enzymic systems for practical applications are also discussed.
- 126Engelen, W.; Janssen, B. M. G.; Merkx, M. DNA-based control of protein activity. Chem. Commun. 2016, 52, 3598– 3610, DOI: 10.1039/C5CC09853JGoogle Scholar126https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC28XhtF2isrg%253D&md5=2205b7f35ee3a448b4a7b09acfa3abd7DNA-based control of protein activityEngelen, W.; Janssen, B. M. G.; Merkx, M.Chemical Communications (Cambridge, United Kingdom) (2016), 52 (18), 3598-3610CODEN: CHCOFS; ISSN:1359-7345. (Royal Society of Chemistry)A review. DNA has emerged as a highly versatile construction material for nanometer-sized structures and sophisticated mol. machines and circuits. The successful application of nucleic acid based systems greatly relies on their ability to autonomously sense and act on their environment. In this feature article, the development of DNA-based strategies to dynamically control protein activity via oligonucleotide triggers is discussed. Depending on the desired application, protein activity can be controlled by directly conjugating them to an oligonucleotide handle, or expressing them as a fusion protein with DNA binding motifs. To control proteins without modifying them chem. or genetically, multivalent ligands and aptamers that reversibly inhibit their function provide valuable tools to regulate proteins in a noncovalent manner. The goal of this feature article is to give an overview of strategies developed to control protein activity via oligonucleotide-based triggers, as well as hurdles yet to be taken to obtain fully autonomous systems that interrogate, process and act on their environments by DNA-based protein control.
- 127Fu, J.; Wang, Z.; Liang, X. H.; Oh, S. W.; St. Iago-McRae, E.; Zhang, T. DNA-scaffolded proximity assembly and confinement of multienzyme reactions. Top. Curr. Chem. 2020, 378, 38, DOI: 10.1007/s41061-020-0299-3Google Scholar127https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB3cXmsVert7k%253D&md5=2ee0e83645bdd98cffc3f83314bb4e00DNA-Scaffolded Proximity Assembly and Confinement of Multienzyme ReactionsFu, Jinglin; Wang, Zhicheng; Liang, Xiao Hua; Oh, Sung Won; St. Iago-McRae, Ezry; Zhang, TingTopics in Current Chemistry (2020), 378 (3), 38CODEN: TPCCAQ; ISSN:2364-8961. (Springer International Publishing AG)A review. Cellular functions rely on a series of organized and regulated multienzyme cascade reactions. The catalytic efficiencies of these cascades depend on the precise spatial organization of the constituent enzymes, which is optimized to facilitate substrate transport and regulate activities. Mimicry of this organization in a non-living, artificial system would be very useful in a broad range of applications-with impacts on both the scientific community and society at large. Self-assembled DNA nanostructures are promising applications to organize biomol. components into prescribed, multidimensional patterns. In this review, we focus on recent progress in the field of DNA-scaffolded assembly and confinement of multienzyme reactions. DNA self-assembly is exploited to build spatially organized multienzyme cascades with control over their relative distance, substrate diffusion paths, compartmentalization and activity actuation. The combination of addressable DNA assembly and multienzyme cascades can deliver breakthroughs toward the engineering of novel synthetic and biomimetic reactors.
- 128Kröll, S.; Niemeyer, C. M. Nucleic acid-based enzyme cascades─current trends and future perspectives. Angew. Chem., Int. Ed. 2024, 63, e202314452, DOI: 10.1002/anie.202314452Google ScholarThere is no corresponding record for this reference.
- 129Liang, J.; Liang, K. Multi-enzyme cascade reactions in metal-organic frameworks. Chem. Rec. 2020, 20, 1100, DOI: 10.1002/tcr.202000067Google Scholar129https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB3cXhsVClt7fF&md5=c3527de6b56da9e62ac78172ce1c6057Multi-enzyme Cascade Reactions in Metal-Organic FrameworksLiang, Jieying; Liang, KangChemical Record (2020), 20 (10), 1100-1116CODEN: CRHEAK; ISSN:1528-0691. (Wiley-VCH Verlag GmbH & Co. KGaA)Multi-enzyme cascade reactions are indispensable in biotechnol. and many industrial (bio)chem. processes. However, most natural enzymes have poor stability and reusability, and tend to inactivate in toxic media or high temp., which significantly limit their broader applications. Metal-org. frameworks (MOFs) are promising candidates for enzymes immobilization to produce nanocomposite structures that not only could shield the enzymes from harsh environments, but also facilitate selective diffusion of substrates and intermediates to the reactive site via their tailorable and ordered pore network. Multi-enzyme cascade reactions in MOFs have recently attracted considerable attention. This Personal Account discusses the different strategies for multi-enzyme-MOF interfaces and their cutting-edge applications from biosensing and catalytic nanomedicine to artificial/hybrid cells. At last, we provide a crit. evaluation and future prospects to outline future research directions.
- 130Dhakshinamoorthy, A.; Asiri, A. M.; Garcia, H. Integration of metal organic frameworks with enzymes as multifunctional solids for cascade catalysis. Dalton Trans. 2020, 49, 11059– 11072, DOI: 10.1039/D0DT02045AGoogle Scholar130https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB3cXht1ClsrbN&md5=63dc33498161488f91cb98a222f2781fIntegration of metal organic frameworks with enzymes as multifunctional solids for cascade catalysisDhakshinamoorthy, Amarajothi; Asiri, Abdullah M.; Garcia, HermenegildoDalton Transactions (2020), 49 (32), 11059-11072CODEN: DTARAF; ISSN:1477-9226. (Royal Society of Chemistry)A review. Enzymes exhibit a large degree of compatibility with metal-org. frameworks (MOFs) which allows the development of multicomponent catalysts consisting of enzymes adsorbed or occluded by MOFs. The combination of enzymes and MOFs in a multicomponent catalyst can be used to promote cascade reactions in which two or more individual reactions are performed in a single step. Cascade reactions take place due to the cooperation of active sites present on the MOF with the enzyme. A survey of the available data establishes that often an enzyme undergoes stabilization by assocn. with a MOF and the system exhibits notable recyclability. In addn., the existence of synergism is obsd. as a consequence of the close proximity of all the required active sites in the multicomponent catalyst. After an introductory section describing the specific features and properties of enzyme-MOF assemblies, the main part of the present review focuses on the description of the cascade reactions that have been reported with com. enzymes assocd. with MOFs, paying special attention to the advantages derived from the multicomponent catalyst. Related to the catalytic activity to metabolize glucose, generating reactive oxygen species (ROS) and decreasing the soln. pH, an independent section describes the recent use of enzyme-MOF catalysts in cancer therapy. The last paragraphs summarize the current state of the art and provide our view on future developments in this field.
- 131Wang, X.; Lan, P. C.; Ma, S. Metal–organic frameworks for enzyme immobilization: beyond host matrix materials. ACS Cent. Sci. 2020, 6 (9), 1497– 1506, DOI: 10.1021/acscentsci.0c00687Google Scholar131https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB3cXhs1OrsLrI&md5=8b6eba4e66b83d884976908803e31e12Metal-Organic Frameworks for Enzyme Immobilization: Beyond Host Matrix MaterialsWang, Xiaoliang; Lan, Pui Ching; Ma, ShengqianACS Central Science (2020), 6 (9), 1497-1506CODEN: ACSCII; ISSN:2374-7951. (American Chemical Society)A review. Enzyme immobilization in metal-org. frameworks (MOFs) as a promising strategy is attracting the interest of scientists from different disciplines with the expansion of MOFs' development. Different from other traditional host materials, their unique strengths of high surface areas, large yet adjustable pore sizes, functionalizable pore walls, and diverse architectures make MOFs an ideal platform to study hosted enzymes, which is crit. to the industrial and com. process. In addn. to the protective function of MOFs, the extensive roles of MOFs in the enzyme immobilization are being well-explored by making full use of their remarkable properties like well-defined structure, high porosity, and tunable functionality. Such development shifts the focus from the exploration of immobilization strategies toward functionalization. Meanwhile, this would undoubtedly contribute to a better understanding of enzymes in regards to the structural transformation after being hosted in a confinement environment, particularly to the orientation and conformation change as well as the interplay between enzyme and matrix MOFs. In this Outlook, the authors target a comprehensive review of the role diversities of the host matrix MOF based on the current enzyme immobilization research, along with proposing an outlook toward the future development of this field, including the representatives of potential techniques and methodologies being capable of studying the hosted enzymes. Current research of role/function diversities of host matrix MOFs is reviewed, noting the importance of understanding enzyme structural alternation and enzyme-MOF interaction after immobilization.
- 132Lian, X.; Fang, Y.; Joseph, E.; Wang, Q.; Li, J.; Banerjee, S.; Lollar, C.; Wang, X.; Zhou, H.-C. Enzyme–MOF (metal–organic framework) composites. Chem. Soc. Rev. 2017, 46, 3386– 3401, DOI: 10.1039/C7CS00058HGoogle Scholar132https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2sXmslOku7s%253D&md5=329ea8161e9200a133c76ba386fc70a7Enzyme-MOF (metal-organic framework) compositesLian, Xizhen; Fang, Yu; Joseph, Elizabeth; Wang, Qi; Li, Jialuo; Banerjee, Sayan; Lollar, Christina; Wang, Xuan; Zhou, Hong-CaiChemical Society Reviews (2017), 46 (11), 3386-3401CODEN: CSRVBR; ISSN:0306-0012. (Royal Society of Chemistry)A review. The ex vivo application of enzymes in various processes, esp. via enzyme immobilization techniques, has been extensively studied in recent years in order to enhance the recyclability of enzymes, to minimize enzyme contamination in the product, and to explore novel horizons for enzymes in biomedical applications. Possessing remarkable amenability in structural design of the frameworks as well as almost unparalelled surface tunability, Metal-Org. Frameworks (MOFs) have been gaining popularity as candidates for enzyme immobilization platforms. Many MOF-enzyme composites have achieved unprecedented results, far outperforming free enzymes in many aspects. This review summarizes recent developments of MOF-enzyme composites with special emphasis on preparative techniques and the synergistic effects of enzymes and MOFs. The applications of MOF-enzyme composites, primarily in transferation, catalysis and sensing, are presented as well. The enhancement of enzymic activity of the composites over free enzymes in biol. incompatible conditions is emphasized in many cases.
- 133Liang, W.; Wied, P.; Carraro, F.; Sumby, C. J.; Nidetzky, B.; Tsung, C.-K.; Falcaro, P.; Doonan, C. J. Metal–organic framework-based enzyme biocomposites. Chem. Rev. 2021, 121 (3), 1077– 1129, DOI: 10.1021/acs.chemrev.0c01029Google Scholar133https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB3MXpvFylsQ%253D%253D&md5=22a04b209c88b1c69ee3e73f8aeba6c3Metal-Organic Framework-Based Enzyme BiocompositesLiang, Weibin; Wied, Peter; Carraro, Francesco; Sumby, Christopher J.; Nidetzky, Bernd; Tsung, Chia-Kuang; Falcaro, Paolo; Doonan, Christian J.Chemical Reviews (Washington, DC, United States) (2021), 121 (3), 1077-1129CODEN: CHREAY; ISSN:0009-2665. (American Chemical Society)A review. Because of their efficiency, selectivity, and environmental sustainability, there are significant opportunities for enzymes in chem. synthesis and biotechnol. However, as the three-dimensional active structure of enzymes is predominantly maintained by weaker noncovalent interactions, thermal, pH, and chem. stressors can modify or eliminate activity. Metal-org. frameworks (MOFs), which are extended porous network materials assembled by a bottom-up building block approach from metal-based nodes and org. linkers, can be used to afford protection to enzymes. The self-assembled structures of MOFs can be used to encase an enzyme in a process called encapsulation when the MOF is synthesized in the presence of the biomol. Alternatively, enzymes can be infiltrated into mesoporous MOF structures or surface bound via covalent or noncovalent processes. Integration of MOF materials and enzymes in this way affords protection and allows the enzyme to maintain activity in challenge conditions (e.g., denaturing agents, elevated temp., non-native pH, and org. solvents). In addn. to forming simple enzyme/MOF biocomposites, other materials can be introduced to the composites to improve recovery or facilitate advanced applications in sensing and fuel cell technol. This review canvasses enzyme protection via encapsulation, pore infiltration, and surface adsorption and summarizes strategies to form multicomponent composites. Also, given that enzyme/MOF biocomposites straddle materials chem. and enzymol., this review provides an assessment of the characterization methodologies used for MOF-immobilized enzymes and identifies some key parameters to facilitate development of the field.
- 134Hu, J.; Zhang, G.; Liu, S. Enzyme-responsive polymeric assemblies, nanoparticles and hydrogels. Chem. Soc. Rev. 2012, 41 (18), 5933– 5949, DOI: 10.1039/c2cs35103jGoogle Scholar134https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC38Xht1aqsbrF&md5=d18156de75b1053ac9e63aadfa9661f5Enzyme-responsive polymeric assemblies, nanoparticles and hydrogelsHu, Jinming; Zhang, Guoqing; Liu, ShiyongChemical Society Reviews (2012), 41 (18), 5933-5949CODEN: CSRVBR; ISSN:0306-0012. (Royal Society of Chemistry)A review. Being responsive and adaptive to external stimuli is an intrinsic feature characteristic of all living organisms and soft matter. Specifically, responsive polymers can exhibit reversible or irreversible changes in chem. structures and/or phys. properties in response to a specific signal input such as pH, temp., ionic strength, light irradn., mech. force, elec. and magnetic fields, and analyte of interest (e.g., ions, bioactive mols., etc.) or an integration of them. The past decade has evidenced tremendous growth in the fundamental research of responsive polymers, and accordingly, diverse applications in fields ranging from drug or gene nanocarriers, imaging, diagnostics, smart actuators, adaptive coatings, to self-healing materials were explored and suggested. Among a variety of external stimuli that were utilized for the design of novel responsive polymers, enzymes have recently emerged to be a promising triggering motif. Enzyme-catalyzed reactions are highly selective and efficient toward specific substrates under mild conditions. They are involved in all biol. and metabolic processes, serving as the prime protagonists in the chem. of living organisms at a mol. level. The integration of enzyme-catalyzed reactions with responsive polymers can further broaden the design flexibility and scope of applications by endowing the latter with enhanced triggering specificity and selectivity. In this tutorial review, the authors describe recent developments concerning enzyme-responsive polymeric assemblies, nanoparticles, and hydrogels by highlighting this research area with selected literature reports. Three different types of systems, namely, enzyme-triggered self-assembly and aggregation of synthetic polymers, enzyme-driven disintegration and structural reorganization of polymeric assemblies and nanoparticles, and enzyme-triggered sol-to-gel and gel-to-sol transitions, are described. Their promising applications in drug controlled release, biocatalysis, imaging, sensing, and diagnostics are also discussed.
- 135Li, P.; Zhong, Y.; Wang, X.; Hao, J. Enzyme-regulated healable polymeric hydrogels. ACS Cent. Sci. 2020, 6 (9), 1507– 1522, DOI: 10.1021/acscentsci.0c00768Google Scholar135https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB3cXhsFWnt73F&md5=1fcff98ac8ba657eece9f626d7f4ebc7Enzyme-Regulated Healable Polymeric HydrogelsLi, Panpan; Zhong, Yuanbo; Wang, Xu; Hao, JingchengACS Central Science (2020), 6 (9), 1507-1522CODEN: ACSCII; ISSN:2374-7951. (American Chemical Society)A review. The enzyme-regulated healable polymeric hydrogels are a kind of emerging soft material capable of repairing the structural defects and recovering the hydrogel properties, wherein their fabrication, self-healing, or degrdn. is mediated by enzymic reactions. Despite achievements that have been made in controllable crosslinking and de-crosslinking of hydrogels by utilizing enzyme-catalyzed reactions in the past few years, this substrate-specific strategy for regulating healable polymeric hydrogels remains in its infancy, because both the intelligence and practicality of current man-made enzyme-regulated healable materials are far below the levels of living organisms. A systematic summary of current achievements and a reasonable prospect at this point can play pos. roles for the future development in this field. This Outlook focuses on the emerging and rapidly developing research area of bioinspired enzyme-regulated self-healing polymeric hydrogel systems. The enzymic fabrication and degrdn. of healable polymeric hydrogels, as well as the enzymically regulated self-healing of polymeric hydrogels, are reviewed. The functions and applications of the enzyme-regulated healable polymeric hydrogels are discussed. A systematic summary of current achievements in the area of enzyme-regulated healable polymeric hydrogels is given by detailing their fabrication, self-healing, degrdn., and applications.
- 136Amir, R. J.; Zhong, S.; Pochan, D. J.; Hawker, C. J. Enzymatically triggered self-assembly of block copolymers. J. Am. Chem. Soc. 2009, 131 (39), 13949– 13951, DOI: 10.1021/ja9060917Google Scholar136https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD1MXhtFWqtLnO&md5=4bd33ad61070f9c98a4359ea77b02664Enzymatically Triggered Self-Assembly of Block CopolymersAmir, Roey J.; Zhong, Sheng; Pochan, Darrin J.; Hawker, Craig J.Journal of the American Chemical Society (2009), 131 (39), 13949-13951CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)The polymn. of vinyl monomers with cleavable enzymic substrates has been shown to lead to water-sol. double-hydrophilic block copolymers which, upon enzymic activation of the diblock copolymers, become amphiphilic and undergo self-assembly into colloidal nanostructures. The ability to change the chem. and phys. characteristics of polymeric materials by an enzymic reaction opens the way for novel and exciting applications such as enzymic-triggered activation of surfaces and formation of nanostructures in vivo in a highly controlled manner.
- 137Kobayashi, S.; Uyama, H.; Kimura, S. Enzymatic polymerization. Chem. Rev. 2001, 101 (12), 3793– 3818, DOI: 10.1021/cr990121lGoogle Scholar137https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD3MXotVOht7Y%253D&md5=6bdfb08b5a7bea461570df0bae3547f4Enzymatic polymerizationKobayashi, Shiro; Uyama, Hiroshi; Kimura, ShunsakuChemical Reviews (Washington, D. C.) (2001), 101 (12), 3793-3818CODEN: CHREAY; ISSN:0009-2665. (American Chemical Society)A review with refs. on recent advances in enzymic polymn. Enzymes discussed included peroxidases, laccases, glycosyltransferases, acyltransferases, glycosidases, lipases, and proteases.
- 138Klemperer, R. G.; Shannon, M. R.; Ross Anderson, J. L.; Perriman, A. W. Bienzymatic generation of interpenetrating polymer networked engineered living materials with shape changing Pproperties. Adv. Mater. Technol. 2023, 8, 2300626, DOI: 10.1002/admt.202300626Google Scholar138https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB3sXhsVKltLfL&md5=3fb13931d0d744a3167696ab4af0b185Bienzymatic Generation of Interpenetrating Polymer Networked Engineered Living Materials with Shape Changing PropertiesKlemperer, R. George; Shannon, Mark R.; Ross Anderson, J. L.; Perriman, Adam W.Advanced Materials Technologies (Weinheim, Germany) (2023), 8 (18), 2300626CODEN: AMTDCM; ISSN:2365-709X. (Wiley-VCH Verlag GmbH & Co. KGaA)The synthesis of a porous shape-changing interpenetrating network (IPN) bioink for the fabrication of large-scale (cm) reversibly thermosensitive structures is described. The poly(N-isopropylacrylamide) (PNIPAm) IPN is generated in situ within an ionically crosslinked alginate hydrogel at room temp. and under aerobic conditions using a horseradish peroxidase (HRP)/glucose oxidase (GOx) bienzymic initiation system. Mech. testing assessment of the IPN hydrogels confirm mech. reinforcement via covalent single network interdigitation. Furthermore, the thermosensitive bioink can be used to print biohybrid reactors contg. genetically engineered phosphotriesterase-expressing E. coli capable of hydrolyzing toxic organophosphorus compds. Herein, increasing the bioink pore size using the contractile-thermosensitive response of the IPN improves the temp.-dependent theor. mass-transfer-limited enzyme catalyzed reaction rate, providing a plausible route to externally regulated enzymic catalysis within bioprinted structures.
- 139Mao, Y.; Su, T.; Wu, Q.; Liao, C.; Wang, Q. Dual enzymatic formation of hybrid hydrogels with supramolecular-polymeric networks. Chem. Commun. 2014, 50 (92), 14429– 14432, DOI: 10.1039/C4CC06472KGoogle Scholar139https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2cXhsFyrurzO&md5=65fed273b072c904d9ac9f48cb433722Dual enzymatic formation of hybrid hydrogels with supramolecular-polymeric networksMao, Yanjie; Su, Teng; Wu, Qing; Liao, Chuanan; Wang, QigangChemical Communications (Cambridge, United Kingdom) (2014), 50 (92), 14429-14432CODEN: CHCOFS; ISSN:1359-7345. (Royal Society of Chemistry)This communication describes a mild construction of hybrid hydrogels with supramol.-polymeric networks via a dual enzymic reaction.
- 140Wei, Q.; Xu, M.; Liao, C.; Wu, Q.; Liu, M.; Zhang, Y.; Wu, C.; Cheng, L.; Wang, Q. Printable hybrid hydrogel by dual enzymatic polymerization with superactivity. Chem. Sci. 2016, 7 (4), 2748– 2752, DOI: 10.1039/C5SC02234GGoogle Scholar140https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC28Xhtl2lsw%253D%253D&md5=d9cbcf00eddb407ca0a7943980ef46aaPrintable hybrid hydrogel by dual enzymatic polymerization with superactivityWei, Qingcong; Xu, Mengchi; Liao, Chuanan; Wu, Qing; Liu, Mingyu; Zhang, Ye; Wu, Chengtie; Cheng, Liming; Wang, QigangChemical Science (2016), 7 (4), 2748-2752CODEN: CSHCCN; ISSN:2041-6520. (Royal Society of Chemistry)A new approach has been developed to fabricate tough hybrid hydrogels by employing dual enzyme-mediated redox initiation to achieve post-self-assembly crosslinking polymn. The resulting hydrogel combines the merits of supramol. hydrogels with polymeric hydrogels to achieve higher mech. strength and porous networks. Designed 3D constructs were fabricated via in situ 3D printing. The in situ immobilized GOx/HRP in Gel II exhibited superactivity compared to free enzymes, which might be attributed to the synergistic effect of co-localized GOx and HRP minimizing the distances for mass transport between the gel and the bulk soln. This mech. strong hybrid hydrogel maintained high reusability and thermal stability as well. In addn., in situ 3D cell culture was demonstrated, thus indicating that this biodegradable hybrid hydrogel is biocompatible with cells. The subsequent 3D cell printing further indicates that the hybrid hydrogel is a promising scaffold for bio-related applications such as biocatalysis and tissue engineering.
- 141Yang, Z.; Liang, G.; Wang, L.; Xu, B. Using a kinase/phosphatase switch to regulate a supramolecular hydrogel and forming the supramolecular hydrogel in vivo. J. Am. Chem. Soc. 2006, 128 (9), 3038– 3043, DOI: 10.1021/ja057412yGoogle Scholar141https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD28Xht1Cjs70%253D&md5=326363d3b9d418c808c8749b2e6592c0Using a Kinase/Phosphatase Switch to Regulate a Supramolecular Hydrogel and Forming the Supramolecular Hydrogel in VivoYang, Zhimou; Liang, Gaolin; Wang, Ling; Xu, BingJournal of the American Chemical Society (2006), 128 (9), 3038-3043CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)We have designed and synthesized a new hydrogelator Nap-FFGEY (1), which forms a supramol. hydrogel. A kinase/phosphatase switch is used to control the phosphorylation and dephosphorylation of the hydrogelator and to regulate the formation of supramol. hydrogels. Adding a kinase to the hydrogel induces a gel-sol phase transition in the presence of adenosine triphosphates (ATP) because the tyrosine residue is converted into tyrosine phosphate by the kinase to give a more hydrophilic mol. of Nap-FFGEY-P(O)(OH)2 (2); treating the resulting soln. with a phosphatase transforms 2 back to 1 and restores the hydrogel. Electron micrographs of the hydrogels indicate that 1 self-assembles into nanofibers. S.c. injection of 2 in mice shows that 80.5±1.2% of 2 turns into 1 and results in the formation of the supramol. hydrogel of 1 in vivo. This simple biomimetic approach for regulating the states of supramol. hydrogels promises a new way to design and construct biomaterials.
- 142Knipe, J. M.; Chen, F.; Peppas, N. A. Enzymatic biodegradation of hydrogels for protein delivery targeted to the small intestine. Biomacromolecules 2015, 16 (3), 962– 972, DOI: 10.1021/bm501871aGoogle Scholar142https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2MXisF2gurk%253D&md5=377318e0f9e887be253719e9b3bf98bfEnzymatic Biodegradation of Hydrogels for Protein Delivery Targeted to the Small IntestineKnipe, Jennifer M.; Chen, Frances; Peppas, Nicholas A.Biomacromolecules (2015), 16 (3), 962-972CODEN: BOMAF6; ISSN:1525-7797. (American Chemical Society)Multiresponsive poly(methacrylic acid-co-N-vinylpyrrolidone) hydrogels were synthesized with biodegradable oligopeptide crosslinks. The oligopeptide crosslinks were incorporated using EDC-NHS zero-length links between the carboxylic acid groups of the polymer and free primary amines on the peptide. The reaction of the peptide was confirmed by primary amine assay and IR spectroscopy. The microgels exhibited pH-responsive swelling as well as enzyme-catalyzed degrdn. targeted by trypsin present in the small intestine, as demonstrated upon incubation with gastrointestinal fluids from rats. Relative turbidity was used to evaluate enzyme-catalyzed degrdn. as a function of time, and initial trypsin concn. controlled both the degrdn. mechanism as well as the extent of degrdn. Trypsin activity was effectively extinguished by incubation at 70 °C, and both the microgels and degrdn. products posed no cytotoxic effect toward two different cell lines. The microgels demonstrated pH-dependent loading of the protein insulin for oral delivery to the small intestine.
- 143Pappas, C. G.; Sasselli, I. R.; Ulijn, R. V. Biocatalytic pathway selection in transient tripeptide nanostructures. Angew. Chem., Int. Ed. 2015, 54 (28), 8119– 8123, DOI: 10.1002/anie.201500867Google Scholar143https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2MXptFert7s%253D&md5=d57863a160d87e7b9d913c2c0d9ebda0Biocatalytic Pathway Selection in Transient Tripeptide NanostructuresPappas, Charalampos G.; Sasselli, Ivan R.; Ulijn, Rein V.Angewandte Chemie, International Edition (2015), 54 (28), 8119-8123CODEN: ACIEF5; ISSN:1433-7851. (Wiley-VCH Verlag GmbH & Co. KGaA)Structural adaptation in living systems is achieved by competing catalytic pathways that drive assembly and disassembly of mol. components under the influence of chem. fuels. We report on a simple mimic of such a system that displays transient, sequence-dependent formation of supramol. nanostructures based on biocatalytic formation and hydrolysis of self-assembling tripeptides. The systems are catalyzed by α-chymotrypsin and driven by hydrolysis of dipeptide aspartyl-phenylalanine-Me ester (the sweetener aspartame, DF-OMe). We obsd. switch-like pathway selection, with the kinetics and consequent lifetime of transient nanostructures controlled by the peptide sequence. In direct competition, kinetic (rather than thermodn.) component selection is obsd.
- 144Cook, A. B.; Decuzzi, P. Harnessing endogenous stimuli for responsive materials in theranostics. ACS Nano 2021, 15 (2), 2068– 2098, DOI: 10.1021/acsnano.0c09115Google Scholar144https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB3MXjt1Kht70%253D&md5=3502531fce5be079450b300c9a292506Harnessing Endogenous Stimuli for Responsive Materials in TheranosticsCook, Alexander B.; Decuzzi, PaoloACS Nano (2021), 15 (2), 2068-2098CODEN: ANCAC3; ISSN:1936-0851. (American Chemical Society)A review. Materials that respond to endogenous stimuli are being leveraged to enhance spatiotemporal control in a range of biomedical applications from drug delivery to diagnostic tools. The design of materials that undergo morphol. or chem. changes in response to specific biol. cues or pathologies will be an important area of research for improving efficacies of existing therapies and imaging agents, while also being promising for developing personalized theranostic systems. Internal stimuli-responsive systems can be engineered across length scales from nanometers to macroscopic and can respond to endogenous signals such as enzymes, pH, glucose, ATP, hypoxia, redox signals, and nucleic acids by incorporating synthetic bio-inspired moieties or natural building blocks. This Review will summarize response mechanisms and fabrication strategies used in internal stimuli-responsive materials with a focus on drug delivery and imaging for a broad range of pathologies, including cancer, diabetes, vascular disorders, inflammation, and microbial infections. We will also discuss obsd. challenges, future research directions, and clin. translation aspects of these responsive materials.
- 145Ku, T. H.; Chien, M. P.; Thompson, M. P.; Sinkovits, R. S.; Olson, N. H.; Baker, T. S.; Gianneschi, N. C. Controlling and switching the morphology of micellar nanoparticles with enzymes. J. Am. Chem. Soc. 2011, 133 (22), 8392– 8395, DOI: 10.1021/ja2004736Google Scholar145https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3MXkt1ylu7c%253D&md5=d326644c6353068e202c39e927798e8fControlling and Switching the Morphology of Micellar Nanoparticles with EnzymesKu, Ti-Hsuan; Chien, Miao-Ping; Thompson, Matthew P.; Sinkovits, Robert S.; Olson, Norman H.; Baker, Timothy S.; Gianneschi, Nathan C.Journal of the American Chemical Society (2011), 133 (22), 8392-8395CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)Micelles were prepd. from polymer-peptide block copolymer amphiphiles contg. substrates for protein kinase A, protein phosphatase-1, and matrix metalloproteinases 2 and 9. The authors examine reversible switching of the morphol. of these micelles through a phosphorylation-dephosphorylation cycle and study peptide-sequence directed changes in morphol. in response to proteolysis. Furthermore, the exceptional uniformity of these polymer-peptide particles makes them amenable to cryo-TEM reconstruction techniques lending insight into their internal structure.
- 146Postma, S. G.; Vialshin, I. N.; Gerritsen, C. Y.; Bao, M.; Huck, W. T. Preprogramming complex hydrogel responses using enzymatic reaction networks. Angew. Chem., Int. Ed. 2017, 56 (7), 1794– 1798, DOI: 10.1002/anie.201610875Google Scholar146https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2sXlsFCjuw%253D%253D&md5=cf0189e888c27ea1641e5dade88eea86Preprogramming Complex Hydrogel Responses using Enzymatic Reaction NetworksPostma, Sjoerd G. J.; Vialshin, Ilia N.; Gerritsen, Casper Y.; Bao, Min; Huck, Wilhelm T. S.Angewandte Chemie, International Edition (2017), 56 (7), 1794-1798CODEN: ACIEF5; ISSN:1433-7851. (Wiley-VCH Verlag GmbH & Co. KGaA)The creation of adaptive matter is heavily inspired by biol. systems. However, it remains challenging to design complex material responses that are governed by reaction networks, which lie at the heart of cellular complexity. The main reason for this slow progress is the lack of a general strategy to integrate reaction networks with materials. Herein we use a systematic approach to preprogram the response of a hydrogel to a trigger, in this case the enzyme trypsin, which activates a reaction network embedded within the hydrogel. A full characterization of all the kinetic rate consts. in the system enabled the construction of a computational model, which predicted different hydrogel responses depending on the input concn. of the trigger. The results of the simulation are in good agreement with exptl. findings. Our methodol. can be used to design new, adaptive materials of which the properties are governed by reaction networks of arbitrary complexity.
- 147Ikeda, M.; Tanida, T.; Yoshii, T.; Kurotani, K.; Onogi, S.; Urayama, K.; Hamachi, I. Installing logic-gate responses to a variety of biological substances in supramolecular hydrogel-enzyme hybrids. Nat. Chem. 2014, 6 (6), 511– 518, DOI: 10.1038/nchem.1937Google Scholar147https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2cXntlyis7c%253D&md5=4b6d233a620c107f2a4ca3484442e945Installing logic-gate responses to a variety of biological substances in supramolecular hydrogel-enzyme hybridsIkeda, Masato; Tanida, Tatsuya; Yoshii, Tatsuyuki; Kurotani, Kazuya; Onogi, Shoji; Urayama, Kenji; Hamachi, ItaruNature Chemistry (2014), 6 (6), 511-518CODEN: NCAHBB; ISSN:1755-4330. (Nature Publishing Group)Soft materials that exhibit stimuli-responsive behavior under aq. conditions (such as supramol. hydrogels composed of self-assembled nanofibres) have many potential biol. applications. However, designing a macroscopic response to structurally complex biochem. stimuli in these materials still remains a challenge. Here we show that redox-responsive peptide-based hydrogels have the ability to encapsulate enzymes and still retain their activities. Moreover, cooperative coupling of enzymic reactions with the gel response enables us to construct unique stimuli-responsive soft materials capable of sensing a variety of disease-related biomarkers. The programmable gel-sol response (even to biol. samples) is visible to the naked eye. Furthermore, we built Boolean logic gates (OR and AND) into the hydrogel-enzyme hybrid materials, which were able to sense simultaneously plural specific biochems. and execute a controlled drug release in accordance with the logic operation. The intelligent soft materials that we have developed may prove valuable in future medical diagnostics or treatments.
- 148Heuser, T.; Merindol, R.; Loescher, S.; Klaus, A.; Walther, A. Photonic devices out of equilibrium: transient memory, signal propagation, and sensing. Adv. Mater. 2017, 29, 1606842, DOI: 10.1002/adma.201606842Google ScholarThere is no corresponding record for this reference.
- 149Hong, Y.; Velegol, D.; Chaturvedi, N.; Sen, A. Biomimetic behavior of synthetic particles: from microscopic randomness to macroscopic control. Phys. Chem. Chem. Phys. 2010, 12 (7), 1423– 1435, DOI: 10.1039/B917741HGoogle Scholar149https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3cXhsVGrtbw%253D&md5=08a39a9ff8e6fa837c98a023e717d636Biomimetic behavior of synthetic particles: from microscopic randomness to macroscopic controlHong, Yiying; Velegol, Darrell; Chaturvedi, Neetu; Sen, AyusmanPhysical Chemistry Chemical Physics (2010), 12 (7), 1423-1435CODEN: PPCPFQ; ISSN:1463-9076. (Royal Society of Chemistry)Randomness is an inherent property of biol. systems. In contrast, randomness has been mostly avoided in designing synthetic or artificial systems. Particularly, in designing micro/nano-motors, some researchers have successfully used external fields to gain deterministic control over the directionality of the objects, which otherwise move in completely random directions due to Brownian motion. However, a partial control that preserves a certain degree of randomness can be very useful in certain applications of micro/nano-motors. In this Perspective we review the current progress in establishing autonomous motion of micro/nano-particles that possess controlled randomness, provide insight into the phenomena where macroscopic order originates from microscopic disorder and discuss the resemblance between these artificial systems and biol. emergent/collective behaviors.
- 150Sengupta, S.; Dey, K. K.; Muddana, H. S.; Tabouillot, T.; Ibele, M. E.; Butler, P. J.; Sen, A. Enzyme molecules as nanomotors. J. Am. Chem. Soc. 2013, 135 (4), 1406– 1414, DOI: 10.1021/ja3091615Google Scholar150https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3sXms1yitQ%253D%253D&md5=962d69545d196b4117014792625d036fEnzyme Molecules as NanomotorsSengupta, Samudra; Dey, Krishna K.; Muddana, Hari S.; Tabouillot, Tristan; Ibele, Michael E.; Butler, Peter J.; Sen, AyusmanJournal of the American Chemical Society (2013), 135 (4), 1406-1414CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)Using fluorescence correlation spectroscopy, we show that the diffusive movements of catalase enzyme mols. increase in the presence of the substrate, hydrogen peroxide, in a concn.-dependent manner. Employing a microfluidic device to generate a substrate concn. gradient, we show that both catalase and urease enzyme mols. spread toward areas of higher substrate concn., a form of chemotaxis at the mol. scale. Using glucose oxidase and glucose to generate a hydrogen peroxide gradient, we induce the migration of catalase toward glucose oxidase, thereby showing that chem. interconnected enzymes can be drawn together.
- 151Zhao, X.; Gentile, K.; Mohajerani, F.; Sen, A. Powering motion with enzymes. Acc. Chem. Res. 2018, 51 (10), 2373– 2381, DOI: 10.1021/acs.accounts.8b00286Google Scholar151https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC1cXhslyjsrbI&md5=55244a7c7b0a2dc5f1f7fb656fcf2e38Powering Motion with EnzymesZhao, Xi; Gentile, Kayla; Mohajerani, Farzad; Sen, AyusmanAccounts of Chemical Research (2018), 51 (10), 2373-2381CODEN: ACHRE4; ISSN:0001-4842. (American Chemical Society)Enzymes are ubiquitous in living systems. Apart from traditional motor proteins, the function of enzymes was assumed to be confined to the promotion of biochem. reactions. Recent work shows that free swimming enzymes, when catalyzing reactions, generate enough mech. force to cause their own movement, typically obsd. as substrate-concn.-dependent enhanced diffusion. Preliminary indication is that the impulsive force generated per turnover is comparable to the force produced by motor proteins and is within the range to activate biol. adhesion mols. responsible for mechanosensation by cells, making force generation by enzymic catalysis a novel mechanobiol.-relevant event. Furthermore, when exposed to a gradient in substrate concn., enzymes move up the gradient: an example of chemotaxis at the mol. level. The driving force for mol. chemotaxis appears to be the lowering of chem. potential due to thermodynamically favorable enzyme-substrate interactions and we suggest that chemotaxis promotes enzymic catalysis by directing the motion of the catalyst and substrates toward each other. Enzymes that are part of a reaction cascade have been shown to assemble through sequential chemotaxis; each enzyme follows its own specific substrate gradient, which in turn is produced by the preceding enzymic reaction. Thus, sequential chemotaxis in catalytic cascades allows time-dependent, self-assembly of specific catalyst particles. This is an example of how information can arise from chem. gradients, and it is tempting to suggest that similar mechanisms underlie the organization of living systems. On a practical level, chemotaxis can be used to sep. out active catalysts from their less active or inactive counterparts in the presence of their resp. substrates and should, therefore, find wide applicability. When attached to bigger particles, enzyme ensembles act as "engines", imparting motility to the particles and moving them directionally in a substrate gradient. The impulsive force generated by enzyme catalysis can also be transmitted to the surrounding fluid and mol. and colloidal tracers, resulting in convective fluid pumping and enhanced tracer diffusion. Enzyme-powered pumps that transport fluid directionally can be fabricated by anchoring enzymes onto a solid support and supplying the substrate. Thus, enzyme pumps constitute a novel platform that combines sensing and microfluidic pumping into a single self-powered microdevice. Taken in its entirety, force generation by active enzymes has potential applications ranging from nanomachinery, nanoscale assembly, cargo transport, drug delivery, micro- and nanofluidics, and chem./biochem. sensing. We also hypothesize that, in vivo, enzymes may be responsible for the stochastic motion of the cytoplasm, the organization of metabolons and signaling complexes, and the convective transport of fluid in cells. A detailed understanding of how enzymes convert chem. energy to directional mech. force can lead us to the basic principles of fabrication, development, and monitoring of biol. and biomimetic mol. machines.
- 152Zhang, Y.; Hess, H. Enhanced diffusion of catalytically active enzymes. ACS Cen. Sci. 2019, 5 (6), 939– 948, DOI: 10.1021/acscentsci.9b00228Google Scholar152https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC1MXpsVejsb4%253D&md5=2f39b427e23ce42ee54b229000e9d7dfEnhanced Diffusion of Catalytically Active EnzymesZhang, Yifei; Hess, HenryACS Central Science (2019), 5 (6), 939-948CODEN: ACSCII; ISSN:2374-7951. (American Chemical Society)A review. The past decade has seen an increasing no. of investigations into enhanced diffusion of catalytically active enzymes. These studies suggested that enzymes are actively propelled as they catalyze reactions or bind with ligands (e.g., substrates or inhibitors). In this Outlook, we chronol. summarize and discuss the exptl. observations and theor. interpretations and emphasize the potential contradictions in these efforts. We point out that the existing multimeric forms of enzymes or isoenzymes may cause artifacts in measurements and that the conformational changes upon substrate binding are usually not sufficient to give rise to a diffusion enhancement greater than 30%. Therefore, more rigorous expts. and a more comprehensive theory are urgently needed to quant. validate and describe the enhanced enzyme diffusion.
- 153Zhang, Y.; Hess, H. Chemically-powered swimming and diffusion in the microscopic world. Nat. Rev. Chem. 2021, 5, 500– 510, DOI: 10.1038/s41570-021-00281-6Google Scholar153https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB3MXhvVGgs73E&md5=b029942d817871f9955deb37d7f0ab0eChemically-powered swimming and diffusion in the microscopic worldZhang, Yifei; Hess, HenryNature Reviews Chemistry (2021), 5 (7), 500-510CODEN: NRCAF7; ISSN:2397-3358. (Nature Portfolio)A review. The past decade has seen intriguing reports and heated debates concerning the chem.-driven enhanced motion of objects ranging from small mols. to millimeter-size synthetic robots. These objects, in solns. in which chem. reactions were occurring, were obsd. to diffuse (spread non-directionally) or swim (move directionally) at rates exceeding those expected from Brownian motion alone. The debates have focused on whether obsd. enhancement is an exptl. artifact or a real phenomenon. If the latter were true, then we would also need to explain how the chem. energy is converted into mech. work. In this Perspective, we summarize and discuss recent observations and theories of active diffusion and swimming. Notably, the chemomech. coupling and magnitude of diffusion enhancement are strongly size-dependent and should vanish as the size of the swimmers approaches the mol. scale. We evaluate the reliability of common techniques to measure diffusion coeffs. and finish by considering the potential applications and chem. to mech. energy conversion efficiencies of typical nanoswimmers and microswimmers.
- 154Patiño, T.; Arqué, X.; Mestre, R.; Palacios, L.; Sánchez, S. Fundamental aspects of enzyme-powered micro- and nanoswimmers. Acc. Chem. Res. 2018, 51 (11), 2662– 2671, DOI: 10.1021/acs.accounts.8b00288Google Scholar154https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC1cXhvVOgs7bK&md5=ea807f80bdec589b2886c1f5e4640ec9Fundamental Aspects of Enzyme-Powered Micro- and NanoswimmersPatino, Tania; Arque, Xavier; Mestre, Rafael; Palacios, Lucas; Sanchez, SamuelAccounts of Chemical Research (2018), 51 (11), 2662-2671CODEN: ACHRE4; ISSN:0001-4842. (American Chemical Society)A review and discussion with 54 refs. Self-propulsion at the nanoscale constitutes a challenge due to the need for overcoming viscous forces and Brownian motion. Inspired by nature, artificial micro- and nanomachines powered by catalytic reactions have been developed. Due to the toxicity of the most commonly used fuels, enzyme catalysis has emerged as a versatile and biocompatible alternative to generate self-propulsion. Different swimmer sizes, ranging from the nanoscale to the microscale, and geometries, including tubular and spherical shapes, have been explored by our group and others. However, there is still a lack of understanding of the mechanisms underlying enzyme-mediated propulsion. Size, shape, and enzyme distribution, as well as their intrinsic enzymic properties, may play a crucial role in motion dynamics. In this Account, we present the efforts carried out by our group and others on the use of enzymes to power micro- and nanoswimmers. We examine the different structures, materials and enzymes reported so far to fabricate biocatalytic micro- and nanoswimmers with special emphasis on their effect in motion dynamics. We discuss the development of tubular microjets and, in particular the biocompatible propulsion of nanotubular jets, after which our group reported bubble-free enzymically propelled tubular structures with different dynamics depending on the tube length and enzyme localization (inside, outside or both). In the case of swimmers of spherical shape, we highlight the role of asymmetry in enzyme coverage and how it can affect their motion dynamics. Different approaches have been described to generate asym. distribution of enzymes, namely Janus particles, polymeric vesicles and the non-Janus with patch-like enzyme distribution that we recently demonstrated which produce motion as well. We also examine the correlation between enzyme kinetics and active motion. Enzyme activity, and consequently speed, can be modulated by modifying substrate concn. or adding specific inhibitors. Finally, we review the theory of active Brownian motion and how the size of the particles can influence the anal. of the results. Fundamentally, nanoscaled swimmers are more affected by Brownian fluctuations than microsized swimmers and, therefore, their motion is an enhanced diffusion with respect to the passive case. Microswimmers, however, can overcome these fluctuations and show propulsive or ballistic trajectories. We provide some considerations on how to analyze the motion of these swimmers from an exptl. point of view. Despite the rapid progress in enzyme-based micro- and nanoswimmers, deeper understanding of the mechanisms of motion is needed and further efforts should be aimed to study their lifetime, long-term stability and their ability to navigate in complex media.
- 155Wang, L.; Song, S.; van Hest, J.; Abdelmohsen, L.; Huang, X.; Sánchez, S. Biomimicry of cellular motility and communication based on synthetic soft-architectures. Small 2020, 16 (27), 1907680, DOI: 10.1002/smll.201907680Google Scholar155https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB3cXmsVSlsLg%253D&md5=0c324d0e4564083add69a7f4cc66e60dBiomimicry of Cellular Motility and Communication Based on Synthetic Soft-ArchitecturesWang, Lei; Song, Shidong; van Hest, Jan; Abdelmohsen, Loai K. E. A.; Huang, Xin; Sanchez, SamuelSmall (2020), 16 (27), 1907680CODEN: SMALBC; ISSN:1613-6810. (Wiley-VCH Verlag GmbH & Co. KGaA)A review. Cells, sophisticated membrane-bound units that contain the fundamental mols. of life, provide a precious library for inspiration and motivation for both society and academia. Scientists from various disciplines have made great endeavors toward the understanding of the cellular evolution by engineering artificial counterparts (protocells) that mimic or initiate structural or functional cellular aspects. In this regard, several works have discussed possible building blocks, designs, functions, or dynamics that can be applied to achieve this goal. Although great progress has been made, fundamental-yet complex-behaviors such as cellular communication, responsiveness to environmental cues, and motility remain a challenge, yet to be resolved. Herein, recent efforts toward utilizing soft systems for cellular mimicry are summarized-following the main outline of cellular evolution, from basic compartmentalization, and biol. reactions for energy prodn., to motility and communicative behaviors between artificial cell communities or between artificial and natural cell communities. Finally, the current challenges and future perspectives in the field are discussed, hoping to inspire more future research and to help the further advancement of this field.
- 156Hermanová, S.; Pumera, M. Biocatalytic micro- and nanomotors. Chem. Eur. J. 2020, 26, 1108, DOI: 10.1002/chem.202084962Google ScholarThere is no corresponding record for this reference.
- 157Muddana, H. S.; Sengupta, S.; Mallouk, T. E.; Sen, A.; Butler, P. J. Substrate catalysis enhances single-enzyme diffusion. J. Am. Chem. Soc. 2010, 132 (7), 2110– 2111, DOI: 10.1021/ja908773aGoogle Scholar157https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3cXht1SgtLo%253D&md5=2f32d56d7eaa33a5c3f8a59e8aac82e6Substrate Catalysis Enhances Single-Enzyme DiffusionMuddana, Hari S.; Sengupta, Samudra; Mallouk, Thomas E.; Sen, Ayusman; Butler, Peter J.Journal of the American Chemical Society (2010), 132 (7), 2110-2111CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)We show that diffusion of single urease enzyme mols. increases in the presence of urea in a concn.-dependent manner and calc. the force responsible for this increase. Urease diffusion measured using fluorescence correlation spectroscopy increased by 16-28% over buffer controls at urea concns. ranging from 0.001 to 1 M. This increase was significantly attenuated when urease was inhibited with pyrocatechol, demonstrating that the increase in diffusion was the result of enzyme catalysis of urea. Local mol. pH changes as measured using the pH-dependent fluorescence lifetime of SNARF-1 conjugated to urease were not sufficient to explain the increase in diffusion. Thus, a force generated by self-electrophoresis remains the most plausible explanation. This force, evaluated using Brownian dynamics simulations, was 12 pN per reaction turnover. These measurements demonstrate force generation by a single enzyme mol. and lay the foundation for a further understanding of biol. force generation and the development of enzyme-driven nanomotors.
- 158Hortelão, A. C.; García-Jimeno, S.; Cano-Sarabia, M.; Patiño, T.; Maspoch, D.; Sanchez, S. LipoBots: Using liposomal vesicles as protective shell of urease-based nanomotors. Adv. Funct. Mater. 2020, 30 (42), 2002767, DOI: 10.1002/adfm.202002767Google Scholar158https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB3cXhs1Wrs7vK&md5=4c05f90974703c5e86e8c291e0908163LipoBots when using Liposomal Vesicles as Protective Shell of Urease-Based NanomotorsHortelao, Ana C.; Garcia-Jimeno, Sonia; Cano-Sarabia, Mary; Patino, Tania; Maspoch, Daniel; Sanchez, SamuelAdvanced Functional Materials (2020), 30 (42), 2002767CODEN: AFMDC6; ISSN:1616-301X. (Wiley-VCH Verlag GmbH & Co. KGaA)Developing self-powered nanomotors made of biocompatible and functional components is of paramount importance in future biomedical applications. Herein, the functional features of LipoBots (LBs) composed of a liposomal carrier contg. urease enzymes for propulsion, including their protective properties against acidic conditions and their on-demand triggered activation, are reported. Given the functional nature of liposomes, enzymes can be either encapsulated or coated on the surface of the vesicles. The influence of the location of urease on motion dynamics is first studied, finding that the surface-urease LBs undergo self-propulsion whereas the encapsulated-urease LBs do not. However, adding a percolating agent present in the bile salts to the encapsulated-urease LBs triggers active motion. Moreover, it is found that when both types of nanomotors are exposed to a medium of similar pH found in the stomach, the surface-urease LBs lose activity and motion capabilities, while the encapsulated-urease LBs retain activity and mobility. The results for the protection enzyme activity through encapsulation within liposomes and in situ triggering of the motion of LBs upon exposure to bile salts may open new avenues for the use of liposome-based nanomotors in drug delivery, for example, in the gastrointestinal tract, where bile salts are naturally present in the intestine.
- 159Choi, H.; Cho, S. H.; Hahn, S. K. Urease-powered polydopamine nanomotors for intravesical therapy of bladder diseases. ACS Nano 2020, 14 (6), 6683– 6692, DOI: 10.1021/acsnano.9b09726Google Scholar159https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB3cXhtVyis77E&md5=e26e764ddceeccee66212cc4ad32d045Urease-Powered Polydopamine Nanomotors for Intravesical Therapy of Bladder DiseasesChoi, Hyunsik; Cho, Seong Hwi; Hahn, Sei KwangACS Nano (2020), 14 (6), 6683-6692CODEN: ANCAC3; ISSN:1936-0851. (American Chemical Society)Intravesical therapeutic delivery has been extensively investigated for various bladder diseases such as bladder cancer, overactive bladder, urinary incontinence, and interstitial cystitis. However, conventional drug carriers have a low therapeutic delivery efficiency because of the passive diffusion of drug mols. in a bladder and the rapid clearance by periodic urination. Here, we report biocompatible and bioavailable enzyme-powered polymer nanomotors which can deeply penetrate into a mucosa layer of the bladder wall and remain for a long-term period in the bladder. The successful fabrication of nanomotors was confirmed by high-resoln. transmission electron microscopy, energy-dispersive X-ray mapping, zeta-potential anal., Fourier transform IR spectroscopy, and urease activity and nanomotor trajectory analyses. After injection into the bladder, urease-immobilized nanomotors became active, moving around in the bladder by converting urea into carbon dioxide and ammonia. The nanomotors resulted in the facilitated penetration to the mucosa layer of the bladder wall and the prolonged retention in the bladder even after repeated urination. The enhanced penetration and retention of the nanomotors as a drug delivery carrier in the bladder would be successfully harnessed for treating a variety of bladder diseases.
- 160Yang, Z.; Wang, L.; Gao, Z.; Hao, X.; Luo, M.; Yu, Z.; Guan, J. Ultrasmall enzyme-powered janus nanomotor working in blood circulation system. ACS Nano 2023, 17 (6), 6023– 6035, DOI: 10.1021/acsnano.3c00548Google Scholar160https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB3sXkvVKrtbY%253D&md5=f5847c9fc47492c4de9ddb183016deb3Ultrasmall Enzyme-Powered Janus Nanomotor Working in Blood Circulation SystemYang, Zili; Wang, Liangmeng; Gao, Zhixue; Hao, Xiaomeng; Luo, Ming; Yu, Zili; Guan, JianguoACS Nano (2023), 17 (6), 6023-6035CODEN: ANCAC3; ISSN:1936-0851. (American Chemical Society)Injectable chem. powered nanomotors may revolutionize biomedical technologies, but to date, it is a challenge for them to move autonomously in the blood circulation system and they are too large in size to break through the biol. barriers therein. Herein, we report a general scalable colloidal chem. synthesis approach for the fabrication of ultrasmall urease-powered Janus nanomotors (UPJNMs) that have a size (100-30 nm) meeting the requirement to break through the biol. barriers in the blood circulation system and can efficiently move in body fluids with only endogenous urea as fuel. In our protocol, the two hemispheroid surfaces of eccentric Au-polystyrene nanoparticles are stepwise grafted with poly(ethylene glycol) brushes and ureases via selective etching and chem. coupling, resp., forming the UPJNMs. The UPJNMs have lasting powerful mobility with ionic tolerance and pos. chemotaxis, while they are able to be dispersed steadily and self-propelled in real body fluids, as well as demonstrate good biosafety and a long circulation time in the blood circulation system of mice. Thus, the as-prepd. UPJNMs are promising as an active theranostics nanosystem for future biomedical applications.
- 161Toebes, B. J.; Cao, F.; Wilson, D. A. Spatial control over catalyst positioning on biodegradable polymeric nanomotors. Nat. Commun. 2019, 10 (1), 5308, DOI: 10.1038/s41467-019-13288-xGoogle Scholar161https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A280%3ADC%252BB3Mfit1egsg%253D%253D&md5=430b34dab0e78d12f9becc3da31918f1Spatial control over catalyst positioning on biodegradable polymeric nanomotorsToebes B Jelle; Cao F; Wilson Daniela ANature communications (2019), 10 (1), 5308 ISSN:.Scientists over the world are inspired by biological nanomotors and try to mimic these complex structures. In recent years multiple nanomotors have been created for various fields, such as biomedical applications or environmental remediation, which require a different design both in terms of size and shape, as well as material properties. So far, only relatively simple designs for synthetic nanomotors have been reported. Herein, we report an approach to create biodegradable polymeric nanomotors with a multivalent design. PEG-PDLLA (poly(ethylene glycol)-b-poly(D,L-lactide)) stomatocytes with azide handles were created that were selectively reduced on the outside surface by TCEP (tris(2-carboxyethyl)phosphine) functionalized beads. Thereby, two different functional handles were created, both on the inner and outer surface of the stomatocytes, providing spatial control for catalyst positioning. Enzymes were coupled on the inside of the stomatocyte to induce motion in the presence of fuel, while fluorophores and other molecules can be attached on the outside.
- 162Qiu, B.; Xie, L.; Zeng, J.; Liu, T.; Yan, M.; Zhou, S.; Liang, Q.; Tang, J.; Liang, K.; Kong, B. Interfacially super-assembled asymmetric and H2O2 sensitive multilayer-sandwich magnetic mesoporous silica nanomotors for detecting and removing heavy metal ions. Adv. Funct. Mater. 2021, 31, 2010694, DOI: 10.1002/adfm.202010694Google Scholar162https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB3MXmvVyjtLw%253D&md5=00fa2e105237342feedcdb693c43f840Interfacially Super-Assembled Asymmetric and H2O2 Sensitive Multilayer-Sandwich Magnetic Mesoporous Silica Nanomotors for Detecting and Removing Heavy Metal IonsQiu, Beilei; Xie, Lei; Zeng, Jie; Liu, Tianyi; Yan, Miao; Zhou, Shan; Liang, Qirui; Tang, Jinyao; Liang, Kang; Kong, BiaoAdvanced Functional Materials (2021), 31 (21), 2010694CODEN: AFMDC6; ISSN:1616-301X. (Wiley-VCH Verlag GmbH & Co. KGaA)Asym. hollow and magnetic mesoporous silica nanocomposites have great potential applications due to their unique structural-functional properties. Here, asym. multilayer-sandwich magnetic mesoporous silica nanobottles (MMSNBs) are presented through an interfacial super-assembly strategy. Asym. hollow silica nanobottles (SNBs) are first prepd., and Fe3O4 nanoparticles monolayers and mesoporous silica layers are uniformly super-assembled on the surfaces of SNBs, resp. The high Fe3O4 nanoparticles loading endows MMSNBs with a high magnetization (8.5 emu g-1), while the mesoporous silica layers exhibit high surface area (613.4 m2 g-1) and large pore size (3.6 nm). MMSNBs can be employed as a novel type of enzyme-powered nanomotors by integrating catalase (Cat-MMSNBs), which show an av. speed of 7.59μm s-1 (≈25 body lengths s-1) at 1.5 wt% H2O2. Accordingly, the water quality can be monitored by evaluating the movement speed of Cat-MMSNBs. Moreover, MMSNBs act as a good adsorbent for removing more than 90% of the heavy metal ions with the advantage of the mesoporous structure. In addn., the good magnetic response enables the MMSNBs with precise directional control and is conducive to recycling for repeated operation. This bottom-up interfacial super-assembly construction strategy allows for a new understanding of the rational design and synthesis of multi-functional nanomotors.
- 163Abdelmohsen, L. K.; Nijemeisland, M.; Pawar, G. M.; Janssen, G. J.; Nolte, R. J.; van Hest, J. C.; Wilson, D. A. Dynamic loading and unloading of proteins in polymeric stomatocytes: formation of an enzyme-loaded supramolecular nanomotor. ACS Nano 2016, 10 (2), 2652– 2660, DOI: 10.1021/acsnano.5b07689Google Scholar163https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC28XhtlymsLs%253D&md5=950ae40c6f7c8ae1a6b6d370c5fd71d5Dynamic Loading and Unloading of Proteins in Polymeric Stomatocytes: Formation of an Enzyme-Loaded Supramolecular NanomotorAbdelmohsen, Loai K. E. A.; Nijemeisland, Marlies; Pawar, Gajanan M.; Janssen, Geert-Jan A.; Nolte, Roeland J. M.; van Hest, Jan C. M.; Wilson, Daniela A.ACS Nano (2016), 10 (2), 2652-2660CODEN: ANCAC3; ISSN:1936-0851. (American Chemical Society)Self-powered artificial nanomotors are currently attracting increased interest as mimics of biol. motors but also as potential components of nanomachinery, robotics, and sensing devices. We have recently described the controlled shape transformation of polymersomes into bowl-shaped stomatocytes and the assembly of platinum-driven nanomotors. However, the platinum encapsulation inside the structures was low; only 50% of the structures contained the catalyst and required both high fuel concns. for the propelling of the nanomotors and harsh conditions for the shape transformation. Application of the nanomotors in a biol. setting requires the nanomotors to be efficiently propelled by a naturally available energy source and at biol. relevant concns. Here we report a strategy for enzyme entrapment and nanomotor assembly via controlled and reversible folding of polymersomes into stomatocytes under mild conditions, allowing the encapsulation of the proteins inside the stomach with almost 100% efficiency and retention of activity. The resulting enzyme-driven nanomotors are capable of propelling these structures at low fuel concns. (hydrogen peroxide or glucose) via a one-enzyme or two-enzyme system. The confinement of the enzymes inside the stomach does not hinder their activity and in fact facilitates the transfer of the substrates, while protecting them from the deactivating influences of the media. This is particularly important for future applications of nanomotors in biol. settings esp. for systems where fast autonomous movement occurs at physiol. concns. of fuel.
- 164Nijemeisland, M.; Abdelmohsen, L. K.; Huck, W. T.; Wilson, D. A.; van Hest, J. C. A compartmentalized out-of-equilibrium enzymatic reaction network for sustained autonomous movement. ACS Cent. Sci. 2016, 2 (11), 843– 849, DOI: 10.1021/acscentsci.6b00254Google Scholar164https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC28Xhsl2gtbfP&md5=c4c80a73fb5d6109de97e97bcb43ce09A Compartmentalized Out-of-Equilibrium Enzymatic Reaction Network for Sustained Autonomous MovementNijemeisland, Marlies; Abdelmohsen, Loai K. E. A.; Huck, Wilhelm T. S.; Wilson, Daniela A.; van Hest, Jan C. M.ACS Central Science (2016), 2 (11), 843-849CODEN: ACSCII; ISSN:2374-7951. (American Chemical Society)Every living cell is a compartmentalized out-of-equil. system exquisitely able to convert chem. energy into function. In order to maintain homeostasis, the flux of metabolites is tightly controlled by regulatory enzymic networks. A crucial prerequisite for the development of life-like materials is the construction of synthetic systems with compartmentalized reaction networks that maintain out-of-equil. function. Here, we aim for autonomous movement as an example of the conversion of feedstock mols. into function. The flux of the conversion is regulated by a rationally designed enzymic reaction network with multiple feed forward loops. By compartmentalizing the network into bowl-shaped nanocapsules the output of the network is harvested as kinetic energy. The entire system shows sustained and tunable microscopic motion resulting from the conversion of multiple external substrates. The successful compartmentalization of an out-of-equil. reaction network is a major first step in harnessing the design principles of life for construction of adaptive and internally regulated life-like systems.
- 165Chatterjee, A.; Ghosh, S.; Ghosh, C.; Das, D. Fluorescent microswimmers based on cross-beta amyloid nanotubes and divergent cascade networks. Angew. Chem., Int. Ed. 2022, 61 (29), e202201547, DOI: 10.1002/anie.202201547Google Scholar165https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB38XhsVCgtL%252FJ&md5=d724d4f4ffe85e8ea7e5fcc1e577582eFluorescent Microswimmers Based on Cross-β Amyloid Nanotubes and Divergent Cascade NetworksChatterjee, Ayan; Ghosh, Souvik; Ghosh, Chandranath; Das, DibyenduAngewandte Chemie, International Edition (2022), 61 (29), e202201547CODEN: ACIEF5; ISSN:1433-7851. (Wiley-VCH Verlag GmbH & Co. KGaA)Shaped through millions of years of evolution, the spatial localization of multiple enzymes in living cells employs extensive cascade reactions to enable highly coordinated multimodal functions. Herein, by utilizing a complex divergent cascade, we exploit the catalytic potential as well as templating abilities of streamlined cross-β amyloid nanotubes to yield two orthogonal roles simultaneously. The short peptide based paracryst. nanotube surfaces demonstrated the generation of fluorescence signals within entangled networks loaded with alc. dehydrogenase (ADH). The nanotubular morphologies were further used to generate cascade-driven microscopic motility through surface entrapment of sarcosine oxidase (SOX) and catalase (Cat). Moreover, a divergent cascade network was initiated by upstream catalysis of the substrate mols. through the surface mutation of catalytic moieties. Notably, the resultant downstream products led to the generation of motile fluorescent microswimmers by utilizing the two sets of orthogonal properties and, thus, mimicked the complex cascade-mediated functionalities of extant biol.
- 166Dey, K. K.; Zhao, X.; Tansi, B. M.; Mendez-Ortiz, W. J.; Cordova-Figueroa, U. M.; Golestanian, R.; Sen, A. Micromotors powered by enzyme catalysis. Nano Lett. 2015, 15 (12), 8311– 8315, DOI: 10.1021/acs.nanolett.5b03935Google Scholar166https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2MXhvVOrtrvJ&md5=cfb4f6a75427fa6c4b9ae436c731d197Micromotors Powered by Enzyme CatalysisDey, Krishna K.; Zhao, Xi; Tansi, Benjamin M.; Mendez-Ortiz, Wilfredo J.; Cordova-Figueroa, Ubaldo M.; Golestanian, Ramin; Sen, AyusmanNano Letters (2015), 15 (12), 8311-8315CODEN: NALEFD; ISSN:1530-6984. (American Chemical Society)Active biocompatible systems are of great current interest for their possible applications in drug or antidote delivery at specific locations. Herein, we report the synthesis and study of self-propelled microparticles powered by enzymic reactions and their directed movement in substrate concn. gradient. Polystyrene microparticles were functionalized with the enzymes urease and catalase using a biotin-streptavidin linkage procedure. The motion of the enzyme-coated particles was studied in the presence of the resp. substrates, using optical microscopy and dynamic light scattering anal. The diffusion of the particles was found to increase in a substrate concn. dependent manner. The directed chemotactic movement of these enzyme-powered motors up the substrate gradient was studied using three-inlet microfluidic channel architecture.
- 167Zhao, X.; Palacci, H.; Yadav, V.; Spiering, M. M.; Gilson, M. K.; Butler, P. J.; Hess, H.; Benkovic, S. J.; Sen, A. Substrate-driven chemotactic assembly in an enzyme cascade. Nat. Chem. 2018, 10 (3), 311– 317, DOI: 10.1038/nchem.2905Google Scholar167https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2sXhvF2lsb3F&md5=3c60afd0a0cbcd0a0f3cefb403e1f20bSubstrate-driven chemotactic assembly in an enzyme cascadeZhao, Xi; Palacci, Henri; Yadav, Vinita; Spiering, Michelle M.; Gilson, Michael K.; Butler, Peter J.; Hess, Henry; Benkovic, Stephen J.; Sen, AyusmanNature Chemistry (2018), 10 (3), 311-317CODEN: NCAHBB; ISSN:1755-4330. (Nature Research)Enzymic catalysis is essential to cell survival. In many instances, enzymes that participate in reaction cascades have been shown to assemble into metabolons in response to the presence of the substrate for the first enzyme. However, what triggers metabolon formation has remained an open question. Through a combination of theory and expts., we show that enzymes in a cascade can assemble via chemotaxis. We apply microfluidic and fluorescent spectroscopy techniques to study the coordinated movement of the first four enzymes of the glycolysis cascade: hexokinase, phosphoglucose isomerase, phosphofructokinase and aldolase. We show that each enzyme independently follows its own specific substrate gradient, which in turn is produced by the preceding enzymic reaction. Furthermore, we find that the chemotactic assembly of enzymes occurs even under cytosolic crowding conditions.
- 168Wang, J.; Toebes, B. J.; Plachokova, A. S.; Liu, Q.; Deng, D.; Jansen, J. A.; Yang, F.; Wilson, D. A. Self-propelled PLGA micromotor with chemotactic response to inflammation. Adv. Healthcare Mater. 2020, 9 (7), 1901710, DOI: 10.1002/adhm.201901710Google Scholar168https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB3cXktlGhurk%253D&md5=230e9df65435e02415eb5cb517cfb1ccSelf-Propelled PLGA Micromotor with Chemotactic Response to InflammationWang, Jiamian; Toebes, B. Jelle; Plachokova, Adelina S.; Liu, Qian; Deng, Dongmei; Jansen, John A.; Yang, Fang; Wilson, Daniela A.Advanced Healthcare Materials (2020), 9 (7), 1901710CODEN: AHMDBJ; ISSN:2192-2640. (Wiley-VCH Verlag GmbH & Co. KGaA)Local drug delivery systems have recently been developed for multiple diseases that have the requirements of site-specific actions, prolonged delivery periods, and decreased drug dosage to reduce undesirable side effects. The challenge for such systems is to achieve directional and precise delivery in inaccessible narrow lesions, such as periodontal pockets or root canals in deeper portions of the dentinal tubules. The primary strategy to tackle this challenge is fabricating a smart tracking delivery system. Here, drug-loaded biodegradable micromotors showing self-propelled directional movement along a hydrogen peroxide concn. gradient produced by phorbol esters-stimulated macrophages are reported. The drug-loaded poly(lactic-co-glycolic acid) micromotors with asym. coverage of enzyme (patch-like enzyme distribution) are prepd. by electrospraying and postfunctionalized with catalase via 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide coupling. Doxycycline, a common drug for the treatment of periodontal disease, is selected as a model drug, and the release study by high-performance liq. chromatog. is shown that both the postfunctionalization step and the presence of hydrogen peroxide have no neg. influence on drug release profiles. The movement behavior in the presence of hydrogen peroxide is confirmed by nanoparticle tracking anal. An in vitro model is designed and confirmed the response efficiency and directional control of the micromotors toward phorbol esters-stimulated macrophages.
- 169Joseph, A.; Contini, C.; Cecchin, D.; Nyberg, S.; Ruiz-Perez, L.; Gaitzsch, J.; Fullstone, G.; Tian, X.; Azizi, J.; Preston, J.; Volpe, G.; Battaglia, G. Chemotactic synthetic vesicles: design and applications in blood-brain barrier crossing. Sci. Adv. 2017, 3, e1700362, DOI: 10.1126/sciadv.1700362Google Scholar169https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC1cXntFGrsbY%253D&md5=0234d41cb96af40a7de3af83d36b1359Chemotactic synthetic vesicles: Design and applications in blood-brain barrier crossingJoseph, Adrian; Contini, Claudia; Cecchin, Denis; Nyberg, Sophie; Ruiz-Perez, Lorena; Gaitzsch, Jens; Fullstone, Gavin; Tian, Xiaohe; Azizi, Juzaili; Preston, Jane; Volpe, Giorgio; Battaglia, GiuseppeScience Advances (2017), 3 (8), e1700362/1-e1700362/12CODEN: SACDAF; ISSN:2375-2548. (American Association for the Advancement of Science)In recent years, scientists have created artificialmicroscopic and nanoscopic self-propelling particles, often referred to as nano- or microswimmers, capable of mimicking biol. locomotion and taxis. This active diffusion enables the engineering of complex operations that so far have not been possible at the micro- and nanoscale. One of the most promising tasks is the ability to engineer nanocarriers that can autonomously navigate within tissues and organs, accessing nearly every site of the human body guided by endogenous chem. gradients.We report a fully synthetic, org., nanoscopic system that exhibits attractive chemotaxisdrivenbyenzymic conversion of glucose.Weachieve this by encapsulating glucose oxidase alone or in combination with catalase into nanoscopic and biocompatible asym. polymer vesicles (known as polymersomes). We show that these vesicles self-propel in response to an external gradient of glucose by inducing a slip velocity on their surface, which makes them move in an extremely sensitive way toward higher-concn. regions. We finally demonstrate that the chemotactic behavior of these nanoswimmers, in combination with LRP-1 (low-d. lipoprotein receptor-related protein 1) targeting, enables a fourfold increase in penetration to the brain compared to nonchemotactic systems.
- 170Kumar, B.; Patil, A. J.; Mann, S. Enzyme-powered motility in buoyant organoclay/DNA protocells. Nat. Chem. 2018, 10 (11), 1154– 116, DOI: 10.1038/s41557-018-0119-3Google Scholar170https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC1cXhsFKqtr7K&md5=a18f59a5d0b21e7a790ea182f37ab3c1Enzyme-powered motility in buoyant organoclay/DNA protocellsKumar, B. V. V. S. Pavan; Patil, Avinash J.; Mann, StephenNature Chemistry (2018), 10 (11), 1154-1163CODEN: NCAHBB; ISSN:1755-4330. (Nature Research)Reconstitution and simulation of cellular motility in microcompartmentalized colloidal objects have important implications for microcapsule-based remote sensing, environmentally induced signaling between artificial cell-like entities and programming spatial migration in synthetic protocell consortia. Here the authors describe the design and construction of catalase-contg. organoclay/DNA semipermeable microcapsules, which in the presence of hydrogen peroxide exhibit enzyme-powered oxygen gas bubble-dependent buoyancy. The authors det. the optimum conditions for single and/or multiple bubble generation per microcapsule, monitor the protocell velocities and resilience, and use remote magnetic guidance to establish reversible changes in the buoyancy. Co-encapsulation of catalase and glucose oxidase is exploited to establish a spatiotemporal response to antagonistic bubble generation and depletion to produce protocells capable of sustained oscillatory vertical movement. The motility of the microcapsules can be used for the flotation of macroscopic objects, self-sorting of mixed protocell communities and the delivery of a biocatalyst from an inert to chem. active environment. These results highlight new opportunities to constructing programmable microcompartmentalized colloids with buoyancy-derived motility.
- 171Gao, N.; Li, M.; Tian, L.; Patil, A. J.; Pavan Kumar, B.; Mann, S. Chemical-mediated translocation in protocell-based microactuators. Nat. Chem. 2021, 13 (9), 868– 879, DOI: 10.1038/s41557-021-00728-9Google Scholar171https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB3MXhsVSmsb3L&md5=8db27f942d6097312a2b8f36a6845274Chemical-mediated translocation in protocell-based microactuatorsGao, Ning; Li, Mei; Tian, Liangfei; Patil, Avinash J.; Pavan Kumar, B. V. V. S.; Mann, StephenNature Chemistry (2021), 13 (9), 868-879CODEN: NCAHBB; ISSN:1755-4330. (Nature Portfolio)Artificial cell-like communities participate in diverse modes of chem. interaction but exhibit minimal interfacing with their local environment. Here we develop an interactive microsystem based on the immobilization of a population of enzyme-active semipermeable proteinosomes within a helical hydrogel filament to implement signal-induced movement. We attach large single-polynucleotide/peptide microcapsules at one or both ends of the helical protocell filament to produce free-standing soft microactuators that sense and process chem. signals to perform mech. work. Different modes of translocation are achieved by synergistic or antagonistic enzyme reactions located within the helical connector or inside the attached microcapsule loads. Mounting the microactuators on a ratchet-like surface produces a directional push-pull movement. Our methodol. opens up a route to protocell-based chem. systems capable of utilizing mech. work and provides a step towards the engineering of soft microscale objects with increased levels of operational autonomy.
- 172Wheeldon, I.; Minteer, S. D.; Banta, S.; Barton, S. C.; Atanassov, P.; Sigman, M. Substrate channelling as an approach to cascade reactions. Nat. Chem. 2016, 8 (4), 299– 309, DOI: 10.1038/nchem.2459Google Scholar172https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC28Xks1Cru7Y%253D&md5=5056edbd76a5aa2266906be9e23c579bSubstrate channelling as an approach to cascade reactionsWheeldon, Ian; Minteer, Shelley D.; Banta, Scott; Barton, Scott Calabrese; Atanassov, Plamen; Sigman, MatthewNature Chemistry (2016), 8 (4), 299-309CODEN: NCAHBB; ISSN:1755-4330. (Nature Publishing Group)A review. Millions of years of evolution have produced biol. systems capable of efficient one-pot multi-step catalysis. The underlying mechanisms that facilitate these reaction processes are increasingly providing inspiration in synthetic chem. Substrate channeling, where intermediates between enzymic steps are not in equil. with the bulk soln., enables increased efficiencies and yields in reaction and diffusion processes. Here, we review different mechanisms of substrate channeling found in nature and provide an overview of the anal. methods used to quantify these effects. The incorporation of substrate channeling into synthetic cascades is a rapidly developing concept, and recent examples of the fabrication of cascades with controlled diffusion and flux of intermediates are presented.
- 173Hinzpeter, F.; Gerland, U.; Tostevin, F. Optimal compartmentalization strategies for metabolic microcompartments. Biophys. J. 2017, 112, 767– 779, DOI: 10.1016/j.bpj.2016.11.3194Google Scholar173https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC28XitVOlur3F&md5=5ec8113d5f4060db6906f9c14160aeecOptimal Compartmentalization Strategies for Metabolic MicrocompartmentsHinzpeter, Florian; Gerland, Ulrich; Tostevin, FilipeBiophysical Journal (2017), 112 (4), 767-779CODEN: BIOJAU; ISSN:0006-3495. (Cell Press)Intracellular compartmentalization of cooperating enzymes is a strategy that is frequently used by cells. Segregation of enzymes that catalyze sequential reactions can alleviate challenges such as toxic pathway intermediates, competing metabolic reactions, and slow reaction rates. Inspired by nature, synthetic biologists also seek to encapsulate engineered metabolic pathways within vesicles or proteinaceous shells to enhance the yield of industrially and pharmaceutically useful products. Although enzymic compartments have been extensively studied exptl., a quant. understanding of the underlying design principles is still lacking. Here, the authors study theor. how the size and enzymic compn. of compartments should be chosen so as to maximize the productivity of a model metabolic pathway. Maximizing productivity requires compartments larger than a certain crit. size. The enzyme d. within each compartment should be tuned according to a power-law scaling in the compartment size. The authors explain these observations using an anal. solvable, well-mixed approxn. The authors also study the qual. different compartmentalization strategies that emerge in parameter regimes where this approxn. breaks down. The authors' results suggest that the different sizes and enzyme packings of α- and β-carboxysomes each constitute an optimal compartmentalization strategy given the properties of their resp. protein shells.
- 174Qiao, Y.; Li, M.; Booth, R.; Mann, S. Predatory behaviour in synthetic protocell communities. Nat. Chem. 2017, 9 (2), 110– 119, DOI: 10.1038/nchem.2617Google Scholar174https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC28Xhs1amtbjJ&md5=b9d48e5a1f98b80c2e32b8cb43c768baPredatory behaviour in synthetic protocell communitiesQiao, Yan; Li, Mei; Booth, Richard; Mann, StephenNature Chemistry (2017), 9 (2), 110-119CODEN: NCAHBB; ISSN:1755-4330. (Nature Publishing Group)Recent progress in the chem. construction of colloidal objects comprising integrated biomimetic functions is paving the way towards rudimentary forms of artificial cell-like entities (protocells). Although several new types of protocells are currently available, the design of synthetic protocell communities and investigation of their collective behavior has received little attention. Here we demonstrate an artificial form of predatory behavior in a community of protease-contg. coacervate microdroplets and protein-polymer microcapsules (proteinosomes) that interact via electrostatic binding. The coacervate microdroplets act as killer protocells for the obliteration of the target proteinosome population by protease-induced lysis of the protein-polymer membrane. As a consequence, the proteinosome payload (dextran, single-stranded DNA, platinum nanoparticles) is trafficked into the attached coacervate microdroplets, which are then released as functionally modified killer protocells capable of rekilling. Our results highlight opportunities for the development of interacting artificial protocell communities, and provide a strategy for inducing collective behavior in soft matter microcompartmentalized systems and synthetic protocell consortia.
- 175Qiao, Y.; Li, M.; Qiu, D.; Mann, S. Response-retaliation behavior in synthetic protocell communities. Angew. Chem., Int. Ed. 2019, 58 (49), 17758– 17763, DOI: 10.1002/anie.201909313Google Scholar175https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC1MXitVais77F&md5=6b6ac4f90fc4f496bcfe91f5e17b4d7aResponse-Retaliation Behavior in Synthetic Protocell CommunitiesQiao, Yan; Li, Mei; Qiu, Dong; Mann, StephenAngewandte Chemie, International Edition (2019), 58 (49), 17758-17763CODEN: ACIEF5; ISSN:1433-7851. (Wiley-VCH Verlag GmbH & Co. KGaA)Two different artificial predation strategies are spatially and temporally coupled to generate a simple tit-for-tat mechanism in a ternary protocell network capable of antagonistic enzyme-mediated interactions. The consortium initially consists of protease-sensitive glucose-oxidase-contg. proteinosomes (1), non-interacting pH-sensitive polypeptide/mononucleotide coacervate droplets contg. proteinase K (2), and proteinosome-adhered pH-resistant polymer/polysaccharide coacervate droplets (3). On receiving a glucose signal, secretion of protons from 1 triggers the disassembly of 2 and the released protease is transferred to 3 to initiate a delayed contact-dependent killing of the proteinosomes and cessation of glucose oxidase activity. Our results provide a step towards complex mesoscale dynamics based on programmable response-retaliation behavior in artificial protocell consortia.
- 176Chakraborty, T.; Wegner, S. V. Cell to cell signaling through light in artificial cell communities: glowing predator lures prey. ACS Nano 2021, 15 (6), 9434– 9444, DOI: 10.1021/acsnano.1c01600Google Scholar176https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB3MXhtlCqu77L&md5=0a547d307006fa725cd1e90d72317dbaCell to Cell Signaling through Light in Artificial Cell Communities: Glowing Predator Lures PreyChakraborty, Taniya; Wegner, Seraphine V.ACS Nano (2021), 15 (6), 9434-9444CODEN: ANCAC3; ISSN:1936-0851. (American Chemical Society)Cells commonly communicate with each other through diffusible mols. but nonchem. communication remains elusive. While bioluminescent organisms communicate through light to find prey or attract mates, it is still under debate if signaling through light is possible at the cellular level. Here, we demonstrate that cell to cell signaling through light is possible in artificial cell communities derived from biomimetic vesicles. In our design, artificial sender cells produce an intracellular light signal, which triggers the adhesion to receiver cells. Unlike sol. mols., the light signal propagates fast, independent of diffusion and without the need for a transporter across membranes. To obtain a predator-prey relationship, the luminescence predator cells is loaded with a secondary diffusible poison, which is transferred to the prey cell upon adhesion and leads to its lysis. This design provides a blueprint for light based intercellular communication, which can be used for programing artificial and natural cell communities.
- 177Mason, A. F.; Buddingh, B. C.; Williams, D. S.; van Hest, J. C. M. Hierarchical self-assembly of a copolymer-stabilized coacervate protocell. J. Am. Chem. Soc. 2017, 139 (48), 17309– 17312, DOI: 10.1021/jacs.7b10846Google Scholar177https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2sXhvVSnurzP&md5=30fde604ab0576771bbc76cbefbd7ccfHierarchical Self-Assembly of a Copolymer-Stabilized Coacervate ProtocellMason, Alexander F.; Buddingh, Bastiaan C.; Williams, David S.; van Hest, Jan C. M.Journal of the American Chemical Society (2017), 139 (48), 17309-17312CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)Complex coacervate microdroplets are finding increased utility in synthetic cell applications due to their cytomimetic properties. However, their intrinsic membrane-free nature results in instability that limits their application in protocell research. Herein, the authors present the development of a new protocell model through the spontaneous interfacial self-assembly of copolymer mols. on biopolymer coacervate microdroplets. This hierarchical protocell model not only incorporates the favorable properties of coacervates (such as spontaneous assembly and macromol. condensation) but also assimilates the essential features of a semipermeable copolymeric membrane (such as discretization and stabilization). This was accomplished by engineering an asym., biodegradable triblock copolymer mol. comprising hydrophilic, hydrophobic, and polyanionic components capable of direct coacervate membranization via electrostatic surface anchoring and chain self-assocn. The resulting hierarchical protocell demonstrated striking integrity as a result of membrane formation, successfully stabilizing enzymic cargo against coalescence and fusion in discrete protocellular populations. The semipermeable nature of the copolymeric membrane enabled the incorporation of a simple enzymic cascade, demonstrating chem. communication between discrete populations of neighboring protocells. In this way, the authors pave the way for the development of new synthetic cell constructs.
- 178Wang, X.; Tian, L.; Du, H.; Li, M.; Mu, W.; Drinkwater, B. W.; Han, X.; Mann, S. Chemical communication in spatially organized protocell colonies and protocell/living cell micro-arrays. Chem. Sci. 2019, 10 (41), 9446– 9453, DOI: 10.1039/C9SC04522HGoogle Scholar178https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC1MXhslyqt7bK&md5=07d8659b3475a5879e1fe01d00d1d227Chemical communication in spatially organized protocell colonies and protocell/living cell micro-arraysWang, Xuejing; Tian, Liangfei; Du, Hang; Li, Mei; Mu, Wei; Drinkwater, Bruce W.; Han, Xiaojun; Mann, StephenChemical Science (2019), 10 (41), 9446-9453CODEN: CSHCCN; ISSN:2041-6520. (Royal Society of Chemistry)Micro-arrays of discrete or hemifused giant unilamellar lipid vesicles (GUVs) with controllable spatial geometries, lattice dimensions, trapped occupancies and compns. are prepd. by acoustic standing wave patterning, and employed as platforms to implement chem. signaling in GUV colonies and protocell/living cell consortia. The methodol. offers an alternative approach to GUV micro-array fabrication and provides new opportunities in protocell research and bottom-up synthetic biol.
- 179Buddingh’, B. C.; Elzinga, J.; van Hest, J. C. M. Intercellular communication between artificial cells by allosteric amplification of a molecular signal. Nat. Commun. 2020, 11 (1), 1652, DOI: 10.1038/s41467-020-15482-8Google Scholar179https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB3cXmsVWitbg%253D&md5=d3384619028f0aac719d9e96dd6f40b7Intercellular communication between artificial cells by allosteric amplification of a molecular signalBuddingh', Bastiaan C.; Elzinga, Janneke; van Hest, Jan C. M.Nature Communications (2020), 11 (1), 1652CODEN: NCAOBW; ISSN:2041-1723. (Nature Research)Multicellular organisms rely on intercellular communication to coordinate the behavior of individual cells, which enables their differentiation and hierarchical organization. Various cell mimics have been developed to establish fundamental engineering principles for the construction of artificial cells displaying cell-like organization, behavior and complexity. However, collective phenomena, although of great importance for a better understanding of life-like behavior, are underexplored. Here, we construct collectives of giant vesicles that can communicate with each other through diffusing chem. signals that are recognized and processed by synthetic enzymic cascades. Similar to biol. cells, the Receiver vesicles can transduce a weak signal originating from Sender vesicles into a strong response by virtue of a signal amplification step, which facilitates the propagation of signals over long distances within the artificial cell consortia. This design advances the development of interconnected artificial cells that can exchange metabolic and positional information to coordinate their higher-order organization.
- 180Ji, Y.; Chakraborty, T.; Wegner, S. V. Self-regulated and bidirectional communication in synthetic cell communities. ACS Nano 2023, 17 (10), 8992– 9002, DOI: 10.1021/acsnano.2c09908Google Scholar180https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB3sXptlyltrY%253D&md5=20a75b01b8ea454dcc31f96dc900a1d1Self-Regulated and Bidirectional Communication in Synthetic Cell CommunitiesJi, Yuhao; Chakraborty, Taniya; Wegner, Seraphine V.ACS Nano (2023), 17 (10), 8992-9002CODEN: ANCAC3; ISSN:1936-0851. (American Chemical Society)Cell-to-cell communication is not limited to a sender releasing a signaling mol. and a receiver perceiving it but is often self-regulated and bidirectional. Yet, in communities of synthetic cells, such features that render communication efficient and adaptive are missing. Here, we report the design and implementation of adaptive two-way signaling with lipid-vesicle-based synthetic cells. The first layer of self-regulation derives from coupling the temporal dynamics of the signal, H2O2, prodn. in the sender to adhesions between sender and receiver cells. This way the receiver stays within the signaling range for the duration sender produces the signal and detaches once the signal fades. Specifically, H2O2 acts as both a forward signal and a regulator of the adhesions by activating photoswitchable proteins at the surface for the duration of the chemiluminescence. The second layer of self-regulation arises when the adhesions render the receiver permeable and trigger the release of a backward signal, resulting in bidirectional exchange. These design rules provide a concept for engineering multicellular systems with adaptive communication.
- 181Taylor, H.; Gao, N.; Mann, S. Chemical communication and protocell-matrix dynamics in segregated colloidosome micro-colonies. Angew. Chem., Int. Ed. 2023, 62 (24), e202300932, DOI: 10.1002/anie.202300932Google Scholar181https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB3sXpslOqsb8%253D&md5=4e838bb2964fb3423458d0acfb4d9a0fChemical Communication and Protocell-Matrix Dynamics in Segregated Colloidosome Micro-ColoniesTaylor, Hannah; Gao, Ning; Mann, StephenAngewandte Chemie, International Edition (2023), 62 (24), e202300932CODEN: ACIEF5; ISSN:1433-7851. (Wiley-VCH Verlag GmbH & Co. KGaA)Despite an emerging catalog of collective behaviors in communities of homogeneously distributed cell-like objects, microscale protocell colonies with spatially segregated populations have received minimal attention. Here, we use microfluidics to fabricate Janus-like calcium alginate hydrogel microspheres with spatially partitioned populations of enzyme-contg. inorg. colloidosomes and investigate their potential as integrated platforms for domain-mediated chem. communication and programmable protocell-matrix dynamics. Diffusive chem. signalling within the segregated communities gives rise to increased initial enzyme kinetics compared with a homogeneous distribution of protocells. We employ competing enzyme-mediated hydrogel crosslinking and decrosslinking reactions in different domains of the partitioned colonies to undertake selective expulsion of a specific protocell population from the community. Our results offer new possibilities for the design and construction of spatially organized cytomimetic consortia capable of endogenous chem. processing and protocell-environment interactivity.
- 182Fusi, G.; Del Giudice, D.; Skarsetz, O.; Di Stefano, S.; Walther, A. Autonomous soft robots empowered by chemical reaction networks. Adv. Mater. 2023, 35 (7), e2209870, DOI: 10.1002/adma.202209870Google Scholar182https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB38XjtFOmur7O&md5=245f47eab56714beed4e2585f71879feAutonomous Soft Robots Empowered by Chemical Reaction NetworksFusi, Giorgio; Del Giudice, Daniele; Skarsetz, Oliver; Di Stefano, Stefano; Walther, AndreasAdvanced Materials (Weinheim, Germany) (2023), 35 (7), 2209870CODEN: ADVMEW; ISSN:0935-9648. (Wiley-VCH Verlag GmbH & Co. KGaA)Hydrogel actuators are important for designing stimuli-sensitive soft robots. They generate mech. motion by exploiting compartmentalized (de)swelling in response to a stimulus. However, classical switching methods, such as manually lowering or increasing the pH, cannot provide more complex autonomous motions. By coupling an autonomously operating pH-flip with programmable lifetimes to a hydrogel system contg. pH-responsive and non-responsive compartments, autoonenomous forward and backward motion as well as more complex tasks, such as interlocking of "puzzle pieces" and collection of objects are realized. All operations are initiated by one simple trigger, and the devices operate in a "fire and forget" mode. More complex self-regulatory behavior is obtained by adding chemo-mechano-chemo feedback mechanisms. Due to its simplicity, this method shows great potential for the autonomous operation of soft grippers and metamaterials.
- 183Elani, Y.; Law, R. V.; Ces, O. Vesicle-based artificial cells as chemical microreactors with spatially segregated reaction pathways. Nat. Commun. 2014, 5, 5305, DOI: 10.1038/ncomms6305Google Scholar183https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2cXitVaksb%252FM&md5=9552fb140fc09ca105b5ba24007aff0bVesicle-based artificial cells as chemical microreactors with spatially segregated reaction pathwaysElani, Yuval; Law, Robert V.; Ces, OscarNature Communications (2014), 5 (), 5305CODEN: NCAOBW; ISSN:2041-1723. (Nature Publishing Group)In the discipline of bottom-up synthetic biol., vesicles define the boundaries of artificial cells and are increasingly being used as biochem. microreactors operating in physiol. environments. As the field matures, there is a need to compartmentalize processes in different spatial localities within vesicles, and for these processes to interact with one another. Here we address this by designing and constructing multi-compartment vesicles within which an engineered multi-step enzymic pathway is carried out. The individual steps are isolated in distinct compartments, and their products traverse into adjacent compartments with the aid of transmembrane protein pores, initiating subsequent steps. Thus, an engineered signalling cascade is recreated in an artificial cellular system. Importantly, by allowing different steps of a chem. pathway to be sepd. in space, this platform bridges the gap between table-top chem. and chem. that is performed within vesicles.
- 184Liu, S.; Zhang, Y.; He, X.; Li, M.; Huang, J.; Yang, X.; Wang, K.; Mann, S.; Liu, J. Signal processing and generation of bioactive nitric oxide in a model prototissue. Nat. Commun. 2022, 13 (1), 5254, DOI: 10.1038/s41467-022-32941-6Google Scholar184https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB38XitlGmu7rJ&md5=bbb2f20a4eb05bec0e3c200942cfd753Signal processing and generation of bioactive nitric oxide in a model prototissueLiu, Songyang; Zhang, Yanwen; He, Xiaoxiao; Li, Mei; Huang, Jin; Yang, Xiaohai; Wang, Kemin; Mann, Stephen; Liu, JianboNature Communications (2022), 13 (1), 5254CODEN: NCAOBW; ISSN:2041-1723. (Nature Portfolio)The design and construction of synthetic prototissues from integrated assemblies of artificial protocells is an important challenge for synthetic biol. and bioengineering. Here we spatially segregate chem. communicating populations of enzyme-decorated phospholipid-enveloped polymer/DNA coacervate protocells in hydrogel modules to construct a tubular prototissue-like vessel capable of modulating the output of bioactive nitric oxide (NO). By decorating the protocells with glucose oxidase, horseradish peroxidase or catalase and arranging different modules concentrically, a glucose/hydroxyurea dual input leads to logic-gate signal processing under reaction-diffusion conditions, which results in a distinct NO output in the internal lumen of the model prototissue. The NO output is exploited to inhibit platelet activation and blood clot formation in samples of plasma and whole blood located in the internal channel of the device, thereby demonstrating proof-of-concept use of the prototissue-like vessel for anticoagulation applications. Our results highlight opportunities for the development of spatially organized synthetic prototissue modules from assemblages of artificial protocells and provide a step towards the organization of biochem. processes in integrated micro-compartmentalized media, micro-reactor technol. and soft functional materials.
- 185Sengupta, S.; Patra, D.; Ortiz-Rivera, I.; Agrawal, A.; Shklyaev, S.; Dey, K. K.; Cordova-Figueroa, U.; Mallouk, T. E.; Sen, A. Self-powered enzyme micropumps. Nat. Chem. 2014, 6 (5), 415– 422, DOI: 10.1038/nchem.1895Google Scholar185https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2cXltFOmsbk%253D&md5=1284c8170f2970206bed7455783c9a08Self-powered enzyme micropumpsSengupta, Samudra; Patra, Debabrata; Ortiz-Rivera, Isamar; Agrawal, Arjun; Shklyaev, Sergey; Dey, Krishna K.; Cordova-Figueroa, Ubaldo; Mallouk, Thomas E.; Sen, AyusmanNature Chemistry (2014), 6 (5), 415-422CODEN: NCAHBB; ISSN:1755-4330. (Nature Publishing Group)Non-mech. nano- and microscale pumps that function without the aid of an external power source and provide precise control over the flow rate in response to specific signals are needed for the development of new autonomous nano- and microscale systems. Here we show that surface-immobilized enzymes that are independent of ATP function as self-powered micropumps in the presence of their resp. substrates. In the four cases studied (catalase, lipase, urease and glucose oxidase), the flow is driven by a gradient in fluid d. generated by the enzymic reaction. The pumping velocity increases with increasing substrate concn. and reaction rate. These rechargeable pumps can be triggered by the presence of specific analytes, which enables the design of enzyme-based devices that act both as sensor and pump. Finally, we show proof-of-concept enzyme-powered devices that autonomously deliver small mols. and proteins in response to specific chem. stimuli, including the release of insulin in response to glucose.
- 186Maiti, S.; Shklyaev, O. E.; Balazs, A. C.; Sen, A. Self-organization of fluids in a multienzymatic pump system. Langmuir 2019, 35 (10), 3724– 3732, DOI: 10.1021/acs.langmuir.8b03607Google Scholar186https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC1MXisFyit7c%253D&md5=0cea9a84182475fa2413607c984bd86dSelf-organization of fluids in a multienzymatic pump systemMaiti, Subhabrata; Shklyaev, Oleg E.; Balazs, Anna C.; Sen, AyusmanLangmuir (2019), 35 (10), 3724-3732CODEN: LANGD5; ISSN:0743-7463. (American Chemical Society)The nascent field of microscale flow chem. focuses on harnessing flowing fluids to optimize chem. reactions in microchambers and establish new routes for chem. synthesis. With enzymes and other catalysts anchored to the surface of microchambers, the catalytic reactions can act as pumps and propel the fluids through the containers. Hence, the flows not only affect the catalytic reactions, but these reactions also affect the flows. Understanding this dynamic interplay is vital to enhancing the accuracy and utility of flow technol. Through expts. and simulation, we design a system of three different enzymes, immobilized in sep. gels, on the surface of a microchamber; with the appropriate reactants in the soln., each enzyme-filled gel acts as a pump. The system also exploits a reaction cascade that controls the temporal interactions between two pumps. With three pumps in a triangular arrangement, the spatio-temporal interactions among the chem. reactions become highly coordinated and produce well-defined fluid streams, which transport chems. and form a fluidic "circuit". The circuit layout and flow direction of each constituent stream can be controlled through the no. and placement of the gels and the types of catalysts localized in the gels. These studies provide a new route for forming self-organizing and bifurcating fluids that can yield fundamental insight into nonequil., dynamical systems. Because the flows and fluidic circuits are generated by internal chem. reactions, the fluids can autonomously transport cargo to specific locations in the device. Hence, the findings also provide guidelines to facilitate further automation of microfluidic devices.
- 187Ortiz-Rivera, I.; Courtney, T. M.; Sen, A. Enzyme micropump-based inhibitor assays. Adv. Funct. Mater. 2016, 26, 2135– 2142, DOI: 10.1002/adfm.201504619Google Scholar187https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC28XivVCls70%253D&md5=8c6b301f04b8ef1825eab2ed3ddc04abEnzyme Micropump-Based Inhibitor AssaysOrtiz-Rivera, Isamar; Courtney, Taylor M.; Sen, AyusmanAdvanced Functional Materials (2016), 26 (13), 2135-2142CODEN: AFMDC6; ISSN:1616-301X. (Wiley-VCH Verlag GmbH & Co. KGaA)Rapid, easy-to-use, and portable devices that can provide a read-out without the need for expensive equipment represent the future of sensing technol., with applications in areas like environmental, food, chem., and biol. safety. Enzymes immobilized on a surface function as micropumps in the presence of species (e.g., substrate, cofactor, or biomarker) that trigger the enzymic reaction. The flow speed in these devices increases with increasing reaction rate. This allows the detection of substances that inhibit the enzymic reaction. Using this principle, sensors for toxic substances, like mercury, cadmium, cyanide, and azide, were designed using urease and catalase-powered pumps, resp., with limits of detection well below the concns. permitted by the Environmental Protection Agency. The study was also extended to other inhibitors for these enzymes. The sensing range of fluid flow-based inhibitor assays depends on the type of inhibition, the enzyme concn. on the sensing platform, and, for competitive inhibition, the concn. of substrate used.
- 188Manna, R. K.; Shklyaev, O. E.; Balazs, A. C. Chemically driven multimodal locomotion of active, flexible sheets. Langmuir 2023, 39 (2), 780– 789, DOI: 10.1021/acs.langmuir.2c02666Google Scholar188https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB3sXjtlSltg%253D%253D&md5=8634c838911bc4433cc43d24ff40b905Chemically Driven Multimodal Locomotion of Active, Flexible SheetsManna, Raj Kumar; Shklyaev, Oleg E.; Balazs, Anna C.Langmuir (2023), 39 (2), 780-789CODEN: LANGD5; ISSN:0743-7463. (American Chemical Society)The inhibitor-promoter feedback loop is a vital component in regulatory pathways that controls functionality in living systems. In this loop, the prodn. of chem. A at one site promotes the prodn. of chem. B at another site, but B inhibits the prodn. of A. In soln., differences in the vols. of the reactants and products of this reaction can generate buoyancy-driven fluid flows, which will deform neighboring soft material. To probe the intrinsic interrelationship among chem., hydrodynamics, and fluid-structure interactions, we model a bio-inspired system where a flexible sheet immersed in soln. encompasses two spatially sepd. catalytic patches, which drive the A-B inhibitor-promotor reaction. The convective rolls of fluid generated above the patches can circulate inward or outward depending on the chem. environment. Within the regime displaying chem. oscillations, the dynamic fluid-structure interactions morph the shape of the sheet to periodically "fly", "crawl", or "swim" along the bottom of the confining chamber, revealing an intimate coupling between form and function in this system. The oscillations in the sheet's motion in turn affect the chem. oscillations in the soln. In the regime with non-oscillatory chem., the induced flow still morphs the shape of the sheet, but now, the fluid simply translates the sheet along the length of the chamber. The findings reveal the potential for enzymic reactions in the body to generate hydrodynamic behavior that modifies the shape of neighboring soft tissue, which in turn modifies both the fluid dynamics and the enzymic reaction. The findings indicate that this non-linear dynamic behavior can be playing a crit. role in the functioning of regulatory pathways in living systems.
- 189Simo, C.; Serra-Casablancas, M.; Hortelao, A. C.; Di Carlo, V.; Guallar-Garrido, S.; Plaza-Garcia, S.; Rabanal, R. M.; Ramos-Cabrer, P.; Yague, B.; Aguado, L.; Bardia, L.; Tosi, S.; Gomez-Vallejo, V.; Martin, A.; Patino, T.; Julian, E.; Colombelli, J.; Llop, J.; Sanchez, S. Urease-powered nanobots for radionuclide bladder cancer therapy. Nat. Nanotechnol. 2024, DOI: 10.1038/s41565-023-01577-y .Google ScholarThere is no corresponding record for this reference.
- 190Rollin, J. A.; Tam, T. K.; Zhang, Y. H. P. New biotechnology paradigm: cell-free biosystems for biomanufacturing. Green Chem. 2013, 15 (7), 1708– 1719, DOI: 10.1039/c3gc40625cGoogle Scholar190https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3sXpvVejtrg%253D&md5=36c724ad829eccb7f6b178b2949dad90New biotechnology paradigm: cell-free biosystems for biomanufacturingRollin, Joseph A.; Tam, Tsz Kin; Zhang, Y.-H. PercivalGreen Chemistry (2013), 15 (7), 1708-1719CODEN: GRCHFJ; ISSN:1463-9262. (Royal Society of Chemistry)A review. Cost-efficient prodn. of sustainably-derived biochems. and biofuels is a crit. goal of modern biotechnol. The current predominant biotransformation method is mainly based on microbial fermn., but this system suffers from a mismatch of engineering and cellular objectives, appropriates a significant fraction of substrate/energy sources for self-replication, and poses significant scaling challenges. Cell-free biosystems for biomanufg. (CFB2)-complex, cell-free systems that catalyze the conversion of renewable substrates to a variety of products-are emerging as an alternative to fermn. Within this burgeoning field, a new application is the prodn. of low-value and high-impact biocommodities by CFB2. In this subset, synthetic enzymic networks are capable of producing numerous desired biocommodities, with the advantages of higher product yields, faster reaction rates, and reduced interference from toxic compds., among others. In this review, CFB2, with an emphasis on biocommodities prodn., is compared with microbial fermn.; current applications are presented, illustrating some of the advantages of the system; and remaining challenges are discussed with the path forward for each.
- 191Zhang, Y.; Hess, H. Toward rational design of high-efficiency enzyme cascades. ACS Catal. 2017, 7 (9), 6018– 6027, DOI: 10.1021/acscatal.7b01766Google Scholar191https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2sXht1eks7zI&md5=ee1f1ce70682bace08e88f77ec132b37Toward Rational Design of High-efficiency Enzyme CascadesZhang, Yifei; Hess, HenryACS Catalysis (2017), 7 (9), 6018-6027CODEN: ACCACS; ISSN:2155-5435. (American Chemical Society)A review. The construction of an enzyme cascade with enhanced activity is desirable in biocatalysis, synthetic biol. and other fields. Although many researchers have found that immobilization of enzyme cascades on scaffolds leads to an enhanced activity, the underlying mechanisms still remain controversial. In this Viewpoint, we describe and discuss the frequently used strategies to achieve activity enhancement. We first reiterate that the proximity does not contribute to the increased overall activity of the sequential nor coenzyme regenerating cascade, and suggest that the reported obsd. enhancements in the majority of publications were caused by the influence of the scaffolds. Then we discuss the benefits and limitations of other strategies including bridging the enzymes, co-immobilization, compartmentalization. Finally, we highlight that balancing the stoichiometry, improving the individual activity, overlapping the operating temp. and pH max. are necessary for achieving a high-efficiency enzyme cascade.
- 192Shi, J.; Wu, Y.; Zhang, S.; Tian, Y.; Yang, D.; Jiang, Z. Bioinspired construction of multi-enzyme catalytic systems. Chem. Soc. Rev. 2018, 47, 4295– 4313, DOI: 10.1039/C7CS00914CGoogle Scholar192https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC1cXptValtbo%253D&md5=c3794d904054aee96bf89e4e2c11392eBioinspired construction of multi-enzyme catalytic systemsShi, Jiafu; Wu, Yizhou; Zhang, Shaohua; Tian, Yu; Yang, Dong; Jiang, ZhongyiChemical Society Reviews (2018), 47 (12), 4295-4313CODEN: CSRVBR; ISSN:0306-0012. (Royal Society of Chemistry)A review. Enzyme catalysis, as a green, efficient process, displays exceptional functionality, adaptivity and sustainability. Multi-enzyme catalysis, which can accomplish the tandem synthesis of valuable materials/chems. from renewable feedstocks, establishes a bridge between single-enzyme catalysis and whole-cell catalysis. Multi-enzyme catalysis occupies a unique and indispensable position in the realm of biol. reactions for energy and environmental applications. Two complementary strategies, i.e., compartmentalization and substrate channeling, have been evolved by living organisms for implementing the complex in vivo multi-enzyme reactions (MERs), which have been applied to construct multi-enzyme catalytic systems (MECSs) with superior catalytic activity and stabilities in practical biocatalysis. This tutorial review aims to present the recent advances and future prospects in this burgeoning research area, stressing the features and applications of the two strategies for constructing MECSs and implementing in vitro MERs. The concluding remarks are presented with a perspective on the construction of MECSs through rational combination of compartmentalization and substrate channeling.
- 193Wang, Z.; Sundara Sekar, B.; Li, Z. Recent advances in artificial enzyme cascades for the production of value-added chemicals. Bioresour. Technol. 2021, 323, 124551, DOI: 10.1016/j.biortech.2020.124551Google Scholar193https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB3cXis12iu7zI&md5=c362ef01381c078529cd552465831127Recent advances in artificial enzyme cascades for the production of value-added chemicalsWang, Zilong; Sundara Sekar, Balaji; Li, ZhiBioresource Technology (2021), 323 (), 124551CODEN: BIRTEB; ISSN:0960-8524. (Elsevier Ltd.)A review. Enzyme cascades are efficient tools to perform multi-step synthesis in one-pot in a green and sustainable manner, enabling non-natural synthesis of valuable chems. from easily available substrates by artificially combining two or more enzymes. Bioprodn. of many high-value chems. such as chiral and highly functionalised mols. have been achieved by developing new enzyme cascades. This review summarizes recent advances on engineering and application of enzyme cascades to produce high-value chems. (alcs., aldehydes, ketones, amines, carboxylic acids, etc) from simple starting materials. While 2-step enzyme cascades are developed for versatile enantioselective synthesis, multi-step enzyme cascades are engineered to functionalise basic chems., such as styrenes, cyclic alkanes, and arom. compds. New cascade reactions have also been developed for producing valuable chems. from bio-based substrates, such as L-phenylalanine, and renewable feedstocks such as glucose and glycerol. The challenges in current process and future outlooks in the development of enzyme cascades are also addressed.
- 194Mordhorst, S.; Andexer, J. N. Round, round we go – strategies for enzymatic cofactor regeneration. Nat. Prod. Rep. 2020, 37, 1316– 1333, DOI: 10.1039/D0NP00004CGoogle Scholar194https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB3cXht1CksrrI&md5=17a3b901eeb97ba98e33b9e27dae7899Round, round we go - strategies for enzymatic cofactor regenerationMordhorst, Silja; Andexer, Jennifer N.Natural Product Reports (2020), 37 (10), 1316-1333CODEN: NPRRDF; ISSN:0265-0568. (Royal Society of Chemistry)Covering: up to the beginning of 2020Enzymes depending on cofactors are essential in many biosynthetic pathways of natural products. They are often involved in key steps: catalytic conversions that are difficult to achieve purely with synthetic org. chem. Hence, cofactor-dependent enzymes have great potential for biocatalysis, on the condition that a corresponding cofactor regeneration system is available. For some cofactors, these regeneration systems require multiple steps; such complex enzyme cascades/multi-enzyme systems are (still) challenging for in vitro biocatalysis. Further, artificial cofactor analogs have been synthesized that are more stable, show an altered reaction range, or act as inhibitors. The development of bio-orthogonal systems that can be used for the prodn. of modified natural products in vivo is an ongoing challenge. In light of the recent progress in this field, this review aims to provide an overview of general strategies involving enzyme cofactors, cofactor analogs, and regeneration systems; highlighting the current possibilities for application of enzymes using some of the most common cofactors.
- 195Bachosz, K.; Zdarta, J.; Bilal, M.; Meyer, A. S.; Jesionowski, T. Enzymatic cofactor regeneration systems: A new perspective on efficiency assessment. Sci. Total Environ. 2023, 868, 161630, DOI: 10.1016/j.scitotenv.2023.161630Google Scholar195https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB3sXhs1Onur0%253D&md5=3f105ac482e65f0bb8daa60544c860dcEnzymatic cofactor regeneration systems: A new perspective on efficiency assessmentBachosz, Karolina; Zdarta, Jakub; Bilal, Muhammad; Meyer, Anne S.; Jesionowski, TeofilScience of the Total Environment (2023), 868 (), 161630CODEN: STENDL; ISSN:0048-9697. (Elsevier B.V.)A review. Nowadays, the specificity of enzymic processes makes them more and more important every year, and their usage on an industrial scale seems to be necessary. Enzymic cofactors, however, play a crucial part in the prospective applications of enzymes, because they are indispensable for conducting highly effective biocatalytic activities. Due to the relatively high cost of these compds. and their consumption during the processes carried out, it has become crucial to develop systems for cofactor regeneration. Therefore, in this review, an attempt was made to summarize current knowledge on enzymic regeneration methods, which are characterized by high specificity, non-toxicity and reported to be highly efficient. The regeneration of cofactors, such as nicotinamide dinucleotides, CoA, ATP and flavin nucleotides, which are necessary for the proper functioning of a large no. of enzymes, is discussed, as well as potential directions for further development of these systems are highlighted. This review discusses a range of highly effective cofactor regeneration systems along with the productive synthesis of many useful chems., including the simultaneous renewal of several cofactors at the same time. Addnl., the impact of the enzyme immobilization process on improving the stability and the potential for multiple uses of the developed cofactor regeneration systems was also presented. Moreover, an attempt was made to emphasize the importance of the presented research, as well as the identification of research gaps, which mainly result from the lack of available literature on this topic.
- 196Monck, C.; Elani, Y.; Ceroni, F. Cell-free protein synthesis: biomedical applications and future perspectives. Chem. Eng. Res. Des. 2022, 177, 653– 658, DOI: 10.1016/j.cherd.2021.11.025Google Scholar196https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB3MXisF2gurnN&md5=6ffac1e510aba2ea50c266eaa681a4f5Cell-free protein synthesis: biomedical applications and future perspectivesMonck, Carolina; Elani, Yuval; Ceroni, FrancescaChemical Engineering Research and Design (2022), 177 (), 653-658CODEN: CERDEE; ISSN:1744-3563. (Elsevier B.V.)A review. The use of cell-free protein synthesis (CFPS) has become increasingly widespread in synthetic biol. over recent years, providing an effective platform for the study and engineering of cellular processes. The versatility and portability of CFPS systems have also boosted their potential for usage outside of the lab. in a wide no. of applications, from construct prototyping to bioprodn. CFPS is particularly well suited to biomedical applications, such as the prodn. of clin. mols. and vaccines. It can also be integrated with addnl. technologies such as microfluidics and liposomal encapsulation to provide a new route for on-demand therapeutic expression. In this we outline the key features of CFPS that make it a powerful platform for biomedical applications. We also discuss existing limitations with respect to the use of CFPS in the prodn. of complex protein products and the limited prodn. capacity of current systems. Addressing these will be integral in expanding the application of CFPS in biotherapy.
- 197Perez, J. G.; Stark, J. C.; Jewett, M. C. Cell-free synthetic biology: engineering beyond the cell. Cold Spring Harb. Perspect. Biol. 2016, 8 (12), a023853, DOI: 10.1101/cshperspect.a023853Google Scholar197https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC1cXhtFKms7bO&md5=7f222c69d86efff730fdafaaa5941469Cell-free synthetic biology: engineering beyond the cellPerez, Jessica G.; Stark, Jessica C.; Jewett, Michael C.Cold Spring Harbor Perspectives in Biology (2016), 8 (12), a023853/1-a023853/26CODEN: CSHPEU; ISSN:1943-0264. (Cold Spring Harbor Laboratory Press)Cell-free protein synthesis (CFPS) technologies have enabled inexpensive and rapid recombinant protein expression. Numerous highly active CFPS platforms are now available and have recently been used for synthetic biol. applications. In this review, we focus on the ability of CFPS to expand our understanding of biol. systems and its applications in the synthetic biol. field. First, we outline a variety of CFPS platforms that provide alternative and complementary methods for expressing proteins from different organisms, compared with in vivo approaches. Next, we review the types of proteins, protein complexes, and protein modifications that have been achieved using CFPS systems. Finally, we introduce recent work on genetic networks in cell-free systems and the use of cell-free systems for rapid prototyping of in vivo networks. Given the flexibility of cell-free systems, CFPS holds promise to be a powerful tool for synthetic biol. as well as a protein prodn. technol. in years to come.
- 198Silverman, A. D.; Karim, A. S.; Jewett, M. C. Cell-free gene expression: an expanded repertoire of applications. Nat. Rev. Genet. 2020, 21, 151– 170, DOI: 10.1038/s41576-019-0186-3Google Scholar198https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC1MXit1OitbbO&md5=e227ddcf47b0b961b2b423d414fa3795Cell-free gene expression: an expanded repertoire of applicationsSilverman, Adam D.; Karim, Ashty S.; Jewett, Michael C.Nature Reviews Genetics (2020), 21 (3), 151-170CODEN: NRGAAM; ISSN:1471-0056. (Nature Research)Cell-free biol. is the activation of biol. processes without the use of intact living cells. It has been used for more than 50 years across the life sciences as a foundational research tool, but a recent tech. renaissance has facilitated high-yielding (grams of protein per L), cell-free gene expression systems from model bacteria, the development of cell-free platforms from non-model organisms and multiplexed strategies for rapidly assessing biol. design. These advances provide exciting opportunities to profoundly transform synthetic biol. by enabling new approaches to the model-driven design of synthetic gene networks, the fast and portable sensing of compds., on-demand biomanufg., building cells from the bottom up, and next-generation educational kits.
- 199Korman, T. P.; Sahachartsiri, B.; Li, D.; Vinokur, J. M.; Eisenberg, D.; Bowie, J. U. A synthetic biochemistry system for the in vitro production of isoprene from glycolysis intermediates. Protein Sci. 2014, 23 (5), 576– 585, DOI: 10.1002/pro.2436Google Scholar199https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2cXmsFWls7o%253D&md5=1c21b2170ee9c47872e24879a980623eA synthetic biochemistry system for the in vitro production of isoprene from glycolysis intermediatesKorman, Tyler P.; Sahachartsiri, Bobby; Li, Dan; Vinokur, Jeffrey M.; Eisenberg, David; Bowie, James U.Protein Science (2014), 23 (5), 576-585CODEN: PRCIEI; ISSN:1469-896X. (Wiley-Blackwell)The high yields required for the economical prodn. of chems. and fuels using microbes can be difficult to achieve due to the complexities of cellular metab. An alternative to performing biochem. transformations in microbes is to build biochem. pathways in vitro, an approach we call synthetic biochem. Here we test whether the full mevalonate pathway can be reconstituted in vitro and used to produce the commodity chem. isoprene. We construct an in vitro synthetic biochem. pathway that uses the carbon and ATP produced from the glycolysis intermediate phosphoenolpyruvate to run the mevalonate pathway. The system involves 12 enzymes to perform the complex transformation, while providing and balancing the ATP, NADPH, and acetyl-CoA cofactors. The optimized system produces isoprene from phosphoenolpyruvate in ∼100% molar yield. Thus, by inserting the isoprene pathway into previously developed glycolysis modules it may be possible to produce isoprene and other acetyl-CoA derived isoprenoids from glucose in vitro.
- 200Korman, T. P.; Opgenorth, P. H.; Bowie, J. U. A synthetic biochemistry platform for cell free production of monoterpenes from glucose. Nat. Commun. 2017, 8, 15526, DOI: 10.1038/ncomms15526Google Scholar200https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2sXot12gs78%253D&md5=d792e214239beb42398f0d503db94bfeA synthetic biochemistry platform for cell free production of monoterpenes from glucoseKorman, Tyler P.; Opgenorth, Paul H.; Bowie, James U.Nature Communications (2017), 8 (), 15526CODEN: NCAOBW; ISSN:2041-1723. (Nature Publishing Group)Cell-free systems designed to perform complex chem. conversions of biomass to biofuels or commodity chems. are emerging as promising alternatives to the metabolic engineering of living cells. Here we design a system comprises 27 enzymes for the conversion of glucose into monoterpenes that generates both NAD(P)H and ATP in a modified glucose breakdown module and utilizes both cofactors for building terpenes. Different monoterpenes are produced in our system by changing the terpene synthase enzyme. The system is stable for the prodn. of limonene, pinene and sabinene, and can operate continuously for at least 5 days from a single addn. of glucose. We obtain conversion yields >95% and titers >15 g l-1. The titers are an order of magnitude over cellular toxicity limits and thus difficult to achieve using cell-based systems. Overall, these results highlight the potential of synthetic biochem. approaches for producing bio-based chems.
- 201Opgenorth, P. H.; Korman, T. P.; Bowie, J. U. A synthetic biochemistry molecular purge valve module that maintains redox balance. Nat. Commun. 2014, 5, 4113, DOI: 10.1038/ncomms5113Google Scholar201https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2cXitVShsb%252FE&md5=030c8d73c345febb8aebb33914fc80b4A synthetic biochemistry molecular purge valve module that maintains redox balanceOpgenorth, Paul H.; Korman, Tyler P.; Bowie, James U.Nature Communications (2014), 5 (), 4113CODEN: NCAOBW; ISSN:2041-1723. (Nature Publishing Group)The greatest potential environmental benefit of metabolic engineering would be the prodn. of high-vol. commodity chems., such as biofuels. Yet, the high yields required for the economic viability of low-value chems. is particularly hard to achieve in microbes owing to the myriad competing biochem. pathways. An alternative approach, which we call synthetic biochem., is to eliminate the organism by constructing biochem. pathways in vitro. Viable synthetic biochem., however, will require simple methods to replace the cellular circuitry that maintains cofactor balance. Here we design a simple purge valve module for maintaining NADP+/NADPH balance. We test the purge valve in the prodn. of polyhydroxybutyryl bioplastic and isoprene-pathways where cofactor generation and utilization are unbalanced. We find that the regulatory system is highly robust to variations in cofactor levels and readily transportable. The mol. purge valve provides a step towards developing continuously operating, sustainable synthetic biochem. systems.
- 202Opgenorth, P. H.; Korman, T. P.; Bowie, J. U. A synthetic biochemistry module for production of bio-based chemicals from glucose. Nat. Chem. Biol. 2016, 12 (6), 393– 395, DOI: 10.1038/nchembio.2062Google Scholar202https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC28XlsFOhsL8%253D&md5=813355b68b089d81528532dc2f004b91A synthetic biochemistry module for production of bio-based chemicals from glucoseOpgenorth, Paul H.; Korman, Tyler P.; Bowie, James U.Nature Chemical Biology (2016), 12 (6), 393-395CODEN: NCBABT; ISSN:1552-4450. (Nature Publishing Group)Synthetic biochem., the cell-free prodn. of biol. based chems., is a potentially high-yield, flexible alternative to in vivo metabolic engineering. To limit costs, cell-free systems must be designed to operate continuously with minimal addn. of feedstock chems. We describe a robust, efficient synthetic glucose breakdown pathway and implement it for the prodn. of bioplastic. The system's performance suggests that synthetic biochem. has the potential to become a viable industrial alternative.
- 203Valliere, M. A.; Korman, T. P.; Woodall, N. B.; Khitrov, G. A.; Taylor, R. E.; Baker, D.; Bowie, J. U. A cell-free platform for the prenylation of natural products and application to cannabinoid production. Nat. Commun. 2019, 10 (1), 565, DOI: 10.1038/s41467-019-08448-yGoogle Scholar203https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC1MXntlKktL8%253D&md5=e43f4406f322ca8307256c80a4c961cdA cell-free platform for the prenylation of natural products and application to cannabinoid productionValliere, Meaghan A.; Korman, Tyler P.; Woodall, Nicholas B.; Khitrov, Gregory A.; Taylor, Robert E.; Baker, David; Bowie, James U.Nature Communications (2019), 10 (1), 565CODEN: NCAOBW; ISSN:2041-1723. (Nature Research)Prenylation of natural compds. adds structural diversity, alters biol. activity, and enhances therapeutic potential. Because prenylated compds. often have a low natural abundance, alternative prodn. methods are needed. Metabolic engineering enables natural product biosynthesis from inexpensive biomass, but is limited by the complexity of secondary metabolite pathways, intermediate and product toxicities, and substrate accessibility. Alternatively, enzyme catalyzed prenyl transfer provides excellent regio- and stereo-specificity, but requires expensive isoprenyl pyrophosphate substrates. Here we develop a flexible cell-free enzymic prenylating system that generates isoprenyl pyrophosphate substrates from glucose to prenylate an array of natural products. The system provides an efficient route to cannabinoid precursors cannabigerolic acid (CBGA) and cannabigerovarinic acid (CBGVA) at >1 g/L, and a single enzymic step converts the precursors into cannabidiolic acid (CBDA) and cannabidivarinic acid (CBDVA). Cell-free methods may provide a powerful alternative to metabolic engineering for chems. that are hard to produce in living organisms.
- 204Sarria, S.; Wong, B.; Martin, H. G.; Keasling, J. D.; Peralta-Yahya, P. Microbial synthesis of pinene. ACS Synth. Biol. 2014, 3 (7), 466– 475, DOI: 10.1021/sb4001382Google Scholar204https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2cXjt1ymsro%253D&md5=b83fc0bf6e20fdedef4cb039eb49ed86Microbial Synthesis of PineneSarria, Stephen; Wong, Betty; Martin, Hector Garcia; Keasling, Jay D.; Peralta-Yahya, PamelaACS Synthetic Biology (2014), 3 (7), 466-475CODEN: ASBCD6; ISSN:2161-5063. (American Chemical Society)The volumetric heating values of today's biofuels are too low to power energy-intensive aircraft, rockets, and missiles. Recently, pinene dimers were shown to have a volumetric heating value similar to that of the tactical fuel JP-10. To provide a sustainable source of pinene, we engineered Escherichia coli for pinene prodn. We combinatorially expressed three pinene synthases (PS) and three geranyl diphosphate synthases (GPPS), with the best combination achieving ∼28 mg/L of pinene. We speculated that pinene toxicity was limiting prodn.; however, toxicity should not be limiting at current titers. Because GPPS is inhibited by geranyl diphosphate (GPP) and to increase flux through the pathway, we combinatorially constructed GPPS-PS protein fusions. The Abies grandis GPPS-PS fusion produced 32 mg/L of pinene, a 6-fold improvement over the highest titer previously reported in engineered E. coli. Finally, we investigated the pinene isomer ratio of our pinene-producing microbe and discovered that the isomer profile is detd. not only by the identity of the PS used but also by the identity of the GPPS with which the PS is paired. We demonstrated that the GPP concn. available to PS for cyclization alters the pinene isomer ratio.
- 205Karim, A. S.; Jewett, M. C. A cell-free framework for rapid biosynthetic pathway prototyping and enzyme discovery. Metab. Eng. 2016, 36, 116– 126, DOI: 10.1016/j.ymben.2016.03.002Google Scholar205https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC28Xls1KrsL8%253D&md5=6d833a0a1996ede9015197d1b48d5793A cell-free framework for rapid biosynthetic pathway prototyping and enzyme discoveryKarim, Ashty S.; Jewett, Michael C.Metabolic Engineering (2016), 36 (), 116-126CODEN: MEENFM; ISSN:1096-7176. (Elsevier B. V.)Speeding up design-build-test (DBT) cycles is a fundamental challenge facing biochem. engineering. To address this challenge, we report a new cell-free protein synthesis driven metabolic engineering (CFPS-ME) framework for rapid biosynthetic pathway prototyping. In our framework, cell-free cocktails for synthesizing target small mols. are assembled in a mix-and-match fashion from crude cell lysates either contg. selectively enriched pathway enzymes from heterologous overexpression or directly producing pathway enzymes in lysates by CFPS. As a model, we apply our approach to n-butanol biosynthesis showing that Escherichia coli lysates support a highly active 17-step CoA-dependent n-butanol pathway in vitro. The elevated degree of flexibility in the cell-free environment allows us to manipulate physiochem. conditions, access enzymic nodes, discover new enzymes, and prototype enzyme sets with linear DNA templates to study pathway performance. We anticipate that CFPS-ME will facilitate efforts to define, manipulate, and understand metabolic pathways for accelerated DBT cycles without the need to reengineer organisms.
- 206Dudley, Q. M.; Anderson, K. C.; Jewett, M. C. Cell-free mixing of Escherichia coli crude extracts to prototype and rationally engineer high-titer mevalonate synthesis. ACS Synth. Biol. 2016, 5 (12), 1578– 1588, DOI: 10.1021/acssynbio.6b00154Google Scholar206https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC28Xht1GmtrbK&md5=c45307dfb3c6873a53e0711d454aba58Cell-Free Mixing of Escherichia coli Crude Extracts to Prototype and Rationally Engineer High-Titer Mevalonate SynthesisDudley, Quentin M.; Anderson, Kim C.; Jewett, Michael C.ACS Synthetic Biology (2016), 5 (12), 1578-1588CODEN: ASBCD6; ISSN:2161-5063. (American Chemical Society)Cell-free metabolic engineering (CFME) is advancing a powerful paradigm for accelerating the design and synthesis of biosynthetic pathways. However, as most cell-free biomol. synthesis systems to date use purified enzymes, energy and cofactor balance can be limiting. To address this challenge, we report a new CFME framework for building biosynthetic pathways by mixing multiple crude lysates, or exts. In our modular approach, cell-free lysates, each selectively enriched with an overexpressed enzyme, are generated in parallel and then combinatorically mixed to construct a full biosynthetic pathway. Endogenous enzymes in the cell-free ext. fuel high-level energy and cofactor regeneration. As a model, we apply our framework to synthesize mevalonate, an intermediate in isoprenoid synthesis. We use our approach to rapidly screen enzyme variants, optimize enzyme ratios, and explore cofactor landscapes for improving pathway performance. Further, we show that genomic deletions in the source strain redirect metabolic flux in resultant lysates. In an optimized system, mevalonate was synthesized at 17.6 g·L-1 (119 mM) over 20 h, resulting in a volumetric productivity of 0.88 g·L-1·hr-1. We also demonstrate that this system can be lyophilized and retain biosynthesis capability. Our system catalyzes ∼1250 turnover events for the cofactor NAD+ and demonstrates the ability to rapidly prototype and debug enzymic pathways in vitro for compelling metabolic engineering and synthetic biol. applications.
- 207Casini, A.; Chang, F. Y.; Eluere, R.; King, A. M.; Young, E. M.; Dudley, Q. M.; Karim, A.; Pratt, K.; Bristol, C.; Forget, A. A pressure test to make 10 molecules in 90 days: external evaluation of methods to engineer biology. J. Am. Chem. Soc. 2018, 140 (12), 4302– 4316, DOI: 10.1021/jacs.7b13292Google Scholar207https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC1cXjt1yls78%253D&md5=9b0d5effbfa11528fb82894e7f59b5b3A Pressure Test to Make 10 Molecules in 90 Days: External Evaluation of Methods to Engineer BiologyCasini, Arturo; Chang, Fang-Yuan; Eluere, Raissa; King, Andrew M.; Young, Eric M.; Dudley, Quentin M.; Karim, Ashty; Pratt, Katelin; Bristol, Cassandra; Forget, Anthony; Ghodasara, Amar; Warden-Rothman, Robert; Gan, Rui; Cristofaro, Alexander; Borujeni, Amin Espah; Ryu, Min-Hyung; Li, Jian; Kwon, Yong-Chan; Wang, He; Tatsis, Evangelos; Rodriguez-Lopez, Carlos; O'Connor, Sarah; Mdema, Marnix H.; Fischbach, Michael A.; Jewett, Michael C.; Voigt, Christopher; Gordon, D. BenjaminJournal of the American Chemical Society (2018), 140 (12), 4302-4316CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)Centralized facilities for genetic engineering, or biofoundries, offer the potential to design organisms to address emerging needs in medicine, agriculture, industry, and defense. The field has seen rapid advances in technol., but it is difficult to gauge current capabilities or identify gaps across projects. To this end, our foundry was assessed via a timed "pressure test," in which 3 mo were given to build organisms to produce 10 mols. unknown to us in advance. By applying a diversity of new approaches, we produced the desired mol. or a closely related one for 6/10 targets during the performance period, and made advances toward prodn. of the others as well. Specifically, we increased the titers of 1-hexadecanol, pyrrolnitrin, and pacidamycin D, found novel routes to the enediyne warhead underlying powerful antimicrobials, established a cell free system for monoterpene prodn., produced an intermediate toward vincristine biosynthesis, and encoded 7,802 individually retrievable pathways to 540 bisindoles in a DNA pool. Pathways to THF and barbamide were designed and constructed but toxicity or anal. tools inhibited further progress. In sum, we constructed 1.2Mb DNA, built 215 strains spanning 5 species (Saccharomyces cerevisiae, Escherichia coli, Streptomyces albidoflavus, Streptomyces coelicolor, and Streptomyces albovinaceus), established two cell free systems, and performed 690 assays developed inhouse for the mols.
- 208Dudley, Q. M.; Nash, C. J.; Jewett, M. C. Cell-free biosynthesis of limonene using enzyme-enriched Escherichia coli lysates. Synthetic Biology 2019, 4 (1), ysz003, DOI: 10.1093/synbio/ysz003Google ScholarThere is no corresponding record for this reference.
- 209Nielsen, D. U.; Hu, X.-M.; Daasbjerg, K.; Skrydstrup, T. Chemically and electrochemically catalysed conversion of CO2 to CO with follow-up utilization to value-added chemicals. Nat. Catal. 2018, 1 (4), 244– 254, DOI: 10.1038/s41929-018-0051-3Google Scholar209https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC1MXpvVyrtbc%253D&md5=beeabaa45d07b5cfb0d698e643c88949Chemically and electrochemically catalysed conversion of CO2 to CO with follow-up utilization to value-added chemicalsNielsen, Dennis U.; Hu, Xin-Ming; Daasbjerg, Kim; Skrydstrup, TroelsNature Catalysis (2018), 1 (4), 244-254CODEN: NCAACP; ISSN:2520-1158. (Nature Research)A review. Carbon dioxide is ubiquitous and a vital mol. for maintaining life on our planet. However, the ever-increasing emission of anthropogenic CO2 into our atm. has provoked dramatic climate changes. In principle, CO2 could represent an important one-carbon building block for the chem. industry, yet its high thermodn. and kinetic stability has limited its applicability to only a handful of industrial applications. On the other hand, carbon monoxide represents a more versatile reagent applied in many industrial transformations. Here we review the different methods for converting CO2 to CO with specific focus on the reverse water gas shift reaction, main element reductants, and electrochem. protocols applying homogeneous and heterogeneous catalysts. Particular emphasis is given to synthetic methods that couple the deoxygenation step with a follow-up carbonylation step for the synthesis of carbonyl-contg. mols., thus avoiding the need to handle or store this toxic but highly synthetically useful diat. gas.
- 210Long, S. P.; Ainsworth, E. A.; Rogers, A.; Ort, D. R. Rising atmospheric carbon dioxide: plants FACE the future. Annu. Rev. Plant Biol. 2004, 55, 591– 628, DOI: 10.1146/annurev.arplant.55.031903.141610Google Scholar210https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD2cXlvFeisb8%253D&md5=b69cfb74635972e8d2139f7d53022c20Rising atmospheric carbon dioxide: Plants FACE the futureLong, Stephen P.; Ainsworth, Elizabeth A.; Rogers, Alistair; Ort, Donald R.Annual Review of Plant Biology (2004), 55 (), 591-628, 3 plates C1-C3CODEN: ARPBDW ISSN:. (Annual Reviews Inc.)A review. Atm. CO2 concn. ([CO2]) is now higher than it was at any time in the past 26 million years and is expected to nearly double during this century. Terrestrial plants with the C3 photosynthetic pathway respond in the short term to increased [CO2] via increased net photosynthesis and decreased transpiration. In the longer term, this increase is often offset by downregulation of photosynthetic capacity. But much of what is currently known about plant responses to elevated [CO2] comes from enclosure studies, where the responses of plants may be modified by size constraints and the limited life-cycle stages that are examd. Free-Air CO2 Enrichment (FACE) was developed as a means to grow plants in the field at controlled elevation of CO2 under fully open-air field conditions. The findings of FACE expts. are quant. summarized via meta-analytic statistics and compared to findings from chamber studies. Although trends agree with parallel summaries of enclosure studies, important quant. differences emerge that have important implications both for predicting the future terrestrial biosphere and understanding how crops may need to be adapted to the changed and changing atm.
- 211Spreitzer, R. J.; Salvucci, M. E. Rubisco: structure, regulatory interactions, and possibilities for a better enzyme. Annu. Rev. Plant Biol. 2002, 53, 449– 475, DOI: 10.1146/annurev.arplant.53.100301.135233Google Scholar211https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD38XlsVWhurk%253D&md5=1dd37bb9e88ffe399e5a2458aa966401Rubisco: Structure, regulatory interactions, and possibilities for a better enzymeSpreitzer, Robert J.; Salvucci, Michael E.Annual Review of Plant Biology (2002), 53 (), 449-475CODEN: ARPBDW ISSN:. (Annual Reviews Inc.)A review with 159 refs. Ribulose 1,5-bisphosphate (RuBP) carboxylase/oxygenase (Rubisco) catalyzes the 1st step in net photosynthetic CO2 assimilation and photorespiratory C oxidn. The enzyme is notoriously inefficient as a catalyst for the carboxylation of RuBP and is subject to competitive inhibition by O2, inactivation by loss of carbamylation, and dead-end inhibition by RuBP. These inadequacies make Rubisco rate-limiting for photosynthesis and an obvious target for increasing agricultural productivity. The resoln. of x-ray crystal structures and detailed anal. of divergent, mutant, and hybrid enzymes have increased insight into the structure-function relations of Rubisco. The interactions and assocns. relatively far from the Rubisco active site, including regulatory interactions with Rubisco activase, may present new approaches and strategies for understanding and ultimately improving this complex enzyme.
- 212Könneke, M.; Schubert, D. M.; Brown, P. C.; Hugler, M.; Standfest, S.; Schwander, T.; Schada von Borzyskowski, L.; Erb, T. J.; Stahl, D. A.; Berg, I. A. Ammonia-oxidizing archaea use the most energy-efficient aerobic pathway for CO2 fixation. Proc. Natl. Acad. Sci. U. S. A. 2014, 111 (22), 8239– 8244, DOI: 10.1073/pnas.1402028111Google Scholar212https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A280%3ADC%252BC2cjktFylsA%253D%253D&md5=3fe1a4dedd0699d28106d30cd201239aAmmonia-oxidizing archaea use the most energy-efficient aerobic pathway for CO2 fixationKonneke Martin; Schubert Daniel M; Brown Philip C; Berg Ivan A; Hugler Michael; Standfest Sonja; Schwander Thomas; Schada von Borzyskowski Lennart; Erb Tobias J; Stahl David AProceedings of the National Academy of Sciences of the United States of America (2014), 111 (22), 8239-44 ISSN:.Archaea of the phylum Thaumarchaeota are among the most abundant prokaryotes on Earth and are widely distributed in marine, terrestrial, and geothermal environments. All studied Thaumarchaeota couple the oxidation of ammonia at extremely low concentrations with carbon fixation. As the predominant nitrifiers in the ocean and in various soils, ammonia-oxidizing archaea contribute significantly to the global nitrogen and carbon cycles. Here we provide biochemical evidence that thaumarchaeal ammonia oxidizers assimilate inorganic carbon via a modified version of the autotrophic hydroxypropionate/hydroxybutyrate cycle of Crenarchaeota that is far more energy efficient than any other aerobic autotrophic pathway. The identified genes of this cycle were found in the genomes of all sequenced representatives of the phylum Thaumarchaeota, indicating the environmental significance of this efficient CO2-fixation pathway. Comparative phylogenetic analysis of proteins of this pathway suggests that the hydroxypropionate/hydroxybutyrate cycle emerged independently in Crenarchaeota and Thaumarchaeota, thus supporting the hypothesis of an early evolutionary separation of both archaeal phyla. We conclude that high efficiency of anabolism exemplified by this autotrophic cycle perfectly suits the lifestyle of ammonia-oxidizing archaea, which thrive at a constantly low energy supply, thus offering a biochemical explanation for their ecological success in nutrient-limited environments.
- 213Scheffen, M.; Marchal, D. G.; Beneyton, T.; Schuller, S. K.; Klose, M.; Diehl, C.; Lehmann, J.; Pfister, P.; Carrillo, M.; He, H. A new-to-nature carboxylation module to improve natural and synthetic CO2 fixation. Nat. Catal. 2021, 4 (2), 105– 115, DOI: 10.1038/s41929-020-00557-yGoogle Scholar213https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB3MXhtFKjsLrE&md5=7f64a1dfca369fd9ca39c4c356a5034eA new-to-nature carboxylation module to improve natural and synthetic CO2 fixationScheffen, Marieke; Marchal, Daniel G.; Beneyton, Thomas; Schuller, Sandra K.; Klose, Melanie; Diehl, Christoph; Lehmann, Jessica; Pfister, Pascal; Carrillo, Martina; He, Hai; Aslan, Selcuk; Cortina, Nina S.; Claus, Peter; Bollschweiler, Daniel; Baret, Jean-Christophe; Schuller, Jan M.; Zarzycki, Jan; Bar-Even, Arren; Erb, Tobias J.Nature Catalysis (2021), 4 (2), 105-115CODEN: NCAACP; ISSN:2520-1158. (Nature Research)The capture of CO2 by carboxylases is key to sustainable biocatalysis and a carbon-neutral bio-economy, yet currently limited to few naturally existing enzymes. Here, we developed glycolyl-CoA carboxylase (GCC), a new-to-nature enzyme, by combining rational design, high-throughput microfluidics and microplate screens. During this process, GCC's catalytic efficiency improved by three orders of magnitude to match the properties of natural CO2-fixing enzymes. We verified our active-site redesign with an at.-resoln., 1.96-Å cryo-electron microscopy structure and engineered two more enzymes that, together with GCC, form a carboxylation module for the conversion of glycolate (C2) to glycerate (C3). We demonstrate how this module can be interfaced with natural photorespiration, ethylene glycol conversion and synthetic CO2 fixation. Based on stoichiometrical calcns., GCC is predicted to increase the carbon efficiency of all of these processes by up to 150% while reducing their theor. energy demand, showcasing how expanding the soln. space of natural metab. provides new opportunities for biotechnol. and agriculture.
- 214Schwander, T.; Schada von Borzyskowski, L.; Burgener, S.; Cortina, N. S.; Erb, T. J. A synthetic pathway for the fixation of carbon dioxide in vitro. Science 2016, 354, 900– 904, DOI: 10.1126/science.aah5237Google Scholar214https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC28XhvFGrtb%252FM&md5=eb266bffa55c2422c2ee78f1be4f2d22A synthetic pathway for the fixation of carbon dioxide in vitroSchwander, Thomas; Schada von Borzyskowski, Lennart; Burgener, Simon; Cortina, Nina Socorro; Erb, Tobias J.Science (Washington, DC, United States) (2016), 354 (6314), 900-904CODEN: SCIEAS; ISSN:0036-8075. (American Association for the Advancement of Science)Carbon dioxide (CO2) is an important carbon feedstock for a future green economy. This requiresthe development of efficient strategies for its conversion into multicarbon compds. We describe a synthetic cycle for the continuous fixation of CO2 in vitro. The crotonyl-CoA (CoA)/ethylmalonyl-CoA/hydroxybutyryl-CoA (CETCH) cycle is a reaction network of 17 enzymes that converts CO2 into org. mols. at a rate of 5 nmol of CO2 per min per mg of protein. The CETCH cycle was drafted by metabolic retrosynthesis, established with enzymes originating from nine different organisms of all three domains of life, and optimizedin several rounds by enzyme engineering and metabolic proofreading. The CETCH cycle adds a seventh, synthetic alternative to the six naturally evolved CO2 fixation pathways, thereby openingthe way for in vitro and in vivo applications.
- 215Gale, N. L.; Beck, J. V. Evidence for the Calvin cycle and hexose monophosphate pathway in Thiobacillus ferrooxidans. J. Bacteriol. 1967, 94, 1052– 1059, DOI: 10.1128/jb.94.4.1052-1059.1967Google Scholar215https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaF2sXltVGmtr4%253D&md5=7433f5b8394c57030679600870078889Evidence for the Calvin cycle and hexose monophosphate pathway in Thiobacillus ferrooxidansGale, Nord L.; Beck, Jay VernJournal of Bacteriology (1967), 94 (4), 1052-9CODEN: JOBAAY; ISSN:0021-9193.The enzymes of the Calvin reductive pentose phosphate cycle and the hexose monophosphate pathway were demonstrated in cell-free exts. of T. ferroxidans. This, together with analyses of the products of CO2 fixation in cell-free systems, suggests that these pathways are operative in whole cells of this bacterium. Nevertheless, the amt. of CO2 fixed in these cell-free systems was limited by the type and amt. of compd. added as substrate. The inability of cell exts. to regenerate pentose phosphates and to perpetuate the cyclic fixation of CO2 is partially attributable to low activity of triosephosphate dehydrogenase under the exptl. conditions optimal for the enzymes involved in the utilization of ribose 5-phosphate or ribulose 1,5-diphosphate as substrate for CO2 incorporation. With the exception of ribulose 1,5-diphosphate, all substrates required the addn. of ATP or ADP for CO2 fixation. Under optimal conditions, with ribose 5-phosphate serving as substrate, each micromole of ATP added resulted in the fixation of 1.5 micromoles of CO2, whereas each micromole of ADP resulted in 0.5 micromole of CO2 fixed. These values reflect the activity of adenylate kinase in the ext. prepns. The Km for ATP in the phosphoribulokinase reaction was 0.91 × 10-3M. Kinetic studies conducted with carboxydismutase showed Km values of 1.15 × 10-4M and 5 × 10-2M for ribulose 1,5-diphosphate and bicarbonate, resp. 32 references.
- 216Miller, T. E.; Beneyton, T.; Schwander, T.; Diehl, C.; Girault, M.; McLean, R.; Chotel, T.; Claus, P.; Cortina, N. S.; Baret, J.-C.; Erb, T. J. Light-powered CO2 fixation in a chloroplast mimic with natural and synthetic parts. Science 2020, 368, 649– 654, DOI: 10.1126/science.aaz6802Google Scholar216https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB3cXovFCqs7Y%253D&md5=e91945625b5292262a7237f8f895858bLight-powered CO2 fixation in a chloroplast mimic with natural and synthetic partsMiller, Tarryn E.; Beneyton, Thomas; Schwander, Thomas; Diehl, Christoph; Girault, Mathias; McLean, Richard; Chotel, Tanguy; Claus, Peter; Cortina, Nina Socorro; Baret, Jean-Christophe; Erb, Tobias J.Science (Washington, DC, United States) (2020), 368 (6491), 649-654CODEN: SCIEAS; ISSN:1095-9203. (American Association for the Advancement of Science)Nature integrates complex biosynthetic and energy-converting tasks within compartments such as chloroplasts and mitochondria. Chloroplasts convert light into chem. energy, driving carbon dioxide fixation. We used microfluidics to develop a chloroplast mimic by encapsulating and operating photosynthetic membranes in cell-sized droplets. These droplets can be energized by light to power enzymes or enzyme cascades and analyzed for their catalytic properties in multiplex and real time. We demonstrate how these microdroplets can be programmed and controlled by adjusting internal compns. and by using light as an external trigger. We showcase the capability of our platform by integrating the crotonyl-CoA (CoA)/ethylmalonyl-CoA/hydroxybutyryl-CoA (CETCH) cycle, a synthetic network for carbon dioxide conversion, to create an artificial photosynthetic system that interfaces the natural and the synthetic biol. worlds.
- 217Sundaram, S.; Diehl, C.; Cortina, N. S.; Bamberger, J.; Paczia, N.; Erb, T. J. A modular in vitro platform for the production of terpenes and polyketides from CO2. Angew. Chem., Int. Ed. 2021, 60 (30), 16420– 16425, DOI: 10.1002/anie.202102333Google Scholar217https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB3MXhsVKmt7nE&md5=70441107ebceb5dc590597e34d390b79A Modular In Vitro Platform for the Production of Terpenes and Polyketides from CO2Sundaram, Srividhya; Diehl, Christoph; Cortina, Nina Socorro; Bamberger, Jan; Paczia, Nicole; Erb, Tobias J.Angewandte Chemie, International Edition (2021), 60 (30), 16420-16425CODEN: ACIEF5; ISSN:1433-7851. (Wiley-VCH Verlag GmbH & Co. KGaA)A long-term goal in realizing a sustainable biocatalysis and org. synthesis is the direct use of the greenhouse gas CO2 as feedstock for the prodn. of bulk and fine chems., such as pharmaceuticals, fragrances and food additives. Here we developed a modular in vitro platform for the continuous conversion of CO2 into complex multi-carbon compds., such as monoterpenes (C10), sesquiterpenes (C15) and polyketides. Combining natural and synthetic metabolic pathway modules, we established a route from CO2 into the key intermediates acetyl- and malonyl-CoA, which can be subsequently diversified through the action of different terpene and polyketide synthases. Our proof-of-principle study demonstrates the simultaneous operation of different metabolic modules comprising of up to 29 enzymes in one pot, which paves the way for developing and optimizing synthesis routes for the generation of complex CO2-based chems. in the future.
- 218Diehl, C.; Gerlinger, P. D.; Paczia, N.; Erb, T. J. Synthetic anaplerotic modules for the direct synthesis of complex molecules from CO2. Nat. Chem. Biol. 2023, 19 (2), 168– 175, DOI: 10.1038/s41589-022-01179-0Google Scholar218https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB38XjtVCjtL%252FE&md5=408c17b934bc0342bde4ba4eb400c1f5Synthetic anaplerotic modules for the direct synthesis of complex molecules from CO2Diehl, Christoph; Gerlinger, Patrick D.; Paczia, Nicole; Erb, Tobias J.Nature Chemical Biology (2023), 19 (2), 168-175CODEN: NCBABT; ISSN:1552-4450. (Nature Portfolio)Abstr.: Anaplerosis is an essential feature of metab. that allows the continuous operation of natural metabolic networks, such as the citric acid cycle, by constantly replenishing drained intermediates. However, this concept has not been applied to synthetic in vitro metabolic networks, thus far. Here we used anaplerotic strategies to directly access the core sequence of the CETCH cycle, a new-to-nature in vitro CO2-fixation pathway that features several C3-C5 biosynthetic precursors. We drafted four different anaplerotic modules that use CO2 to replenish the CETCH cycle's intermediates and validated our designs by producing 6-deoxyerythronolide B (6-DEB), the C21-macrolide backbone of erythromycin. Our best design allowed the carbon-pos. synthesis of 6-DEB via 54 enzymic reactions in vitro at yields comparable to those with isolated 6-DEB polyketide synthase (DEBS). Our work showcases how new-to-nature anaplerotic modules can be designed and tailored to enhance and expand the synthetic capabilities of complex catalytic in vitro reaction networks. [graphic not available: see fulltext].
- 219Xiao, L.; Liu, G.; Gong, F.; Zhu, H.; Zhang, Y.; Cai, Z.; Li, Y. A minimized synthetic carbon fixation cycle. ACS Catal. 2022, 12 (1), 799– 808, DOI: 10.1021/acscatal.1c04151Google Scholar219https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB3MXivVeks7jP&md5=72345a4b3a3b1f07f55d553527d4386dA minimized synthetic carbon fixation cycleXiao, Lu; Liu, Guoxia; Gong, Fuyu; Zhu, Huawei; Zhang, Yanping; Cai, Zhen; Li, YinACS Catalysis (2022), 12 (1), 799-808CODEN: ACCACS; ISSN:2155-5435. (American Chemical Society)Natural CO2 fixation cycles usually comprise multiple reactions, which may reduce the efficiency of the cycle. Here, we report the design and exptl. demonstration of a minimized synthetic CO2 fixation cycle which contains only four reactions. The cycle comprises pyruvate carboxylase, oxaloacetate acetylhydrolase, acetate-CoA ligase, and pyruvate synthase and is named the POAP cycle. The POAP cycle can condense two mols. of CO2 into one mol. of oxalate in each step at the expense of two mols. of ATP and one reducing equiv. in the form of NAD(P)H. By identifying a ferredoxin from Hydrogenobacter thermophilus that can efficiently drive the rate-limiting reductive carboxylation step, the POAP cycle can be operated at 50°C under anaerobic conditions, reaching a CO2 fixation rate of 8.0 nmol CO2 min-1 mg-1 CO2-fixing enzymes. The design and demonstration of the POAP cycle may provide a model to study CO2 fixation in the earliest organisms.
- 220Bierbaumer, S.; Nattermann, M.; Schulz, L.; Zschoche, R.; Erb, T. J.; Winkler, C. K.; Tinzl, M.; Glueck, S. M. Enzymatic conversion of CO2: from natural to artificial utilization. Chem. Rev. 2023, 123 (9), 5702– 5754, DOI: 10.1021/acs.chemrev.2c00581Google Scholar220https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB3sXhs1GntL4%253D&md5=be4bc4e4b9fe5d5d7b2ba233dafdab61Enzymatic Conversion of CO2: From Natural to Artificial UtilizationBierbaumer, Sarah; Nattermann, Maren; Schulz, Luca; Zschoche, Reinhard; Erb, Tobias J.; Winkler, Christoph K.; Tinzl, Matthias; Glueck, Silvia M.Chemical Reviews (Washington, DC, United States) (2023), 123 (9), 5702-5754CODEN: CHREAY; ISSN:0009-2665. (American Chemical Society)A review. Enzymic carbon dioxide fixation is one of the most important metabolic reactions as it allows the capture of inorg. carbon from the atm. and converting it into org. biomass. However, due to the often unfavorable thermodn. and the difficulties assocd. with the utilization of CO2, a gaseous substrate that is found in comparatively low concns. in the atm., such reactions remain challenging for biotechnol. applications. Nature has tackled these problems by evolution of dedicated CO2-fixing enzymes, i.e., carboxylases, and embedding them in complex metabolic pathways. Biotechnol. employs such carboxylating and decarboxylating enzymes for the carboxylation of arom. and aliph. substrates either by embedding them into more complex reaction cascades or by shifting the reaction equil. via reaction engineering. This review aims to provide an overview of natural CO2-fixing enzymes and their mechanistic similarities. We also discuss biocatalytic applications of carboxylases and decarboxylases for the synthesis of valuable products and provide a sep. summary of strategies to improve the efficiency of such processes. We briefly summarize natural CO2 fixation pathways, provide a roadmap for the design and implementation of artificial carbon fixation pathways, and highlight examples of biocatalytic cascades involving carboxylases. Addnl., we suggest that biochem. utilization of reduced CO2 homologues, such as formate or methanol represents a suitable alternative to direct use of CO2 and provide several examples. Our discussion closes with a techno-economic perspective on enzymic CO2 fixation and its potential to reduce CO2-emissions.
- 221Cai, T.; Sun, H.; Qiao, J.; Zhu, L.; Zhang, F.; Zhang, J.; Tang, Z.; Wei, X.; Yang, J.; Yuan, Q. Cell-free chemo-enzymatic starch synthesis from carbon dioxide. Science 2021, 373, 1523– 152, DOI: 10.1126/science.abh4049Google Scholar221https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB3MXitFOisbnK&md5=81fb33a622016cbc14aacff7170dc3a1Cell-free chemoenzymatic starch synthesis from carbon dioxideCai, Tao; Sun, Hongbing; Qiao, Jing; Zhu, Leilei; Zhang, Fan; Zhang, Jie; Tang, Zijing; Wei, Xinlei; Yang, Jiangang; Yuan, Qianqian; Wang, Wangyin; Yang, Xue; Chu, Huanyu; Wang, Qian; You, Chun; Ma, Hongwu; Sun, Yuanxia; Li, Yin; Li, Can; Jiang, Huifeng; Wang, Qinhong; Ma, YanheScience (Washington, DC, United States) (2021), 373 (6562), 1523-1527CODEN: SCIEAS; ISSN:1095-9203. (American Association for the Advancement of Science)Starches, a storage form of carbohydrates, are a major source of calories in the human diet and a primary feedstock for bioindustry. We report a chem.-biochem. hybrid pathway for starch synthesis from carbon dioxide (CO2) and hydrogen in a cell-free system. The artificial starch anabolic pathway (ASAP), consisting of 11 core reactions, was drafted by computational pathway design, established through modular assembly and substitution, and optimized by protein engineering of three bottleneck-assocd. enzymes. In a chemoenzymic system with spatial and temporal segregation, ASAP, driven by hydrogen, converts CO2 to starch at a rate of 22 nmol of CO2 per min per mg of total catalyst, an ~ 8.5-fold higher rate than starch synthesis in maize. This approach opens the way toward future chemo-biohybrid starch synthesis from CO2.
- 222Zhou, J.; Tian, X.; Yang, Q.; Zhang, Z.; Chen, C.; Cui, Z.; Ji, Y.; Schwaneberg, U.; Chen, B.; Tan, T. Three multi-enzyme cascade pathways for conversion of C1 to C2/C4 compounds. Chem. Catal. 2022, 2 (10), 2675– 2690, DOI: 10.1016/j.checat.2022.07.011Google Scholar222https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB3sXjslOqt78%253D&md5=645bccc9f0ec5b17e95c3f31815b92e1Three multi-enzyme cascade pathways for conversion of C1 to C2/C4 compoundsZhou, Junhui; Tian, Xinyu; Yang, Qian; Zhang, Zixuan; Chen, Changjing; Cui, Ziheng; Ji, Yu; Schwaneberg, Ulrich; Chen, Biqiang; Tan, TianweiChem Catalysis (2022), 2 (10), 2675-2690CODEN: CCHAE9; ISSN:2667-1093. (Elsevier Inc.)A feasible and promising carbon redn. strategy is to convert CO2 into C1 compds. through renewable energy-driven first and then use a multi-enzyme cascade to obtain compds. with higher carbon nos. and market value. In this study, three multi-enzyme cascade pathways for prepg. important C2/C4 compds. (ethylene glycol [EG], glycolic acid [GA], and D-erythrose) from methanol were proposed. All pathways use glycolaldehyde as the key intermediate, which can be obtained from methanol through glycolaldehyde synthase and alc. oxidase cascade. Then, the target compds. can be acquired by adding different enzymes. As a result, 0.90 g/L EG, 0.33 g/L GA, and 53.65 mg/L D-erythrose were produced from methanol. Compared with the titers of EG (6.6 mM) and GA (1.2 g/L) synthesis routes from formaldehyde reported in the literature, the titers of this study (27.58 mM EG and 1.59 g/L GA from formaldehyde) were significantly improved.
- 223Zhang, J.; Liu, D.; Liu, Y.; Chu, H.; Bai, J.; Cheng, J.; Zhao, H.; Fu, S.; Liu, H.; Fu, Y. Hybrid synthesis of polyhydroxybutyrate bioplastics from carbon dioxide. Green Chem. 2023, 25 (8), 3247– 3255, DOI: 10.1039/D3GC00387FGoogle Scholar223https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB3sXmvVWltLo%253D&md5=5becb0e5eeb8c4d64bdcc5b2a6bc3510Hybrid synthesis of polyhydroxybutyrate bioplastics from carbon dioxideZhang, Jie; Liu, Dingyu; Liu, Yuwan; Chu, Huanyu; Bai, Jie; Cheng, Jian; Zhao, Haodong; Fu, Shaoping; Liu, Huihong; Fu, YuE.; Ma, Yanhe; Jiang, HuifengGreen Chemistry (2023), 25 (8), 3247-3255CODEN: GRCHFJ; ISSN:1463-9262. (Royal Society of Chemistry)Global sustainable development has intensified the demand for switching to a renewable economy with a reduced carbon footprint. Here, we report a hybrid system, coupling a chem. process for CO2 redn. with hydrogen, and a biol. process for polyhydroxybutyrate (PHB) synthesis, capable of converting CO2 into bioplastics with a theor. carbon yield of 100%. The synthetic pathway from CO2 to PHB was modularly optimized by improving the catalytic efficiency of key enzymes, avoiding the kinetic trap of metabolic flux and optimizing the whole catalytic process, resulting in 5.96 g L-1 PHB with a productivity of 1.19 g L-1 h-1 and a molar CO2 utilization efficiency of 71.8%. These results represent a promising closed-loop prodn. process from CO2 to biodegradable plastics.
- 224Li, F.; Wei, X.; Zhang, L.; Liu, C.; You, C.; Zhu, Z. Installing a green engine to drive an enzyme cascade: a light-powered in vitro biosystem for poly(3-hydroxybutyrate) synthesis. Angew. Chem., Int. Ed. 2022, 61 (1), e202111054, DOI: 10.1002/anie.202111054Google Scholar224https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB3MXisFOitL3M&md5=33486d9a6a565d88773c9986adecdc8fInstalling a Green Engine To Drive an Enzyme Cascade: A Light-Powered In Vitro Biosystem for Poly(3-hydroxybutyrate) SynthesisLi, Fei; Wei, Xinlei; Zhang, Lin; Liu, Cheng; You, Chun; Zhu, ZhiguangAngewandte Chemie, International Edition (2022), 61 (1), e202111054CODEN: ACIEF5; ISSN:1433-7851. (Wiley-VCH Verlag GmbH & Co. KGaA)Many existing in vitro biosystems harness power from the chem. energy contained in substrates and co-substrates, and light or elec. energy provided from abiotic parts, leading to a compromise in atom economy, incompatibility between biol. and abiotic parts, and most importantly, incapability to spatiotemporally co-regenerate ATP and NADPH. In this study, we developed a light-powered in vitro biosystem for poly(3-hydroxybutyrate) (PHB) synthesis using natural thylakoid membranes (TMs) to regenerate ATP and NADPH for a five-enzyme cascade. Through effective coupling of cofactor regeneration and mass conversion, 20 mM PHB was yielded from 50 mM sodium acetate with a molar conversion efficiency of carbon of 80.0% and a light-energy conversion efficiency of 3.04%, which are much higher than the efficiencies of similar in vitro PHB synthesis biosystems. This suggests the promise of installing TMs as a green engine to drive more enzyme cascades.
- 225Liu, H.; Arbing, M. A.; Bowie, J. U. Expanding the use of ethanol as a feedstock for cell-free synthetic biochemistry by implementing acetyl-CoA and ATP generating pathways. Sci. Rep. 2022, 12 (1), 7700, DOI: 10.1038/s41598-022-11653-3Google Scholar225https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB38Xht1OiurjJ&md5=0575f6683e35f5090a75b81d9d52a81cExpanding the use of ethanol as a feedstock for cell-free synthetic biochemistry by implementing acetyl-CoA and ATP generating pathwaysLiu, Hongjiang; Arbing, Mark A.; Bowie, James U.Scientific Reports (2022), 12 (1), 7700CODEN: SRCEC3; ISSN:2045-2322. (Nature Portfolio)Ethanol is a widely available carbon compd. that can be increasingly produced with a net neg. carbon balance. Carbon-neg. ethanol might therefore provide a feedstock for building a wider range of sustainable chems. Here we show how ethanol can be converted with a cell free system into acetyl-CoA, a central precursor for myriad biochems., and how we can use the energy stored in ethanol to generate ATP, another key mol. important for powering biochem. pathways. The ATP generator produces acetone as a value-added side product. Our ATP generator reached titers of 27 ± 6 mM ATP and 59 ± 15 mM acetone with max. ATP synthesis rate of 2.8 ± 0.6 mM/h and acetone of 7.8 ± 0.8 mM/h. We illustrated how the ATP generating module can power cell-free biochem. pathways by converting mevalonate into isoprenol at a titer of 12.5 ± 0.8 mM and a max. productivity of 1.0 ± 0.05 mM/h. These proof-of-principle demonstrations may ultimately find their way to the manuf. of diverse chems. from ethanol and other simple carbon compds.
- 226Bailoni, E.; Partipilo, M.; Coenradij, J.; Grundel, D. A. J.; Slotboom, D. J.; Poolman, B. Minimal out-of-equilibrium metabolism for synthetic cells: a membrane perspective. ACS Synth. Biol. 2023, 12 (4), 922– 946, DOI: 10.1021/acssynbio.3c00062Google Scholar226https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB3sXntVSrsLc%253D&md5=cb72d2883fc86ce12e323b5f2d8dc842Minimal Out-of-Equilibrium Metabolism for Synthetic Cells: A Membrane PerspectiveBailoni, Eleonora; Partipilo, Michele; Coenradij, Jelmer; Grundel, Douwe A. J.; Slotboom, Dirk J.; Poolman, BertACS Synthetic Biology (2023), 12 (4), 922-946CODEN: ASBCD6; ISSN:2161-5063. (American Chemical Society)A review. Life-like systems need to maintain a basal metab., which includes importing a variety of building blocks required for macromol. synthesis, exporting dead-end products, and recycling cofactors and metabolic intermediates, while maintaining steady internal phys. and chem. conditions (physicochem. homeostasis). A compartment, such as a unilamellar vesicle, functionalized with membrane-embedded transport proteins and metabolic enzymes encapsulated in the lumen meets these requirements. Here, we identify four modules designed for a minimal metab. in a synthetic cell with a lipid bilayer boundary: energy provision and conversion, physicochem. homeostasis, metabolite transport, and membrane expansion. We review design strategies that can be used to fulfill these functions with a focus on the lipid and membrane protein compn. of a cell. We compare our bottom-up design with the equiv. essential modules of JCVI-syn3a, a top-down genome-minimized living cell with a size comparable to that of large unilamellar vesicles. Finally, we discuss the bottlenecks related to the insertion of a complex mixt. of membrane proteins into lipid bilayers and provide a semiquant. est. of the relative surface area and lipid-to-protein mass ratios (i.e., the minimal no. of membrane proteins) that are required for the construction of a synthetic cell.
- 227Lee, K. Y.; Park, S. J.; Lee, K. A.; Kim, S. H.; Kim, H.; Meroz, Y.; Mahadevan, L.; Jung, K. H.; Ahn, T. K.; Parker, K. K. Photosynthetic artificial organelles sustain and control ATP-dependent reactions in a protocellular system. Nat. Biotechnol. 2018, 36 (6), 530– 535, DOI: 10.1038/nbt.4140Google Scholar227https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC1cXhtVWktrzM&md5=38cc9b7aa604f285b5d84c793c9e26cfPhotosynthetic artificial organelles sustain and control ATP-dependent reactions in a protocellular systemLee, Keel Yong; Park, Sung-Jin; Lee, Keon Ah; Kim, Se-Hwan; Kim, Heeyeon; Meroz, Yasmine; Mahadevan, L.; Jung, Kwang-Hwan; Ahn, Tae Kyu; Parker, Kevin Kit; Shin, KwanwooNature Biotechnology (2018), 36 (6), 530-535CODEN: NABIF9; ISSN:1087-0156. (Nature Research)Inside cells, complex metabolic reactions are distributed across the modular compartments of organelles. Reactions in organelles have been recapitulated in vitro by reconstituting functional protein machineries into membrane systems. However, maintaining and controlling these reactions is challenging. Here we designed, built, and tested a switchable, light-harvesting organelle that provides both a sustainable energy source and a means of directing intravesicular reactions. An ATP (ATP) synthase and two photoconverters (plant-derived photosystem II and bacteria-derived proteorhodopsin) enable ATP synthesis. Independent optical activation of the two photoconverters allows dynamic control of ATP synthesis: red light facilitates and green light impedes ATP synthesis. We encapsulated the photosynthetic organelles in a giant vesicle to form a protocellular system and demonstrated optical control of two ATP-dependent reactions, carbon fixation and actin polymn., with the latter altering outer vesicle morphol. Switchable photosynthetic organelles may enable the development of biomimetic vesicle systems with regulatory networks that exhibit homeostasis and complex cellular behaviors.
- 228Berhanu, S.; Ueda, T.; Kuruma, Y. Artificial photosynthetic cell producing energy for protein synthesis. Nat. Commun. 2019, 10 (1), 1325, DOI: 10.1038/s41467-019-09147-4Google Scholar228https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A280%3ADC%252BB3cbnt1yjsw%253D%253D&md5=706ef019ff2af417bb362cf8bb29d3b0Artificial photosynthetic cell producing energy for protein synthesisBerhanu Samuel; Ueda Takuya; Kuruma Yutetsu; Kuruma YutetsuNature communications (2019), 10 (1), 1325 ISSN:.Attempts to construct an artificial cell have widened our understanding of living organisms. Many intracellular systems have been reconstructed by assembling molecules, however the mechanism to synthesize its own constituents by self-sufficient energy has to the best of our knowledge not been developed. Here, we combine a cell-free protein synthesis system and small proteoliposomes, which consist of purified ATP synthase and bacteriorhodopsin, inside a giant unilamellar vesicle to synthesize protein by the production of ATP by light. The photo-synthesized ATP is consumed as a substrate for transcription and as an energy for translation, eventually driving the synthesis of bacteriorhodopsin or constituent proteins of ATP synthase, the original essential components of the proteoliposome. The de novo photosynthesized bacteriorhodopsin and the parts of ATP synthase integrate into the artificial photosynthetic organelle and enhance its ATP photosynthetic activity through the positive feedback of the products. Our artificial photosynthetic cell system paves the way to construct an energetically independent artificial cell.
- 229Bailoni, E.; Poolman, B. ATP recycling fuels sustainable glycerol 3-phosphate formation in synthetic cells fed by dynamic dialysis. ACS Synth. Biol. 2022, 11 (7), 2348– 2360, DOI: 10.1021/acssynbio.2c00075Google Scholar229https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB38Xos1KitLo%253D&md5=24e104c5d768995c265e6adac1cc131eATP Recycling Fuels Sustainable Glycerol 3-Phosphate Formation in Synthetic Cells Fed by Dynamic DialysisBailoni, Eleonora; Poolman, BertACS Synthetic Biology (2022), 11 (7), 2348-2360CODEN: ASBCD6; ISSN:2161-5063. (American Chemical Society)The bottom-up construction of an autonomously growing, self-reproducing cell represents a great challenge for synthetic biol. Synthetic cellular systems are envisioned as out-of-equil. enzymic networks encompassed by a selectively open phospholipid bilayer allowing for protein-mediated communication; internal metabolite recycling is another key aspect of a sustainable metab. Importantly, gaining tight control over the external medium is essential to avoid thermodn. equil. due to nutrient depletion or waste buildup in a closed compartment (e.g., a test tube). Implementing a sustainable strategy for phospholipid biosynthesis is key to expanding the cellular boundaries. However, phospholipid biosynthesis is currently limited by substrate availability, e.g., of glycerol 3-phosphate, the essential core of phospholipid headgroups. Here, we reconstitute an enzymic network for sustainable glycerol 3-phosphate synthesis inside large unilamellar vesicles. We exploit the Escherichia coli glycerol kinase GlpK to synthesize glycerol 3-phosphate from externally supplied glycerol. We fuel phospholipid headgroup formation by sustainable L-arginine breakdown. In addn., we design and characterize a dynamic dialysis setup optimized for synthetic cells, which is used to control the external medium compn. and to achieve sustainable glycerol 3-phosphate synthesis.
- 230Blanken, D.; Foschepoth, D.; Serrao, A. C.; Danelon, C. Genetically controlled membrane synthesis in liposomes. Nat. Commun. 2020, 11 (1), 4317, DOI: 10.1038/s41467-020-17863-5Google Scholar230https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB3cXhslejtL3L&md5=cf776e04404b4d227a23041a0acef3c8Genetically controlled membrane synthesis in liposomesBlanken, Duco; Foschepoth, David; Serrao, Adriana Calaca; Danelon, ChristopheNature Communications (2020), 11 (1), 4317CODEN: NCAOBW; ISSN:2041-1723. (Nature Research)Lipid membranes, nucleic acids, proteins, and metab. are essential for modern cellular life. Synthetic systems emulating the fundamental properties of living cells must therefore be built upon these functional elements. In this work, phospholipid-producing enzymes encoded in a synthetic minigenome are cell-free expressed within liposome compartments. The de novo synthesized metabolic pathway converts precursors into a variety of lipids, including the constituents of the parental liposome. Balanced prodn. of phosphatidylethanolamine and phosphatidylglycerol is realized, owing to transcriptional regulation of the activity of specific genes combined with a metabolic feedback mechanism. Fluorescence-based methods are developed to image the synthesis and membrane incorporation of phosphatidylserine at the single liposome level. Our results provide exptl. evidence for DNA-programed membrane synthesis in a minimal cell model. Strategies are discussed to alleviate current limitations toward effective liposome growth and self-reprodn.
- 231Partipilo, M.; Ewins, E. J.; Frallicciardi, J.; Robinson, T.; Poolman, B.; Slotboom, D. J. Minimal pathway for the regeneration of redox cofactors. JACS Au 2021, 1 (12), 2280– 2293, DOI: 10.1021/jacsau.1c00406Google Scholar231https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB3MXisVKnsrjJ&md5=cd8bcc68d0494b701fad84d2f816f644Minimal Pathway for the Regeneration of Redox CofactorsPartipilo, Michele; Ewins, Eleanor J.; Frallicciardi, Jacopo; Robinson, Tom; Poolman, Bert; Slotboom, Dirk JanJACS Au (2021), 1 (12), 2280-2293CODEN: JAAUCR; ISSN:2691-3704. (American Chemical Society)Effective metabolic pathways are essential for the construction of in vitro systems mimicking the biochem. complexity of living cells. Such pathways require the inclusion of a metabolic branch that ensures the availability of reducing equiv. Here, we built a minimal enzymic pathway confinable in the lumen of liposomes, in which the redox status of the nicotinamide cofactors NADH and NADPH is controlled by an externally provided formate. Formic acid permeates the membrane where a luminal formate dehydrogenase uses NAD+ to form NADH and carbon dioxide. Carbon dioxide diffuses out of the liposomes, leaving only the reducing equiv. in the lumen. A sol. transhydrogenase subsequently utilizes NADH for redn. of NADP+ thereby making NAD+ available again for the first reaction. The pathway is functional in liposomes ranging from a few hundred nanometers in diam. (large unilamellar vesicles) up to several tens of micrometers (giant unilamellar vesicles) and remains active over a period of 7 days. We demonstrate that the downstream biochem. process of redn. of glutathione disulfide can be driven by the transfer of reducing equiv. from formate via NAD(P)H, thereby providing a versatile set of electron donors for reductive metab.
- 232Pols, T.; Sikkema, H. R.; Gaastra, B. F.; Frallicciardi, J.; Smigiel, W. M.; Singh, S.; Poolman, B. A synthetic metabolic network for physicochemical homeostasis. Nat. Commun. 2019, 10 (1), 4239, DOI: 10.1038/s41467-019-12287-2Google Scholar232https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A280%3ADC%252BB3MngtVOrtA%253D%253D&md5=9738665e6507488772d12472354a902eA synthetic metabolic network for physicochemical homeostasisPols Tjeerd; Sikkema Hendrik R; Gaastra Bauke F; Frallicciardi Jacopo; Smigiel Wojciech M; Singh Shubham; Poolman BertNature communications (2019), 10 (1), 4239 ISSN:.One of the grand challenges in chemistry is the construction of functional out-of-equilibrium networks, which are typical of living cells. Building such a system from molecular components requires control over the formation and degradation of the interacting chemicals and homeostasis of the internal physical-chemical conditions. The provision and consumption of ATP lies at the heart of this challenge. Here we report the in vitro construction of a pathway in vesicles for sustained ATP production that is maintained away from equilibrium by control of energy dissipation. We maintain a constant level of ATP with varying load on the system. The pathway enables us to control the transmembrane fluxes of osmolytes and to demonstrate basic physicochemical homeostasis. Our work demonstrates metabolic energy conservation and cell volume regulatory mechanisms in a cell-like system at a level of complexity minimally needed for life.
- 233Katz, E.; Privman, V. Enzyme-based logic systems for information processing. Chem. Soc. Rev. 2010, 39 (5), 1835– 1857, DOI: 10.1039/b806038jGoogle Scholar233https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3cXltFKgsb8%253D&md5=8761554ed19bd8f3938689902d6b78c0Enzyme-based logic systems for information processingKatz, Evgeny; Privman, VladimirChemical Society Reviews (2010), 39 (5), 1835-1857CODEN: CSRVBR; ISSN:0306-0012. (Royal Society of Chemistry)A review. In this crit. review the authors review enzymic systems which involve biocatalytic reactions utilized for information processing (biocomputing). Extensive ongoing research in biocomputing, mimicking Boolean logic gates has been motivated by potential applications in biotechnol. and medicine. Furthermore, novel sensor concepts have been contemplated with multiple inputs processed biochem. before the final output is coupled to transducing "smart-material" electrodes and other systems. These applications have warranted recent emphasis on networking of biocomputing gates. First few-gate networks have been exptl. realized, including coupling, for instance, to signal-responsive electrodes for signal readout. To achieve scalable, stable network design and functioning, considerations of noise propagation and control have been initiated as a new research direction. Optimization of single enzyme-based gates for avoiding analog noise amplification has been explored, as were certain network-optimization concepts. The authors review and exemplify these developments, as well as offer an outlook for possible future research foci. The latter include design and uses of non-Boolean network elements, e.g., filters, as well as other developments motivated by potential novel sensor and biotechnol. applications (136 refs.).
- 234Benenson, Y. Biomolecular computing systems: principles, progress and potential. Nat. Rev. Genet. 2012, 13 (7), 455– 468, DOI: 10.1038/nrg3197Google Scholar234https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC38XotlCku7Y%253D&md5=df2cf7bb6f50782328b62cdcccdd2160Biomolecular computing systems: principles, progress and potentialBenenson, YaakovNature Reviews Genetics (2012), 13 (7), 455-468CODEN: NRGAAM; ISSN:1471-0056. (Nature Publishing Group)A review. The task of information processing, or computation, can be performed by natural and man-made 'devices'. Man-made computers are made from silicon chips, whereas natural 'computers', such as the brain, use cells and mols. Computation also occurs on a much smaller scale in regulatory and signalling pathways in individual cells and even within single biomols. Indeed, much of what we recognize as life results from the remarkable capacity of biol. building blocks to compute in highly sophisticated ways. Rational design and engineering of biol. computing systems can greatly enhance our ability to study and to control biol. systems. Potential applications include tissue engineering and regeneration and medical treatments. This Review introduces key concepts and discusses recent progress that has been made in biomol. computing.
- 235Grozinger, L.; Amos, M.; Gorochowski, T. E.; Carbonell, P.; Oyarzun, D. A.; Stoof, R.; Fellermann, H.; Zuliani, P.; Tas, H.; Goni-Moreno, A. Pathways to cellular supremacy in biocomputing. Nat. Commun. 2019, 10 (1), 5250, DOI: 10.1038/s41467-019-13232-zGoogle Scholar235https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A280%3ADC%252BB3Mfht12rtA%253D%253D&md5=3890719db11fbfe37974c513492740baPathways to cellular supremacy in biocomputingGrozinger Lewis; Stoof Ruud; Fellermann Harold; Zuliani Paolo; Goni-Moreno Angel; Amos Martyn; Gorochowski Thomas E; Gorochowski Thomas E; Carbonell Pablo; Oyarzun Diego A; Oyarzun Diego A; Tas HuseyinNature communications (2019), 10 (1), 5250 ISSN:.Synthetic biology uses living cells as the substrate for performing human-defined computations. Many current implementations of cellular computing are based on the "genetic circuit" metaphor, an approximation of the operation of silicon-based computers. Although this conceptual mapping has been relatively successful, we argue that it fundamentally limits the types of computation that may be engineered inside the cell, and fails to exploit the rich and diverse functionality available in natural living systems. We propose the notion of "cellular supremacy" to focus attention on domains in which biocomputing might offer superior performance over traditional computers. We consider potential pathways toward cellular supremacy, and suggest application areas in which it may be found.
- 236Ivanov, N. M.; Baltussen, M. G.; Regueiro, C. L. F.; Derks, M.; Huck, W. T. S. Computing arithmetic functions using immobilised enzymatic reaction networks. Angew. Chem., Int. Ed. 2023, 62 (7), e202215759, DOI: 10.1002/anie.202215759Google Scholar236https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB3sXhtFGitrg%253D&md5=d8a6cb44f9286028dba214a8be43f00bComputing Arithmetic Functions Using Immobilised Enzymatic Reaction NetworksIvanov, Nikita M.; Baltussen, Mathieu G.; Regueiro, Cristina Lia Fernandez; Derks, Max T. G. M.; Huck, Wilhelm T. S.Angewandte Chemie, International Edition (2023), 62 (7), e202215759CODEN: ACIEF5; ISSN:1433-7851. (Wiley-VCH Verlag GmbH & Co. KGaA)Living systems use enzymic reaction networks to process biochem. information and make decisions in response to external or internal stimuli. Herein, we present a modular and reusable platform for mol. information processing using enzymes immobilized in hydrogel beads and compartmentalised in a continuous stirred tank reactor. We demonstrate how this setup allows us to perform simple arithmetic operations, such as addn., subtraction and multiplication, using various concns. of substrates or inhibitors as inputs and the prodn. of a fluorescent mol. as the readout.
- 237Genot, A. J.; Fujii, T.; Rondelez, Y. Computing with competition in biochemical networks. Phys. Rev. Lett. 2012, 109 (20), 208102, DOI: 10.1103/PhysRevLett.109.208102Google Scholar237https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC38XhvVCmurrL&md5=df4657c49b9599ee143da5f25b160f72Computing with competition in biochemical networksGenot, Anthony J.; Fujii, Teruo; Rondelez, YannickPhysical Review Letters (2012), 109 (20), 208102/1-208102/5CODEN: PRLTAO; ISSN:0031-9007. (American Physical Society)Cells rely on limited resources such as enzymes or transcription factors to process signals and make decisions. However, independent cellular pathways often compete for a common mol. resource. Competition is difficult to analyze because of its nonlinear global nature, and its role remains unclear. Here we show how decision pathways such as transcription networks may exploit competition to process information. Competition for one resource leads to the recognition of convex sets of patterns, whereas competition for several resources (overlapping or cascaded regulons) allows even more general pattern recognition. Competition also generates surprising couplings, such as correlating species that share no resource but a common competitor. The mechanism we propose relies on three primitives that are ubiquitous in cells: multiinput motifs, competition for a resource, and pos. feedback loops.
- 238Pandi, A.; Koch, M.; Voyvodic, P. L.; Soudier, P.; Bonnet, J.; Kushwaha, M.; Faulon, J. L. Metabolic perceptrons for neural computing in biological systems. Nat. Commun. 2019, 10 (1), 3880, DOI: 10.1038/s41467-019-11889-0Google Scholar238https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A280%3ADC%252BB3MrivFCkuw%253D%253D&md5=709c8f35b4f3ed72a901ad0bfefd03ecMetabolic perceptrons for neural computing in biological systemsPandi Amir; Koch Mathilde; Soudier Paul; Kushwaha Manish; Faulon Jean-Loup; Voyvodic Peter L; Bonnet Jerome; Soudier Paul; Faulon Jean-Loup; Faulon Jean-LoupNature communications (2019), 10 (1), 3880 ISSN:.Synthetic biological circuits are promising tools for developing sophisticated systems for medical, industrial, and environmental applications. So far, circuit implementations commonly rely on gene expression regulation for information processing using digital logic. Here, we present a different approach for biological computation through metabolic circuits designed by computer-aided tools, implemented in both whole-cell and cell-free systems. We first combine metabolic transducers to build an analog adder, a device that sums up the concentrations of multiple input metabolites. Next, we build a weighted adder where the contributions of the different metabolites to the sum can be adjusted. Using a computational model fitted on experimental data, we finally implement two four-input perceptrons for desired binary classification of metabolite combinations by applying model-predicted weights to the metabolic perceptron. The perceptron-mediated neural computing introduced here lays the groundwork for more advanced metabolic circuits for rapid and scalable multiplex sensing.
- 239Okumura, S.; Gines, G.; Lobato-Dauzier, N.; Baccouche, A.; Deteix, R.; Fujii, T.; Rondelez, Y.; Genot, A. J. Nonlinear decision-making with enzymatic neural networks. Nature 2022, 610 (7932), 496– 501, DOI: 10.1038/s41586-022-05218-7Google Scholar239https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB38Xis1Onu7rJ&md5=e6485c7b357ad006c2f01ab78a791e80Nonlinear decision-making with enzymatic neural networksOkumura, S.; Gines, G.; Lobato-Dauzier, N.; Baccouche, A.; Deteix, R.; Fujii, T.; Rondelez, Y.; Genot, A. J.Nature (London, United Kingdom) (2022), 610 (7932), 496-501CODEN: NATUAS; ISSN:1476-4687. (Nature Portfolio)Atificial neural networks have revolutionized electronic computing. Similarly, mol. networks with neuromorphic architectures may enable mol. decision-making on a level comparable to gene regulatory networks1,2. Non-enzymic networks could in principle support neuromorphic architectures, and seminal proofs-of-principle have been reported3,4. However, leakages (i.e., the unwanted release of species), as well as issues with sensitivity, speed, prepn. and the lack of strong nonlinear responses, make the compn. of layers delicate, and mol. classifications equiv. to a multilayer neural network remain elusive (for example, the partitioning of a concn. space into regions that cannot be linearly sepd.). Here we introduce DNA-encoded enzymic neurons with tuneable wts. and biases, and which are assembled in multilayer architectures to classify nonlinearly separable regions. We first leverage the sharp decision margin of a neuron to compute various majority functions on 10 bits. We then compose neurons into a two-layer network and synthesize a parametric family of rectangular functions on a microRNA input. Finally, we connect neural and logical computations into a hybrid circuit that recursively partitions a concn. plane according to a decision tree in cell-sized droplets. This computational power and extreme miniaturization open avenues to query and manage mol. systems with complex contents, such as liq. biopsies or DNA databases.
- 240Hathcock, D.; Sheehy, J.; Weisenberger, C.; Ilker, E.; Hinczewski, M. Noise filtering and prediction in biological signaling networks. IEEE Transactions on Molecular, Biological and Multi-Scale Communications 2016, 2 (1), 16– 30, DOI: 10.1109/TMBMC.2016.2633269Google ScholarThere is no corresponding record for this reference.
- 241O’Brien, J.; Murugan, A. Temporal pattern recognition through analog molecular computation. ACS Synth. Biol. 2019, 8 (4), 826– 832, DOI: 10.1021/acssynbio.8b00503Google Scholar241https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC1MXktFahsr8%253D&md5=ac507d10d6d2c3ab067d76b0859dde63Temporal Pattern Recognition through Analog Molecular ComputationO'Brien, Jackson; Murugan, ArvindACS Synthetic Biology (2019), 8 (4), 826-832CODEN: ASBCD6; ISSN:2161-5063. (American Chemical Society)Living cells communicate information about physiol. conditions by producing signaling mols. in a specific timed manner. Different conditions can result in the same total amt. of a signaling mol., differing only in the pattern of the mol. concn. over time. Such temporally coded information can be completely invisible to even state-of-the-art mol. sensors with high chem. specificity that respond only to the total amt. of the signaling mol. Here, we demonstrate design principles for circuits with temporal specificity, i.e., mol. circuits that respond to specific temporal patterns in a mol. concn. We consider pulsatile patterns in a mol. concn. characterized by three fundamental temporal features: time period, duty fraction, and no. of pulses. We develop circuits that respond to each one of these features while being insensitive to the others. We demonstrate our design principles using general chem. reaction networks and with explicit simulations of DNA strand displacement reactions. In this way, our work develops building blocks for temporal pattern recognition through mol. computation.
- 242McGrath, T.; Jones, N. S.; Ten Wolde, P. R.; Ouldridge, T. E. Biochemical machines for the interconversion of mutual information and work. Phys. Rev. Lett. 2017, 118 (2), 028101 DOI: 10.1103/PhysRevLett.118.028101Google Scholar242https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2sXpt1Cktbg%253D&md5=f366eb71b2d50e88c3253359c37127b7Biochemical machines for the interconversion of mutual information and workMcGrath, Thomas; Jones, Nick S.; ten Wolde, Pieter Rein; Ouldridge, Thomas E.Physical Review Letters (2017), 118 (2), 028101/1-028101/5CODEN: PRLTAO; ISSN:1079-7114. (American Physical Society)We propose a phys. realizable information-driven device consisting of an enzyme in a chem. bath, interacting with pairs of mols. prepd. in correlated states. These correlations persist without direct interaction and thus store free energy equal to the mutual information. The enzyme can harness this free energy, and that stored in the individual mol. states, to do chem. work. Alternatively, the enzyme can use the chem. driving to create mutual information. A modified system can function without external intervention, approaching biol. systems more closely.
- 243Stern, M.; Murugan, A. Learning without neurons in physical systems. Annu. Rev. Condens. Matter Phys. 2023, 14, 417– 41, DOI: 10.1146/annurev-conmatphys-040821-113439Google ScholarThere is no corresponding record for this reference.
- 244Shklyaev, O. E.; Balazs, A. C. Interlinking spatial dimensions and kinetic processes in dissipative materials to create synthetic systems with lifelike functionality. Nat. Nanotechnol. 2024, 19, 146, DOI: 10.1038/s41565-023-01530-zGoogle Scholar244https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB3sXisFGgsrvF&md5=267ff1ebfaaa2263ab3cae55a61cd399Interlinking spatial dimensions and kinetic processes in dissipative materials to create synthetic systems with lifelike functionalityShklyaev, Oleg E.; Balazs, Anna C.Nature Nanotechnology (2024), 19 (2), 146-159CODEN: NNAABX; ISSN:1748-3387. (Nature Portfolio)Abstr.: Biol. systems spontaneously convert energy input into the actions necessary to survive. Motivated by the efficacy of these processes, researchers aim to forge materials systems that exhibit the self-sustained and autonomous functionality found in nature. Success in this effort will require synthetic analogs of the following: a metab. to generate energy, a vasculature to transport energy and materials, a nervous system to transmit 'commands', a musculoskeletal system to translate commands into phys. action, regulatory networks to monitor the entire enterprise, and a mechanism to convert 'nutrients' into growing materials. Design rules must interconnect the material's structural and kinetic properties over ranges of length (that can vary from the nano- to mesoscale) and timescales to enable local energy dissipations to power global functionality. Moreover, by harnessing dynamic interactions intrinsic to the material, the system itself can perform the work needed for its own functionality. Here, we assess the advances and challenges in dissipative materials design and at the same time aim to spur developments in next-generation functional, 'living' materials.
- 245Bray, D. Protein molecules as computational elements in living cells. Nature 1995, 376, 307– 312, DOI: 10.1038/376307a0Google Scholar245https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaK2MXnt1Sjsrg%253D&md5=f124baf6c54d1697e41fb0f9f57a81b2Protein molecules as computational elements in living cellsBray, DennisNature (London) (1995), 376 (6538), 307-12CODEN: NATUAS; ISSN:0028-0836. (Macmillan Magazines)A review with 40 refs. Many proteins in living cells appear to have as their primary function the transfer and processing of information, rather than the chem. transformation of metabolic intermediates or the building of cellular structures. Such protein are functionally linked through allosteric or other mechanisms into biochem. 'circuits' that perform a variety of simple computational tasks including amplification, integration and information storage.
- 246van Sluijs, B.; Zhou, T.; Helwig, B.; Baltussen, M. G.; Nelissen, F. H. T.; Heus, H. A.; Huck, W. T. S. Inverse design of enzymatic reaction network states. Nat. Commun. 2024, 15, 1602, DOI: 10.1038/s41467-024-45886-9Google Scholar246https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB2cXksVeitL0%253D&md5=b30fd58feb0af469782485178069090aIterative design of training data to control intricate enzymatic reaction networksvan Sluijs, Bob; Zhou, Tao; Helwig, Britta; Baltussen, Mathieu G.; Nelissen, Frank H. T.; Heus, Hans A.; Huck, Wilhelm T. S.Nature Communications (2024), 15 (1), 1602CODEN: NCAOBW; ISSN:2041-1723. (Nature Portfolio)Abstr.: Kinetic modeling of in vitro enzymic reaction networks is vital to understand and control the complex behaviors emerging from the nonlinear interactions inside. However, modeling is severely hampered by the lack of training data. Here, we introduce a methodol. that combines an active learning-like approach and flow chem. to efficiently create optimized datasets for a highly interconnected enzymic reactions network with multiple sub-pathways. The optimal exptl. design (OED) algorithm designs a sequence of out-of-equil. perturbations to maximize the information about the reaction kinetics, yielding a descriptive model that allows control of the output of the network towards any cost function. We exptl. validate the model by forcing the network to produce different product ratios while maintaining a min. level of overall conversion efficiency. Our workflow scales with the complexity of the system and enables the optimization of previously unobtainable network outputs.
- 247Zaikin, A. N.; Zhabotinsky, A. M. Concentration wave propagation in two-dimensional liquid-phase self-oscillating system. Nature 1970, 225, 535– 537, DOI: 10.1038/225535b0Google Scholar247https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaE3cXhtVCgsLc%253D&md5=7ef8bca1d462b0284614ebd74939b503Concentration wave propagation in two-dimensional liquid-phase self-oscillating systemZaikin, A. N.; Zhabotinskii, A. M.Nature (London, United Kingdom) (1970), 225 (5232), 535-7CODEN: NATUAS; ISSN:0028-0836.A study on oscillating chem. reactions in the system bromate-bromomalonic acid-ferroin (indicator and catalyst) was made. The reaction was carried out in a thin layer of soln. at 20°. Photographs were taken at 1 min intervals. In the 1st photograph, the catalyst is completely reduced, and subsequent photographs show it starting to be oxidized at particular points (leading points) from which circular waves of oxidn. are propagated. The 4th photograph shows oxidn. taking place in areas not reached by these waves. The next photographs show waves coming from leading centers oxidizing all the space step by step. Radial sym. patterns are also obsd. The obsd. phenomenon is characterized by the occurrence of progressive concn. waves and by a space structure supported at the expense of redox reaction energy. A model for the wave propagation is proposed.
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Abstract
Figure 1
Figure 1. Overview of the topics discussed in the review. The image in “building synthetic cell” shows a cartoon representation of a synthetic cell (Credit: Graham Johnson/BaSyC consortium).
Figure 2
Figure 2. (a) A schematic representation of the modular organization of enzymatic networks in the cell. Strongly connected modules with their own specific functions share limited connections to different modules. In this network representation, enzymes are represented by nodes and substrates by edges. (b) An example of typical scaling relationships found in the connectedness of enzymes. Here, dots represent types of enzymes, with colors denoting different modules. k represents the number of connections an enzyme has in the ERN, while P(k) represents the frequency of that number of connections occurring. Inside cellular ERNs, these follow a power-law relationship P(k) ≈ k–1. (c) Examples of network motifs often encountered in ERNs. Again, enzymes are represented by nodes and substrates by edges. Edges with arrows indicate a positive interaction (e.g., a substrate that can be used as a reactant), while a flat end indicates an inhibitory effect. (d). Example of the difference in reaction velocity v as a function of substrate concentration between an enzyme with Michaelis–Menten (MM) kinetics and Hill-type kinetics with substrate affinity KM and turnover number kcat. (e) (Left) Example schematic of a full ERN where all interactions are included. (Right) A reduced model form where only essential interactions are maintained. (f) (Left) Bayesian analysis results in parameter estimate probability distributions instead of point estimates, allowing for nonsymmetric errors and standard deviations. (Right) Bayesian estimates can be used to generate a full probabilistic picture of possible enzymatic behavior.
Figure 3
Figure 3. Design of artificial ERNs with complex nonlinear dynamic behavior. (a) General design principle for ERNs with complex dynamic behavior. (b) Central reactions and motifs providing core nonlinearity. (c) Additional processes serving to alter the kinetics of the core motifs and connect different nodes. (d) Types of reactor or setup. (e) Examples of complex nonlinear dynamic behavior available in artificial ERNs. Illustrations: (e7) 50 μm polyacrylamide beads with immobilized urease in a solution containing 5 mM acetic buffer, 50 mM urea, and fluorescent dye SNARF-5,6, scale bar 100 μm, own data; (e8) redox forms of the dye ABTS in the solution containing glucose oxidase (GOx) and horseradish peroxidase (HRP). Adapted with permission from ref (78). Copyright 2018 Springer Nature.
Figure 4
Figure 4. Overview of enzymatic reaction networks that are controlled by different external stimuli. (a) Schematic representation of the trypsin oscillator where the enzyme activity is regulated by a photocleavable inhibitor. Adapted with permission from ref (86). Copyright 2020 John Wiley and Sons. (b) Photoswitchable inhibitor that can reversibly control the enzyme activity in an out-of-equilibrium system using light. Adapted with permission from ref (88). Copyright 2020 licensed under CC 4.0 American Chemical Society. (c) DASA-based polymersomes enable reversible control over the accessibility of substrate toward the active site of an enzyme by light. Adapted with permission from ref (93). Copyright 2022 licensed under CC 4.0 Springer Nature. (d) Ur–Est-based network where Ur increases the pH and Est decreases the pH. Adapted with permission from ref (65). Copyright 2021 John Wiley and sons. (e) Haber–Bosch process where NH3 can be generated from H2 and N2 using enzymes, hydrogenase, and nitrogenase by applying an electrochemical potential. Adapted with permission from ref (106). Copyright 2017 John Wiley and Sons. (f) Cascade network that is spatiotemporally controlled by sound. Adapted with permission from ref (111). Copyright 2022 licensed under CC 4.0 Springer Nature.
Figure 5
Figure 5. Overview of dynamic materials designed with enzymatic reaction networks. (a) Enzymatic polymerization of hydrogels. Adapted with permission from ref (140). Copyright 2016 licensed under CC 3.0 Royal Society of Chemistry. (b) Schematic representation of cross-link degradation of a hydrogel controlled by an enzymatic network. Adapted with permission from ref (146). Copyright 2017 John Wiley and sons.
Figure 6
Figure 6. Overview of enzyme-powered motile systems designed with enzymatic reaction networks. (a) Self-propulsion of stomatocytes by generating oxygen from glucose using an encapsulated metabolic enzymatic network. Adapted with permission from ref (162). Copyright 2022 American Chemical Society. (b) Illustration demonstrating expansion and contraction of a spring made of calcium alginate by antagonistic interaction of two enzymes, i.e., Ur and GOx. Adapted with permission from ref (169). Copyright 2021 Springer Nature.
Figure 7
Figure 7. Overview of communication in compartmentalized bioreactors facilitated by enzymatic reaction networks. (a) Communication between sender and receiver GUVs where the sender produced AMP which allosterically activated ERN within receiver GUVs. Adapted with permission from ref (179). Copyright 2020 licensed under CC 4.0 Springer Nature. (b) Cartoon representation of artificial response–retaliation behavior among protocell communities. In the presence of glucose, GOx containing protease-sensitive proteinosome (P) released acid (2) which disassembled pH-sensitive protease containing coacervate (CT) and released protease (3). Coacervate CK recaptured protease and eventually destroyed P. Adapted with permission from ref (175). Copyright 2019 John Wiley and sons.
Figure 8
Figure 8. (a) A diagram of low-cost substrate glucose being converted into pyruvate or acetyl-CoA as an intermediate and then depending on the selected pathway to isoprene, (199) monoterpenes, (200) PHB, (202) or cannabinoids (203) using purified enzymes (in orange) or to n-butanol, (205) mevalonate, (206) or limonene (208) using crude cell lysate (in yellow). (b) The purge valve concept, where the purge valve is “OFF” when there is low NADPH concentration and “ON” at high NADPH concentration. Adapted with permission from ref (201). Copyright 2014 Springer Nature.
Figure 9
Figure 9. Synthetic CO2 fixation pathways. (a) CETCH cycle converting CO2 to malate. Adapted with permission from ref (214). Copyright 2016 The American Association for the Advancement of Science. (b) POAP cycle converting CO2 to oxalate. Adapted with permission from ref (219). Copyright 2022 American Chemical Society. (c) Chemoenzymatic ASAS pathway converting CO2 to starch. Adapted with permission from ref (221). Copyright 2021 The American Association for the Advancement of Science. (d) Chemoenzymatic pathway converting CO2 to bioplastic polyhydroxybutyrate (PHB). Adapted with permission from ref (223). Copyright 2023 Royal Society of Chemistry.
Figure 10
Figure 10. (a) Schematic of the artificial organelle harvesting light to produce ATP and to drive endergonic reactions. Adapted with permission from ref (227). Copyright 2018 Springer Nature. (b) Schematic of self-constituting protein synthesis in artificial photosynthetic cells. Adapted with permission from ref (228). Copyright 2019 licensed under CC 4.0 Springer Nature. (c) Schematic of the production of phospholipids PE and PG by de novo-synthesized enzymes. Adapted with permission from ref (230). Copyright 2020 licensed under CC 4.0 Springer Nature.
Figure 11
Figure 11. (a) Schematic overview of commonly used ERN designs for two Boolean logic gates (AND and XOR). The computational output is based on an arbitrary threshold for the concentration of a product molecule. (b) A design for an ERN resulting in a multi-input perceptron, investigated in Pandi et al. (238) The design uses so-called transduction reactions to convert a range of different inputs into the same “transducer” substrate. Weights can be set by modifying enzyme concentrations. The final addition results in a roughly sigmoidal response. (c) Design for a chemical neuron, as investigated by Okumura et al. (239) A single neuron is constructed from specific DNA templates (curved lines) enabled by components of the polymerase–exonuclease–nickase (PEN) toolbox (shaded circle). (d) Design for a chemical multilayer perceptron. Individual neurons can be combined by replacing fluorescence-generating reporter strands by input strands for other neurons.
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- 1Colin, R.; Sourjik, V. Emergent properties of bacterial chemotaxis pathway. Curr. Opin. Microbiol. 2017, 39, 24– 33, DOI: 10.1016/j.mib.2017.07.0041https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2sXhtlSgtrvF&md5=7fe30bfe80a983a547e1e630bb85302cEmergent properties of bacterial chemotaxis pathwayColin, Remy; Sourjik, VictorCurrent Opinion in Microbiology (2017), 39 (), 24-33CODEN: COMIF7; ISSN:1369-5274. (Elsevier Ltd.)The chemotaxis pathway of Escherichia coli is the most studied sensory system in prokaryotes. The highly conserved general architecture of this pathway consists of two modules which mediate signal transduction and adaptation. The signal transduction module detects and amplifies changes in environmental conditions and rapidly transmits these signals to control bacterial swimming behavior. The adaptation module gradually resets the activity and sensitivity of the first module after initial stimulation and thereby enables the temporal comparisons necessary for bacterial chemotaxis. Recent exptl. and theor. work has unraveled multiple quant. features emerging from the interplay between these two modules. This has laid the groundwork for rationalization of these emerging properties in the context of the evolutionary optimization of the chemotactic behavior.
- 2Typas, A.; Sourjik, V. Bacterial protein networks: properties and functions. Nat. Rev. Microbiol. 2015, 13 (9), 559– 572, DOI: 10.1038/nrmicro35082https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2MXhtlSntrjF&md5=af4c069d9c63e1bd796b4ce3b4413822Bacterial protein networks: properties and functionsTypas, Athanasios; Sourjik, VictorNature Reviews Microbiology (2015), 13 (9), 559-572CODEN: NRMACK; ISSN:1740-1526. (Nature Publishing Group)A review. Distinct cellular functions are executed by sep. groups of proteins, organized into complexes or functional modules, which are ultimately interconnected in cell-wide protein networks. Understanding the structures and operational modes of these networks is one of the next great challenges in biol., and microorganisms are at the forefront of research in this field. In this Review, the authors present the current understanding of bacterial protein networks, their general properties and the tools that were used for systematically mapping and characterizing them. The authors then discuss two well-studied examples, the chemotaxis network and the cell cycle network in Escherichia coli, to illustrate how network architecture promotes function.
- 3Hunter, T. Protein Kinases and Phosphatases: The yin and yang of protein phosphorylation andsignaling. Cell 1995, 80, 225– 236, DOI: 10.1016/0092-8674(95)90405-03https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaK2MXjtlGnsrs%253D&md5=5262cb6e7a5175a97a11c8d87d8993c0Protein kinases and phosphatases: the Yin and Yang of protein phosphorylation and signalingHunter, TonyCell (Cambridge, Massachusetts) (1995), 80 (2), 225-36CODEN: CELLB5; ISSN:0092-8674. (Cell Press)A review, with 127 refs. Protein phosphorylation plays a cardinal role in regulating many cellular processes in eukaryotes. In particular, protein phosphorylation is a major factor in signal transduction pathways. Processes that are reversibly controlled by protein phosphorylation require not only a protein kinase but also a protein phosphatase. Target proteins are phosphorylated at specific sites by one or more protein kinases, and these phosphates are removed by specific protein phosphatases.
- 4Junttila, M. R.; Li, S. P.; Westermarck, J. Phosphatase-mediated crosstalk between MAPK signaling pathways in the regulation of cell survival. FASEB J. 2008, 22, 954– 965, DOI: 10.1096/fj.06-7859rev4https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD1cXktlWnsLs%253D&md5=574abc2eb18f7708967a8b606b027c79Phosphatase-mediated crosstalk between MAPK signaling pathways in the regulation of cell survivalJunttila, Melissa R.; Li, Song-Ping; Westermarck, JukkaFASEB Journal (2008), 22 (4), 954-965CODEN: FAJOEC; ISSN:0892-6638. (Federation of American Societies for Experimental Biology)A review. Mitogen-activated protein kinase (MAPK) pathways constitute a large modular network that regulates a variety of physiol. processes, such as cell growth, differentiation, and apoptotic cell death. The function of the ERK pathway has been depicted as survival-promoting, in essence by opposing the proapoptotic activity of the stress-activated c-Jun N-terminal kinase (JNK)/p38 MAPK pathways. However, recently published work suggests that extracellular regulated kinase (ERK) pathway activity is suppressed by JNK/p38 kinases during apoptosis induction. In this review, we will summarize the current knowledge about JNK/p38-mediated mechanisms that neg. regulate the ERK pathway. In particular, we will focus on phosphatases (PP2A, MKPs) as inhibitors of ERK pathway activity in regulating apoptosis. A model proposed in this review places the neg. regulation of the ERK pathway in a central position for the cellular decision-making process that dets. whether cells will live or die in response to apoptosis-promoting signals. In addn., we will discuss the potential functional relevance of neg. regulation of ERK pathway activity, for physiol. and pathol. conditions (e.g., cellular transformation).
- 5Saravia, J.; Raynor, J. L.; Chapman, N. M.; Lim, S. A.; Chi, H. Signaling networks in immunometabolism. Cell Res. 2020, 30 (4), 328– 342, DOI: 10.1038/s41422-020-0301-15https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB3cXlslGnsLg%253D&md5=3544210c937ed97ad5562107fff326cdSignaling networks in immunometabolismSaravia, Jordy; Raynor, Jana L.; Chapman, Nicole M.; Lim, Seon Ah; Chi, HongboCell Research (2020), 30 (4), 328-342CODEN: CREEB6; ISSN:1001-0602. (Nature Research)A review. Adaptive immunity is essential for pathogen and tumor eradication, but may also trigger uncontrolled or pathol. inflammation. T cell receptor, co-stimulatory and cytokine signals coordinately dictate specific signaling networks that trigger the activation and functional programming of T cells. In addn., cellular metab. promotes T cell responses and is dynamically regulated through the interplay of serine/threonine kinases, immunol. cues and nutrient signaling networks. In this review, we summarize the upstream regulators and signaling effectors of key serine/threonine kinase-mediated signaling networks, including PI3K-AGC kinases, mTOR and LKB1-AMPK pathways that regulate metab., esp. in T cells. We also provide our perspectives about the pending questions and clin. applicability of immunometabolic signaling. Understanding the regulators and effectors of immunometabolic signaling networks may uncover therapeutic targets to modulate metabolic programming and T cell responses in human disease.
- 6Fink, T.; Lonzaric, J.; Praznik, A.; Plaper, T.; Merljak, E.; Leben, K.; Jerala, N.; Lebar, T.; Strmsek, Z.; Lapenta, F. Design of fast proteolysis-based signaling and logic circuits in mammalian cells. Nat. Chem. Biol. 2019, 15 (2), 115– 122, DOI: 10.1038/s41589-018-0181-66https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC1cXisVyhurjE&md5=c4085d55b59081ee346c5d14650b709eDesign of fast proteolysis-based signaling and logic circuits in mammalian cellsFink, Tina; Lonzaric, Jan; Praznik, Arne; Plaper, Tjasa; Merljak, Estera; Leben, Katja; Jerala, Nina; Lebar, Tina; Strmsek, Ziga; Lapenta, Fabio; Bencina, Mojca; Jerala, RomanNature Chemical Biology (2019), 15 (2), 115-122CODEN: NCBABT; ISSN:1552-4450. (Nature Research)Cellular signal transduction is predominantly based on protein interactions and their post-translational modifications, which enable a fast response to input signals. Owing to difficulties in designing new unique protein-protein interactions, designed cellular logic has focused on transcriptional regulation; however, that process has a substantially slower response, because it requires transcription and translation. Here, the authors present de novo design of modular, scalable signaling pathways based on proteolysis and designed coiled coils (CC) and implemented in mammalian cells. A set of split proteases with highly specific orthogonal cleavage motifs was constructed and combined with strategically positioned cleavage sites and designed orthogonal CC dimerizing domains with tunable affinity for competitive displacement after proteolytic cleavage. This framework enabled the implementation of Boolean logic functions and signaling cascades in mammalian cells. The designed split-protease-cleavable orthogonal-CC-based (SPOC) logic circuits enable response to chem. or biol. signals within minutes rather than hours and should be useful for diverse medical and nonmedical applications.
- 7Feng, Z.; Wang, H.; Wang, F.; Oh, Y.; Berciu, C.; Cui, Q.; Egelman, E. H.; Xu, B. Artificial intracellular filaments. Cell Rep. Phys. Sci. 2020, 1 (7), 100085, DOI: 10.1016/j.xcrp.2020.100085There is no corresponding record for this reference.
- 8Kohyama, S.; Merino-Salomon, A.; Schwille, P. In vitro assembly, positioning and contraction of a division ring in minimal cells. Nat. Commun. 2022, 13 (1), 6098, DOI: 10.1038/s41467-022-33679-x8https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB38Xis1Oru7jO&md5=3c59deeb8978156ebc6202f7d269eb0cIn vitro assembly, positioning and contraction of a division ring in minimal cellsKohyama, Shunshi; Merino-Salomon, Adrian; Schwille, PetraNature Communications (2022), 13 (1), 6098CODEN: NCAOBW; ISSN:2041-1723. (Nature Portfolio)Constructing a minimal machinery for autonomous self-division of synthetic cells is a major goal of bottom-up synthetic biol. One paradigm has been the E. coli divisome, with the MinCDE protein system guiding assembly and positioning of a presumably contractile ring based on FtsZ and its membrane adaptor FtsA. Here, we demonstrate the full in vitro reconstitution of this machinery consisting of five proteins within lipid vesicles, allowing to observe the following sequence of events in real time: 1 Assembly of an isotropic filamentous FtsZ network, 2 its condensation into a ring-like structure, along with pole-to-pole mode selection of Min oscillations resulting in equatorial positioning, and 3 onset of ring constriction, deforming the vesicles from spherical shape. Besides demonstrating these essential features, we highlight the importance of decisive exptl. factors, such as macromol. crowding. Our results provide an exceptional showcase of the emergence of cell division in a minimal system, and may represent a step towards developing a synthetic cell.
- 9Fisher, A. K.; Freedman, B. G.; Bevan, D. R.; Senger, R. S. A review of metabolic and enzymatic engineering strategies for designing and optimizing performance of microbial cell factories. Comput. Struct. Biotechnol. J. 2014, 11 (18), 91– 99, DOI: 10.1016/j.csbj.2014.08.0109https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A280%3ADC%252BC2M3ms1Crsg%253D%253D&md5=a57acb850382c134d91c29404e683258A review of metabolic and enzymatic engineering strategies for designing and optimizing performance of microbial cell factoriesFisher Amanda K; Freedman Benjamin G; Senger Ryan S; Bevan David RComputational and structural biotechnology journal (2014), 11 (18), 91-9 ISSN:2001-0370.Microbial cell factories (MCFs) are of considerable interest to convert low value renewable substrates to biofuels and high value chemicals. This review highlights the progress of computational models for the rational design of an MCF to produce a target bio-commodity. In particular, the rational design of an MCF involves: (i) product selection, (ii) de novo biosynthetic pathway identification (i.e., rational, heterologous, or artificial), (iii) MCF chassis selection, (iv) enzyme engineering of promiscuity to enable the formation of new products, and (v) metabolic engineering to ensure optimal use of the pathway by the MCF host. Computational tools such as (i) de novo biosynthetic pathway builders, (ii) docking, (iii) molecular dynamics (MD) and steered MD (SMD), and (iv) genome-scale metabolic flux modeling all play critical roles in the rational design of an MCF. Genome-scale metabolic flux models are of considerable use to the design process since they can reveal metabolic capabilities of MCF hosts. These can be used for host selection as well as optimizing precursors and cofactors of artificial de novo biosynthetic pathways. In addition, recent advances in genome-scale modeling have enabled the derivation of metabolic engineering strategies, which can be implemented using the genomic tools reviewed here as well.
- 10Hirschi, S.; Ward, T. R.; Meier, W. P.; Muller, D. J.; Fotiadis, D. Synthetic biology: bottom-up assembly of molecular systems. Chem. Rev. 2022, 122 (21), 16294– 16328, DOI: 10.1021/acs.chemrev.2c0033910https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB38XisFWmtrjF&md5=598a8e43708beb84e525bd06c876310bSynthetic Biology: Bottom-Up Assembly of Molecular SystemsHirschi, Stephan; Ward, Thomas R.; Meier, Wolfgang P.; Muller, Daniel J.; Fotiadis, DimitriosChemical Reviews (Washington, DC, United States) (2022), 122 (21), 16294-16328CODEN: CHREAY; ISSN:0009-2665. (American Chemical Society)A review. The bottom-up assembly of biol. and chem. components opens exciting opportunities to engineer artificial vesicular systems for applications with previously unmet requirements. The modular combination of scaffolds and functional building blocks enables the engineering of complex systems with biomimetic or new-to-nature functionalities. Inspired by the compartmentalized organization of cells and organelles, lipid or polymer vesicles are widely used as model membrane systems to investigate the translocation of solutes and the transduction of signals by membrane proteins. The bottom-up assembly and functionalization of such artificial compartments enables full control over their compn. and can thus provide specifically optimized environments for synthetic biol. processes. This review aims to inspire future endeavors by providing a diverse toolbox of mol. modules, engineering methodologies, and different approaches to assemble artificial vesicular systems. Important tech. and practical aspects are addressed and selected applications are presented, highlighting particular achievements and limitations of the bottom-up approach. Complementing the cutting-edge technol. achievements, fundamental aspects are also discussed to cater to the inherently diverse background of the target audience, which results from the interdisciplinary nature of synthetic biol. The engineering of proteins as functional modules and the use of lipids and block copolymers as scaffold modules for the assembly of functionalized vesicular systems are explored in detail. Particular emphasis is placed on ensuring the controlled assembly of these components into increasingly complex vesicular systems. Finally, all descriptions are presented in the greater context of engineering valuable synthetic biol. systems for applications in biocatalysis, biosensing, bioremediation, or targeted drug delivery.
- 11Boguñá, M.; Bonamassa, I.; De Domenico, M.; Havlin, S.; Krioukov, D.; Serrano, M. Á. Network geometry. Nat. Rev. Phys. 2021, 3 (2), 114– 135, DOI: 10.1038/s42254-020-00264-4There is no corresponding record for this reference.
- 12Ravasz, E.; Somera, A. L.; Mongru, D. A.; Oltvai, Z. N.; Barabási, A.-L. Hierarchical organization of modularity in metabolic networks. Science 2002, 297, 1551– 1555, DOI: 10.1126/science.107337412https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD38XmslSjtLs%253D&md5=a7cf905a9f21a5fc614694635efa1f14Hierarchical organization of modularity in metabolic networksRavasz, E.; Somera, A. L.; Mongru, D. A.; Oltvai, Z. N.; Barabasi, A.-L.Science (Washington, DC, United States) (2002), 297 (5586), 1551-1555CODEN: SCIEAS; ISSN:0036-8075. (American Association for the Advancement of Science)Spatially or chem. isolated functional modules composed of several cellular components and carrying discrete functions are considered fundamental building blocks of cellular organization, but their presence in highly integrated biochem. networks lacks quant. support. Here, we show that the metabolic networks of 43 distinct organisms are organized into many small, highly connected topol. modules that combine in a hierarchical manner into larger, less cohesive units, with their no. and degree of clustering following a power law. Within Escherichia coli, the uncovered hierarchical modularity closely overlaps with known metabolic functions. The identified network architecture may be generic to system-level cellular organization.
- 13Barabási, A.-L.; Oltvai, Z. N. Network biology: understanding the cell’s functional organization. Nat. Rev. Genet. 2004, 5 (2), 101– 113, DOI: 10.1038/nrg127213https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD2cXovV2mtg%253D%253D&md5=2ac9082dfaf88d56da1fdd75b9edddb4Network biology: Understanding the cell's functional organizationBarabasi, Albert-Laszlo; Oltvai, Zoltan N.Nature Reviews Genetics (2004), 5 (2), 101-113CODEN: NRGAAM; ISSN:1471-0056. (Nature Publishing Group)A key aim of postgenomic biomedical research is to systematically catalog all mols. and their interactions within a living cell. There is a clear need to understand how these mols. and the interactions between them det. the function of this enormously complex machinery, both in isolation and when surrounded by other cells. Rapid advances in network biol. indicate that cellular networks are governed by universal laws and offer a new conceptual framework that could potentially revolutionize our view of biol. and disease pathologies in the twenty-first century.
- 14Serrano, M. A.; Boguñá, M.; Sagués, F. Uncovering the hidden geometry behind metabolic networks. Mol. Biosyst. 2012, 8 (3), 843– 850, DOI: 10.1039/c2mb05306c14https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC38XitVSmtrk%253D&md5=7f0d358828c360215e2e49c1fc362131Uncovering the hidden geometry behind metabolic networksSerrano, M. Angeles; Boguna, Marian; Sagues, FrancescMolecular BioSystems (2012), 8 (3), 843-850CODEN: MBOIBW; ISSN:1742-2051. (Royal Society of Chemistry)Metab. is a fascinating cell machinery underlying life and disease and genome-scale reconstructions provide us with a captivating view of its complexity. However, deciphering the relationship between metabolic structure and function remains a major challenge. In particular, turning obsd. structural regularities into organizing principles underlying systemic functions is a crucial task that can be significantly addressed after endowing complex network representations of metab. with the notion of geometric distance. Here, we design a cartog. map of metabolic networks by embedding them into a simple geometry that provides a natural explanation for their obsd. network topol. and that codifies node proximity as a measure of hidden structural similarities. We assume a simple and general connectivity law that gives more probability of interaction to metabolite/reaction pairs which are closer in the hidden space. Remarkably, we find an astonishing congruency between the architecture of E. coli and human cell metabs. and the underlying geometry. In addn., the formalism unveils a backbone-like structure of connected biochem. pathways on the basis of a quant. cross-talk. Pathways thus acquire a new perspective which challenges their classical view as self-contained functional units.
- 15Kim, H.; Smith, H. B.; Mathis, C.; Raymond, J.; Walker, S. Universal scaling across biochemical networks on Earth. Sci. Adv. 2019, 5, eaau0149, DOI: 10.1126/sciadv.aau0149There is no corresponding record for this reference.
- 16Gagler, D. C.; Karas, B.; Kempes, C. P.; Malloy, J.; Mierzejewski, V.; Goldman, A. D.; Kim, H.; Walker, S. I. Scaling laws in enzyme function reveal a new kind of biochemical universality. Proc. Natl. Acad. Sci. U. S. A. 2022, 119 (9), e2106655119, DOI: 10.1073/pnas.210665511916https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB38Xns1Kltrg%253D&md5=d18d6cb44a63dc6882871166265aa65aScaling laws in enzyme function reveal a new kind of biochemical universalityGagler, Dylan C.; Karas, Bradley; Kempes, Christopher P.; Malloy, John; Mierzejewski, Veronica; Goldman, Aaron D.; Kim, Hyunju; Walker, Sara I.Proceedings of the National Academy of Sciences of the United States of America (2022), 119 (9), e2106655119CODEN: PNASA6; ISSN:0027-8424. (National Academy of Sciences)All life on Earth is unified by its use of a shared set of component chem. compds. and reactions, providing a detailed model for universal biochem. However, this notion of universality is specific to known biochem. and does not allow quant. predictions about examples not yet obsd. Here, we introduce a more generalizable concept of biochem. universality that is more akin to the kind of universality found in physics. Using annotated genomic datasets including an ensemble of 11,955 metagenomes, 1,282 archaea, 11,759 bacteria, and 200 eukaryotic taxa, we show how enzyme functions form universality classes with common scaling behavior in their relative abundances across the datasets. We verify that these scaling laws are not explained by the presence of compds., reactions, and enzyme functions shared across known examples of life. We demonstrate how these scaling laws can be used as a tool for inferring properties of ancient life by comparing their predictions with a consensus model for the last universal common ancestor (LUCA). We also illustrate how network analyses shed light on the functional principles underlying the obsd. scaling behaviors. Together, our results establish the existence of a new kind of biochem. universality, independent of the details of life on Earth's component chem., with implications for guiding our search for missing biochem. diversity on Earth or for biochemistries that might deviate from the exact chem. makeup of life as we know it, such as at the origins of life, in alien environments, or in the design of synthetic life.
- 17Guell, O.; Sagues, F.; Serrano, M. A. Essential plasticity and redundancy of metabolism unveiled by synthetic lethality analysis. PLoS Comput. Biol. 2014, 10 (5), e1003637, DOI: 10.1371/journal.pcbi.100363717https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2cXhsVGkt7jP&md5=a89b8a58deb751d9506ef1432446fcddEssential plasticity and redundancy of metabolism unveiled by synthetic lethality analysisGuell, Oriol; Sagues, Francesc; Serrano, M. AngelesPLoS Computational Biology (2014), 10 (5), e1003637/1-e1003637/10, 10 pp.CODEN: PCBLBG; ISSN:1553-7358. (Public Library of Science)We unravel how functional plasticity and redundancy are essential mechanisms underlying the ability to survive of metabolic networks. We perform an exhaustive computational screening of synthetic lethal reaction pairs in Escherichia coli in a minimal medium and we find that synthetic lethal pairs divide in two different groups depending on whether the synthetic lethal interaction works as a backup or as a parallel use mechanism, the first corresponding to essential plasticity and the second to essential redundancy. In E. coli, the anal. of pathways entanglement through essential redundancy supports the view that synthetic lethality affects preferentially a single function or pathway. In contrast, essential plasticity, the dominant class, tends to be inter-pathway but strongly localized and unveils Cell Envelope Biosynthesis as an essential backup for Membrane Lipid Metab. When comparing E. coli and Mycoplasma pneumoniae, we find that the metabolic networks of the two organisms exhibit a large difference in the relative importance of plasticity and redundancy which is consistent with the conjecture that plasticity is a sophisticated mechanism that requires a complex organization. Finally, coessential reaction pairs are explored in different environmental conditions to uncover the interplay between the two mechanisms. We find that synthetic lethal interactions and their classification in plasticity and redundancy are basically insensitive to medium compn., and are highly conserved even when the environment is enriched with nonessential compds. or overconstrained to decrease max. biomass formation.
- 18Sambamoorthy, G.; Raman, K. Understanding the evolution of functional redundancy in metabolic networks. Bioinformatics 2018, 34 (17), i981– i987, DOI: 10.1093/bioinformatics/bty60418https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC1MXhtVamu77N&md5=dc84099605e8635841180083ca9daca9Understanding the evolution of functional redundancy in metabolic networksSambamoorthy, Gayathri; Raman, KarthikBioinformatics (2018), 34 (17), i981-i987CODEN: BOINFP; ISSN:1367-4811. (Oxford University Press)Metabolic networks have evolved to reduce the disruption of key metabolic pathways by the establishment of redundant genes/reactions. Synthetic lethals in metabolic networks provide a window to study these functional redundancies. While synthetic lethals have been previously studied in different organisms, there has been no study on how the synthetic lethals are shaped during adaptation/evolution. To understand the adaptive functional redundancies that exist in metabolic networks, we here explore a vast space of 'random' metabolic networks evolved on a glucose environment. We examine essential and synthetic lethal reactions in these random metabolic networks, evaluating over 39 billion phenotypes using an efficient algorithm previously developed in our lab, Fast-SL. We establish that nature tends to harbor higher levels of functional redundancies compared with random networks. We then examd. the propensity for different reactions to compensate for one another and show that certain key metabolic reactions that are necessary for growth in a particular growth medium show much higher redundancies, and can partner with hundreds of different reactions across the metabolic networks that we studied. We also observe that certain redundancies are unique to environments while some others are obsd. in all environments. Interestingly, we observe that even very diverse reactions, such as those belonging to distant pathways, show synthetic lethality, illustrating the distributed nature of robustness in metab. Our study paves the way for understanding the evolution of redundancy in metabolic networks, and sheds light on the varied compensation mechanisms that serve to enhance robustness.
- 19Alon, U. Network motifs: theory and experimental approaches. Nat. Rev. Genet. 2007, 8, 450– 461, DOI: 10.1038/nrg210219https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD2sXlsVSktro%253D&md5=ad292568100bf626f7076dee816826ffNetwork motifs: theory and experimental approachesAlon, UriNature Reviews Genetics (2007), 8 (6), 450-461CODEN: NRGAAM; ISSN:1471-0056. (Nature Publishing Group)A review. Transcription regulation networks control the expression of genes. The transcription networks of well-studied microorganisms appear to be made up of a small set of recurring regulation patterns, called network motifs. The same network motifs have recently been found in diverse organisms from bacteria to humans, suggesting that they serve as basic building blocks of transcription networks. Here, the author reviews network motifs and their functions, with an emphasis on exptl. studies. Network motifs in other biol. networks are also mentioned, including signaling and neuronal networks.
- 20Goldbeter, A.; Koshland, D. E. An amplified sensitivity arising from covalent modification in biological systems. Proc. Natl. Acad. Sci. U. S. A. 1981, 78, 6840– 6844, DOI: 10.1073/pnas.78.11.684020https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaL38XivVOi&md5=949ebb2ad47f4839b5e72c05abbc19f9An amplified sensitivity arising from covalent modification in biological systemsGoldbeter, Albert; Koshland, Daniel E., Jr.Proceedings of the National Academy of Sciences of the United States of America (1981), 78 (11), 6840-4CODEN: PNASA6; ISSN:0027-8424.The transient and steady-state behavior of a reversible covalent modification system is examd. When the modifying enzymes operate outside the region of 1st-order kinetics, small percentage changes in the concn. of the effector controlling either of the modifying enzymes can give much larger percentage changes in the amt. of modified protein. This amplification of the response to a stimulus can provide addnl. sensitivity in biol. control, equiv. to that of allosteric proteins with high Hill coeffs.
- 21Koshland, D. E., Jr.; Goldbeter, A.; Stock, J. B. Amplification and adaptation in regulatory and sensory systems. Science 1982, 217, 220– 225, DOI: 10.1126/science.708955621https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaL38XkvVOisLs%253D&md5=e2492db5ae8999e0775f3bd576e1cc98Amplification and adaptation in regulatory and sensory systemsKoshland, Daniel E., Jr.; Goldbeter, Albert; Stock, Jeffry B.Science (Washington, DC, United States) (1982), 217 (4556), 220-5CODEN: SCIEAS; ISSN:0036-8075.A review with 31 refs. on how biol. systems respond to sensory inputs and changing metabolic conditions both by amplifying signals and by adapting to them.
- 22Barkai, N.; Leibler, S. Robustness in simple biochemical networks. Nature 1997, 387, 913– 917, DOI: 10.1038/4319922https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaK2sXkt1Kiur8%253D&md5=76427ddf0b596f4c18e024acb21182cfRobustness in simple biochemical networksBarkai, N.; Leibler, S.Nature (London) (1997), 387 (6636), 913-917CODEN: NATUAS; ISSN:0028-0836. (Macmillan Magazines)A review, with 21 refs. Cells use complex networks of interacting mol. components to transfer and process information. These "computational devices of living cells" are responsible for many important cellular processes, including cell-cycle regulation and signal transduction. Here we address the issue of the sensitivity of the networks to variations in their biochem. parameters. We propose a mechanism for robust adaptation in simple signal transduction networks. We show that this mechanism applies in particular to bacterial chemotaxis. This is demonstrated within a quant. model which explains, in a unified way, many aspects of chemotaxis, including proper responses to chem. gradients. The adaptation property is a consequence of the networks's connectivity and does not require the 'fine-tuning of parameters. We argue that the key properties of biochem. networks should be robust to ensure their proper functioning.
- 23Milo, R.; Shen-Orr, S.; Itzkovitz, S.; Kashtan, N.; Chklovskii, D.; Alon, U. Network motifs: simple building blocks of complex networks. Science. 2002, 298, 824– 827, DOI: 10.1126/science.298.5594.82423https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD38XotFSntb4%253D&md5=a83828b13c33bee8c7528922d91502b7Network Motifs: Simple Building Blocks of Complex NetworksMilo, R.; Shen-Orr, S.; Itzkovitz, S.; Kashtan, N.; Chklovskii, D.; Alon, U.Science (Washington, DC, United States) (2002), 298 (5594), 824-827CODEN: SCIEAS; ISSN:0036-8075. (American Association for the Advancement of Science)Complex networks are studied across many fields of science. To uncover their structural design principles, we defined "network motifs," patterns of interconnections occurring in complex networks at nos. that are significantly higher than those in randomized networks. We found such motifs in networks from biochem., neurobiol., ecol., and engineering. The motifs shared by ecol. food webs were distinct from the motifs shared by the genetic networks of Escherichia coli and Saccharomyces cerevisiae or from those found in the World Wide Web. Similar motifs were found in networks that perform information processing, even though they describe elements as different as biomols. within a cell and synaptic connections between neurons in Caenorhabditis elegans. Motifs may thus define universal classes of networks. This approach may uncover the basic building blocks of most networks.
- 24Tyson, J. J.; Novak, B. Functional motifs in biochemical reaction networks. Annu. Rev. Phys. Chem. 2010, 61, 219– 240, DOI: 10.1146/annurev.physchem.012809.10345724https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3cXmt1ajsL8%253D&md5=8b514d486aa599e24f0185cfface6067Functional motifs in biochemical reaction networksTyson, John J.; Novak, BelaAnnual Review of Physical Chemistry (2010), 61 (), 219-240CODEN: ARPLAP; ISSN:0066-426X. (Annual Reviews Inc.)A review. The signal-response characteristics of a living cell are detd. by complex networks of interacting genes, proteins, and metabolites. Understanding how cells respond to specific challenges, how these responses are contravened in diseased cells, and how to intervene pharmacol. in the decision-making processes of cells requires an accurate theory of the information-processing capabilities of macromol. regulatory networks. Adopting an engineer's approach to control systems, we ask whether realistic cellular control networks can be decompd. into simple regulatory motifs that carry out specific functions in a cell. We show that such functional motifs exist and review the exptl. evidence that they control cellular responses as expected.
- 25Novak, B.; Tyson, J. J. Design principles of biochemical oscillators. Nat. Rev. Mol. Cell Biol. 2008, 9 (12), 981– 991, DOI: 10.1038/nrm253025https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD1cXhsVWhurnL&md5=2014e5749f715415e81b8afeec1b6008Design principles of biochemical oscillatorsNovak, Bela; Tyson, John J.Nature Reviews Molecular Cell Biology (2008), 9 (12), 981-991CODEN: NRMCBP; ISSN:1471-0072. (Nature Publishing Group)A review. Cellular rhythms are generated by complex interactions among genes, proteins and metabolites. They are used to control every aspect of cell physiol., from signaling, motility and development to growth, division and death. We consider specific examples of oscillatory processes and discuss four general requirements for biochem. oscillations: neg. feedback, time delay, sufficient nonlinearity of the reaction kinetics and proper balancing of the timescales of opposing chem. reactions. Pos. feedback is one mechanism to delay the neg.-feedback signal. Biol. oscillators can be classified according to the topol. of the pos.- and neg.-feedback loops in the underlying regulatory mechanism.
- 26Araujo, R. P.; Liotta, L. A. Universal structures for adaptation in biochemical reaction networks. Nat. Commun. 2023, 14 (1), 2251, DOI: 10.1038/s41467-023-38011-926https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB3sXos1yqsrk%253D&md5=51df0502a1ff9903b4006ae2e353234eUniversal structures for adaptation in biochemical reaction networksAraujo, Robyn P.; Liotta, Lance A.Nature Communications (2023), 14 (1), 2251CODEN: NCAOBW; ISSN:2041-1723. (Nature Portfolio)At the mol. level, the evolution of life is driven by the generation and diversification of adaptation mechanisms. A universal description of adaptation-capable chem. reaction network (CRN) structures has remained elusive until now, since currently-known criteria for adaptation apply only to a tiny subset of possible CRNs. Here we identify the definitive structural requirements that characterize all adaptation-capable collections of interacting mols., however large or complex. We show that these network structures implement a form of integral control in which multiple independent integrals can collaborate to confer the capacity for adaptation on specific mols. Using an algebraic algorithm informed by these findings, we demonstrate the existence of embedded integrals in a variety of biol. important CRNs that have eluded previous methods, and for which adaptation has been obsd. exptl. This definitive picture of biol. adaptation at the level of intermol. interactions represents a blueprint for adaptation-capable signaling networks across all domains of life, and for the design of synthetic biosystems.
- 27Shellman, E. R.; Burant, C. F.; Schnell, S. Network motifs provide signatures that characterize metabolism. Mol. Biosyst. 2013, 9 (3), 352– 360, DOI: 10.1039/c2mb25346a27https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3sXitVequ70%253D&md5=d1d810be1fa3c4459ff0e74fdb3d2f5fNetwork motifs provide signatures that characterize metabolismShellman, Erin R.; Burant, Charles F.; Schnell, SantiagoMolecular BioSystems (2013), 9 (3), 352-360CODEN: MBOIBW; ISSN:1742-2051. (Royal Society of Chemistry)Motifs are repeating patterns that det. the local properties of networks. In this work, we characterized all 3-node motifs using enzyme commission nos. of the International Union of Biochem. and Mol. Biol. to show that motif abundance is related to biochem. function. Further, we present a comparative anal. of motif distributions in the metabolic networks of 21 species across six kingdoms of life. We found the distribution of motif abundances to be similar between species, but unique across cellular organelles. Finally, we show that motifs are able to capture inter-species differences in metabolic networks and that mol. differences between some biol. species are reflected by the distribution of motif abundances in metabolic networks.
- 28Beber, M. E.; Fretter, C.; Jain, S.; Sonnenschein, N.; Muller-Hannemann, M.; Hutt, M. T. Artefacts in statistical analyses of network motifs: general framework and application to metabolic networks. J. R. Soc. Interface 2012, 9 (77), 3426– 3435, DOI: 10.1098/rsif.2012.049028https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A280%3ADC%252BC38fotl2rtg%253D%253D&md5=e778ca3b28d1d464a65eaaaf70e21e5bArtefacts in statistical analyses of network motifs: general framework and application to metabolic networksBeber Moritz Emanuel; Fretter Christoph; Jain Shubham; Sonnenschein Nikolaus; Muller-Hannemann Matthias; Hutt Marc-ThorstenJournal of the Royal Society, Interface (2012), 9 (77), 3426-35 ISSN:.Few-node subgraphs are the smallest collective units in a network that can be investigated. They are beyond the scale of individual nodes but more local than, for example, communities. When statistically over- or under-represented, they are called network motifs. Network motifs have been interpreted as building blocks that shape the dynamic behaviour of networks. It is this promise of potentially explaining emergent properties of complex systems with relatively simple structures that led to an interest in network motifs in an ever-growing number of studies and across disciplines. Here, we discuss artefacts in the analysis of network motifs arising from discrepancies between the network under investigation and the pool of random graphs serving as a null model. Our aim was to provide a clear and accessible catalogue of such incongruities and their effect on the motif signature. As a case study, we explore the metabolic network of Escherichia coli and show that only by excluding ever more artefacts from the motif signature a strong and plausible correlation with the essentiality profile of metabolic reactions emerges.
- 29Piephoff, D. E.; Wu, J.; Cao, J. Conformational nonequilibrium enzyme kinetics: generalized Michaelis-Menten equation. J. Phys. Chem. Lett. 2017, 8 (15), 3619– 3623, DOI: 10.1021/acs.jpclett.7b0121029https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2sXht1Sgu7rF&md5=9f5fa6ac5ff779cfe901d56b6dd7eaf0Conformational Nonequilibrium Enzyme Kinetics: Generalized Michaelis-Menten EquationPiephoff, D. Evan; Wu, Jianlan; Cao, JianshuJournal of Physical Chemistry Letters (2017), 8 (15), 3619-3623CODEN: JPCLCD; ISSN:1948-7185. (American Chemical Society)In a conformational nonequil. steady state (cNESS), enzyme turnover is modulated by the underlying conformational dynamics. On the basis of a discrete kinetic network model, we use the integrated probability flux balance method to derive the cNESS turnover rate for a conformation-modulated enzymic reaction. The traditional Michaelis-Menten (MM) rate equation is extended to a generalized form, which includes non-MM corrections induced by conformational population currents within combined cyclic kinetic loops. When conformational detailed balance is satisfied, the turnover rate reduces to the MM functional form, explaining its validity for many enzymic systems. For the first time, a one-to-one correspondence is established between non-MM terms and combined cyclic loops with unbalanced conformational currents. Cooperativity resulting from nonequil. conformational dynamics can be achieved in enzymic reactions, and we provide a novel, rigorous means of predicting and characterizing such behavior. Our generalized MM equation affords a systematic approach for exploring cNESS enzyme kinetics.
- 30Rohwer, J. M.; Hanekom, A. J.; Crous, C.; Snoep, J. L.; Hofmeyr, J. H. Evaluation of a simplified generic bi-substrate rate equation for computational systems biology. Syst. Biol. (Stevenage) 2006, 153 (5), 338– 41, DOI: 10.1049/ip-syb:2006002630https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A280%3ADC%252BD28rmtVyktQ%253D%253D&md5=a8759b27f01cd966cccbffc6dd2085a4Evaluation of a simplified generic bi-substrate rate equation for computational systems biologyRohwer J M; Hanekom A J; Crous C; Snoep J L; Hofmeyr J H SSystems biology (2006), 153 (5), 338-41 ISSN:1741-2471.The evaluation of a generic simplified bi-substrate enzyme kinetic equation, whose derivation is based on the assumption of equilibrium binding of substrates and products in random order, is described. This equation is much simpler than the mechanistic (ordered and ping-pong) models, in that it contains fewer parameters (that is, no K(i) values for the substrates and products). The generic equation fits data from both the ordered and the ping-pong models well over a wide range of substrate and product concentrations. In the cases where the fit is not perfect, an improved fit can be obtained by considering the rate equation for only a single set of product concentrations. Due to its relative simplicity in comparison to the mechanistic models, this equation will be useful for modelling bi-substrate reactions in computational systems biology.
- 31Transtrum, M. K.; Machta, B. B.; Brown, K. S.; Daniels, B. C.; Myers, C. R.; Sethna, J. P. Perspective: sloppiness and emergent theories in physics, biology, and beyond. J. Chem. Phys. 2015, 143 (1), 010901 DOI: 10.1063/1.492306631https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2MXhtFSktr3N&md5=558fce9562787043593223a854d25541Perspective: Sloppiness and emergent theories in physics, biology, and beyondTranstrum, Mark K.; Machta, Benjamin B.; Brown, Kevin S.; Daniels, Bryan C.; Myers, Christopher R.; Sethna, James P.Journal of Chemical Physics (2015), 143 (1), 010901/1-010901/13CODEN: JCPSA6; ISSN:0021-9606. (American Institute of Physics)Large scale models of phys. phenomena demand the development of new statistical and computational tools in order to be effective. Many such models are "sloppy," i.e., exhibit behavior controlled by a relatively small no. of parameter combinations. We review an information theoretic framework for analyzing sloppy models. This formalism is based on the Fisher information matrix, which is interpreted as a Riemannian metric on a parameterized space of models. Distance in this space is a measure of how distinguishable two models are based on their predictions. Sloppy model manifolds are bounded with a hierarchy of widths and extrinsic curvatures. The manifold boundary approxn. can ext. the simple, hidden theory from complicated sloppy models. We attribute the success of simple effective models in physics as likewise emerging from complicated processes exhibiting a low effective dimensionality. We discuss the ramifications and consequences of sloppy models for biochem. and science more generally. We suggest that the reason our complex world is understandable is due to the same fundamental reason: simple theories of macroscopic behavior are hidden inside complicated microscopic processes. (c) 2015 American Institute of Physics.
- 32Gutenkunst, R. N.; Waterfall, J. J.; Casey, F. P.; Brown, K. S.; Myers, C. R.; Sethna, J. P. Universally sloppy parameter sensitivities in systems biology models. PLoS Comput. Biol. 2007, 3 (10), e189, DOI: 10.1371/journal.pcbi.0030189There is no corresponding record for this reference.
- 33Transtrum, M. K.; Qiu, P. Model reduction by manifold boundaries. Phys. Rev. Lett. 2014, 113 (9), 098701 DOI: 10.1103/PhysRevLett.113.09870133https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2cXhslSht7nO&md5=00bb730d154a553d6ae135f505f0c54fModel reduction by manifold boundariesTranstrum, Mark K.; Qiu, PengPhysical Review Letters (2014), 113 (9), 098701/1-098701/6CODEN: PRLTAO; ISSN:0031-9007. (American Physical Society)Understanding the collective behavior of complex systems from their basic components is a difficult yet fundamental problem in science. Existing model redn. techniques are either applicable under limited circumstances or produce "black boxes" disconnected from the microscopic physics. We propose a new approach by translating the model redn. problem for an arbitrary statistical model into a geometric problem of constructing a low-dimensional, submanifold approxn. to a high-dimensional manifold. When models are overly complex, we use the observation that the model manifold is bounded with a hierarchy of widths and propose using the boundaries as submanifold approxns. We refer to this approach as the manifold boundary approxn. method. We apply this method to several models, including a sum of exponentials, a dynamical systems model of protein signaling, and a generalized Ising model. By focusing on parameters rather than phys. degrees of freedom, the approach unifies many other model redn. techniques, such as singular limits, equil. approxns., and the renormalization group, while expanding the domain of tractable models. The method produces a series of approxns. that decrease the complexity of the model and reveal how microscopic parameters are systematically "compressed" into a few macroscopic degrees of freedom, effectively building a bridge between the microscopic and the macroscopic descriptions.
- 34van de Schoot, R.; Depaoli, S.; King, R.; Kramer, B.; Märtens, K.; Tadesse, M. G.; Vannucci, M.; Gelman, A.; Veen, D.; Willemsen, J.; Yau, C. Bayesian statistics and modelling. Nat. Rev. Methods Primers 2021, 1, 16, DOI: 10.1038/s43586-020-00001-234https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB38XjsVGgur0%253D&md5=fc3e76187d5fa0268f12409a1960dacfPublisher Correction: Bayesian statistics and modellingvan de Schoot, Rens; Depaoli, Sarah; King, Ruth; Kramer, Bianca; Maertens, Kaspar; Tadesse, Mahlet G.; Vannucci, Marina; Gelman, Andrew; Veen, Duco; Willemsen, Joukje; Yau, ChristopherNature Reviews Methods Primers (2021), 1 (1), 16CODEN: NRMPAT; ISSN:2662-8449. (Nature Portfolio)A Correction to this paper has been published: https://doi.org/10.1038/s43586-021-00017-2.
- 35Choi, B.; Rempala, G. A.; Kim, J. K. Beyond the Michaelis-Menten equation: accurate and efficient estimation of enzyme kinetic parameters. Sci. Rep. 2017, 7 (1), 17018, DOI: 10.1038/s41598-017-17072-z35https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A280%3ADC%252BC1M3otlWmug%253D%253D&md5=24801e3eb36b6bd649d17583ae5db895Beyond the Michaelis-Menten equation: Accurate and efficient estimation of enzyme kinetic parametersChoi Boseung; Rempala Grzegorz A; Kim Jae KyoungScientific reports (2017), 7 (1), 17018 ISSN:.Examining enzyme kinetics is critical for understanding cellular systems and for using enzymes in industry. The Michaelis-Menten equation has been widely used for over a century to estimate the enzyme kinetic parameters from reaction progress curves of substrates, which is known as the progress curve assay. However, this canonical approach works in limited conditions, such as when there is a large excess of substrate over enzyme. Even when this condition is satisfied, the identifiability of parameters is not always guaranteed, and often not verifiable in practice. To overcome such limitations of the canonical approach for the progress curve assay, here we propose a Bayesian approach based on an equation derived with the total quasi-steady-state approximation. In contrast to the canonical approach, estimates obtained with this proposed approach exhibit little bias for any combination of enzyme and substrate concentrations. Importantly, unlike the canonical approach, an optimal experiment to identify parameters with certainty can be easily designed without any prior information. Indeed, with this proposed design, the kinetic parameters of diverse enzymes with disparate catalytic efficiencies, such as chymotrypsin, fumarase, and urease, can be accurately and precisely estimated from a minimal amount of timecourse data. A publicly accessible computational package performing such accurate and efficient Bayesian inference for enzyme kinetics is provided.
- 36Linden, N. J.; Kramer, B.; Rangamani, P. Bayesian parameter estimation for dynamical models in systems biology. PLoS Comput. Biol. 2022, 18 (10), e1010651, DOI: 10.1371/journal.pcbi.101065136https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB38XivVanu7bF&md5=06868bf56a1deae8d2eceeb29755e8b1Bayesian parameter estimation for dynamical models in systems biologyLinden, Nathaniel J.; Kramer, Boris; Rangamani, PadminiPLoS Computational Biology (2022), 18 (10), e1010651CODEN: PCBLBG; ISSN:1553-7358. (Public Library of Science)Dynamical systems modeling, particularly via systems of ordinary differential equations, has been used to effectively capture the temporal behavior of different biochem. components in signal transduction networks. Despite the recent advances in exptl. measurements, including sensor development and '-omics' studies that have helped populate protein-protein interaction networks in great detail, modeling in systems biol. lacks systematic methods to est. kinetic parameters and quantify assocd. uncertainties. This is because of multiple reasons, including sparse and noisy exptl. measurements, lack of detailed mol. mechanisms underlying the reactions, and missing biochem. interactions. Addnl., the inherent nonlinearities with respect to the states and parameters assocd. with the system of differential equations further compd. the challenges of parameter estn. In this study, we propose a comprehensive framework for Bayesian parameter estn. and complete quantification of the effects of uncertainties in the data and models. We apply these methods to a series of signaling models of increasing math. complexity. Systematic anal. of these dynamical systems showed that parameter estn. depends on data sparsity, noise level, and model structure, including the existence of multiple steady states. These results highlight how focused uncertainty quantification can enrich systems biol. modeling and enable addnl. quant. analyses for parameter estn.
- 37Baltussen, M. G.; van de Wiel, J.; Fernandez Regueiro, C. L.; Jakstaite, M.; Huck, W. T. S. A Bayesian Approach to Extracting kinetic information from artificial enzymatic networks. Anal. Chem. 2022, 94 (20), 7311– 7318, DOI: 10.1021/acs.analchem.2c0065937https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB38Xht1Ghu7fL&md5=68b8f5735f0b650622ff30064c2ea456A Bayesian approach to extracting kinetic information from artificial enzymatic networksBaltussen, Mathieu G.; van de Wiel, Jeroen; Fernandez Regueiro, Cristina Lia; Jakstaite, Migle; Huck, Wilhelm T. S.Analytical Chemistry (Washington, DC, United States) (2022), 94 (20), 7311-7318CODEN: ANCHAM; ISSN:0003-2700. (American Chemical Society)In order to create artificial enzymic networks capable of increasingly complex behavior, an improved methodol. in understanding and controlling the kinetics of these networks is needed. Here, we introduce a Bayesian anal. method allowing for the accurate inference of enzyme kinetic parameters and detn. of most likely reaction mechanisms, by combining data from different expts. and network topologies in a single probabilistic anal. framework. This Bayesian approach explicitly allows us to continuously improve our parameter ests. and behavior predictions by iteratively adding new data to our models, while automatically taking into account uncertainties introduced by the exptl. setups or the chem. processes in general. We demonstrate the potential of this approach by characterizing systems of enzymes compartmentalized in beads inside flow reactors. The methods we introduce here provide a new approach to the design of increasingly complex artificial enzymic networks, making the design of such networks more efficient, and robust against the accumulation of exptl. errors.
- 38Gábor, A.; Villaverde, A. F.; Banga, J. R. Parameter identifiability analysis and visualization in large-scale kinetic models of biosystems. BMC Syst. Biol. 2017, 11 (1), 54, DOI: 10.1186/s12918-017-0428-y38https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC1cXhtlSjurzP&md5=502fde6dca6c0ea22c784ac57f79f396Parameter identifiability analysis and visualization in large-scale kinetic models of biosystemsGabor, Attila; Villaverde, Alejandro F.; Banga, Julio R.BMC Systems Biology (2017), 11 (), 54/1-54/16CODEN: BSBMCC; ISSN:1752-0509. (BioMed Central Ltd.)Kinetic models of biochem. systems usually consist of ordinary differential equations that have many unknown parameters. Some of these parameters are often practically unidentifiable, i.e., their values cannot be uniquely detd. from the available data. Possible causes are lack of influence on the measured outputs, interdependence among parameters, and poor data quality. Uncorrelated parameters can be seen as the key tuning knobs of a predictive model. Therefore, before attempting to perform parameter estn. (model calibration) it is important to characterize the subset(s) of identifiable parameters and their interplay. Once this is achieved, it is still necessary to perform parameter estn., which poses addnl. challenges. We present a methodol. that (i) detects high-order relationships among parameters, and (ii) visualizes the results to facilitate further anal. We use a collinearity index to quantify the correlation between parameters in a group in a computationally efficient way. Then we apply integer optimization to find the largest groups of uncorrelated parameters. We also use the collinearity index to identify small groups of highly correlated parameters. The results files can be visualized using Cytoscape, showing the identifiable and non-identifiable groups of parameters together with the model structure in the same graph. Our contributions alleviate the difficulties that appear at different stages of the identifiability anal. and parameter estn. process. We show how to combine global optimization and regularization techniques for calibrating medium and large scale biol. models with moderate computation times. Then we evaluate the practical identifiability of the estd. parameters using the proposed methodol. The identifiability anal. techniques are implemented as a MATLAB toolbox called VisId, which is freely available as open source from GitHub. Our approach is geared towards scalability. It enables the practical identifiability anal. of dynamic models of large size, and accelerates their calibration. The visualization tool allows modellers to detect parts that are problematic and need refinement or reformulation, and provides experimentalists with information that can be helpful in the design of new expts.
- 39Villaverde, A. F.; Evans, N. D.; Chappell, M. J.; Banga, J. R. Input-dependent structural identifiability of nonlinear systems. IEEE Control Systems Letters 2019, 3 (2), 272– 277, DOI: 10.1109/LCSYS.2018.2868608There is no corresponding record for this reference.
- 40van Sluijs, B.; Maas, R. J. M.; van der Linden, A. J.; de Greef, T. F. A.; Huck, W. T. S. A microfluidic optimal experimental design platform for forward design of cell-free genetic networks. Nat. Commun. 2022, 13 (1), 3626, DOI: 10.1038/s41467-022-31306-340https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB38XhslWqurfF&md5=ededa61719057fc65d2e305c7ec2287aA microfluidic optimal experimental design platform for forward design of cell-free genetic networksvan Sluijs, Bob; Maas, Roel J. M.; van der Linden, Ardjan J.; de Greef, Tom F. A.; Huck, Wilhelm T. S.Nature Communications (2022), 13 (1), 3626CODEN: NCAOBW; ISSN:2041-1723. (Nature Portfolio)Abstr.: Cell-free protein synthesis has been widely used as a "breadboard" for design of synthetic genetic networks. However, due to a severe lack of modularity, forward engineering of genetic networks remains challenging. Here, we demonstrate how a combination of optimal exptl. design and microfluidics allows us to devise dynamic cell-free gene expression expts. providing max. information content for subsequent non-linear model identification. Importantly, we reveal that applying this methodol. to a library of genetic circuits, that share common elements, further increases the information content of the data resulting in higher accuracy of model parameters. To show modularity of model parameters, we design a pulse decoder and bistable switch, and predict their behavior both qual. and quant. Finally, we update the parameter database and indicate that network topol. affects parameter estn. accuracy. Utilizing our methodol. provides us with more accurate model parameters, a necessity for forward engineering of complex genetic networks.
- 41Hold, C.; Billerbeck, S.; Panke, S. Forward design of a complex enzyme cascade reaction. Nat. Commun. 2016, 7, 12971, DOI: 10.1038/ncomms1297141https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC28Xhs1Smu73E&md5=dddec507e4149c520cf922bc32e42344Forward design of a complex enzyme cascade reactionHold, Christoph; Billerbeck, Sonja; Panke, SvenNature Communications (2016), 7 (), 12971CODEN: NCAOBW; ISSN:2041-1723. (Nature Publishing Group)Enzymic reaction networks are unique in that one can operate a large no. of reactions under the same set of conditions concomitantly in one pot, but the nonlinear kinetics of the enzymes and the resulting system complexity have so far defeated rational design processes for the construction of such complex cascade reactions. Here we demonstrate the forward design of an in vitro 10-membered system using enzymes from highly regulated biol. processes such as glycolysis. For this, we adapt the characterization of the biochem. system to the needs of classical engineering systems theory: we combine online mass spectrometry and continuous system operation to apply std. system theory input functions and to use the detailed dynamic system responses to parameterize a model of sufficient quality for forward design. This allows the facile optimization of a 10-enzyme cascade reaction for fine chem. prodn. purposes.
- 42Wong, A. S. Y.; Huck, W. T. S. Grip on complexity in chemical reaction networks. Beilstein J. Org. Chem. 2017, 13, 1486– 1497, DOI: 10.3762/bjoc.13.14742https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2sXhs1eksbnI&md5=d7e6fbaf3b1e405476e35c2a85845c69Grip on complexity in chemical reaction networksWong, Albert S. Y.; Huck, Wilhelm T. S.Beilstein Journal of Organic Chemistry (2017), 13 (), 1486-1497CODEN: BJOCBH; ISSN:1860-5397. (Beilstein-Institut zur Foerderung der Chemischen Wissenschaften)A new discipline of "systems chem." is emerging, which aims to capture the complexity obsd. in natural systems within a synthetic chem. framework. Living systems rely on complex networks of chem. reactions to control the concn. of mols. in space and time. Despite the enormous complexity in biol. networks, it is possible to identify network motifs that lead to functional outputs such as bistability or oscillations. To truly understand how living systems function, we need a complete understanding of how chem. reaction networks (CRNs) create function. We propose the development of a bottom-up approach to design and construct CRNs where we can follow the influence of single chem. entities on the properties of the network as a whole. Ultimately, this approach should allow us to not only understand such complex networks but also to guide and control their behavior.
- 43Hanopolskyi, A. I.; Smaliak, V. A.; Novichkov, A. I.; Semenov, S. N. Autocatalysis: Kinetics, mechanisms and design. ChemSystemsChem. 2021, 3 (1), e2000026, DOI: 10.1002/syst.20200002643https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB3MXovFyltrw%253D&md5=374e03d4de2ae8956a15e2072fd7bd87Autocatalysis: Kinetics, Mechanisms and DesignHanopolskyi, Anton I.; Smaliak, Viktoryia A.; Novichkov, Alexander I.; Semenov, Sergey N.ChemSystemsChem (2021), 3 (1), e2000026CODEN: CSCHCD; ISSN:2570-4206. (Wiley-VCH Verlag GmbH & Co. KGaA)The importance of autocatalysis spans from practical applications such as in chem. amplified photoresists, to autocatalysis playing a fundamental role in evolution as well as a plausible key role in the origin of life. The phenomenon of autocatalysis is characterized by its kinetic signature rather than by its mechanistic aspects. The mols. that form autocatalytic systems and the mechanisms underlying autocatalytic reactions are very diverse. This chem. diversity, combined with the strong involvement of chem. kinetics, creates a formidable barrier for entrance to the field. Understanding these challenges, we wrote this Review with three main goals in mind: (i) To provide a basic introduction to the kinetics of autocatalytic systems and its relation to the role of autocatalysis in evolution, (ii) To provide a comprehensive overview, including tables, of synthetic chem. autocatalytic systems, and (iii) To provide an in-depth anal. of the concept of autocatalytic reaction networks, their design, and perspectives for their development.
- 44Bánsági, T.; Taylor, A. F. Exploitation of feedback in enzyme-catalysed reactions. Isr. J. Chem. 2018, 58 (6–7), 706– 713, DOI: 10.1002/ijch.20170014144https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC1cXmvVehsbo%253D&md5=61c49644fc12c9f8eeedc356273fb44aExploitation of Feedback in Enzyme-catalysed ReactionsBansagi, Tamas, Jr.; Taylor, Annette F.Israel Journal of Chemistry (2018), 58 (6-7), 706-713CODEN: ISJCAT; ISSN:0021-2148. (Wiley-VCH Verlag GmbH & Co. KGaA)A review. Some cellular systems, such as yeast, bacteria and slime mold, display dynamic behavior including switches and rhythms driven by feedback in enzyme-catalyzed reactions. The mechanisms of these processes have been well investigated and recent attention has turned to generating similar responses in synthetic biocatalytic systems, with a view to creating bioinspired analogs for applications. Here we discuss how feedback arises in the reaction mechanisms of some enzyme-catalyzed reactions in vitro, the behavior obtained and the emerging applications. These autocatalytic reactions may provide insights into behavior in cellular systems as well as new methods for drug delivery, sensing and repair that can be exploited in living systems.
- 45Plasson, R.; Brandenburg, A.; Jullien, L.; Bersini, H. Autocatalyses. J. Phys. Chem. A 2011, 115 (28), 8073– 8085, DOI: 10.1021/jp110079p45https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3MXnvFKitLs%253D&md5=e25b4c31569eb18c58e325585c868cbcAutocatalysesPlasson, Raphael; Brandenburg, Axel; Jullien, Ludovic; Bersini, HuguesJournal of Physical Chemistry A (2011), 115 (28), 8073-8085CODEN: JPCAFH; ISSN:1089-5639. (American Chemical Society)Autocatalysis is a fundamental concept, used in a wide range of domains. From the most general definition of autocatalysis, i.e., a process in which a chem. compd. is able to catalyze its own formation, several different systems can be described. We detail the different categories of autocatalyzes and compare them on the basis of their mechanistic, kinetic, and dynamic properties. It is shown how autocatalytic patterns can be generated by different systems of chem. reactions. With the notion of autocatalysis covering a large variety of mechanistic realizations with very similar behaviors, it is proposed that the key signature of autocatalysis is its kinetic pattern expressed in a math. form.
- 46Budroni, M. A.; Rossi, F.; Rongy, L. From transport phenomena to systems chemistry: chemohydrodynamic oscillations in A+B⃗C systems. ChemSystemsChem. 2022, 4 (1), e202100023, DOI: 10.1002/syst.20210002346https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB38Xot1aktA%253D%253D&md5=d37da696a0bfb395198c3468512bf6d7From Transport Phenomena to Systems Chemistry: Chemohydrodynamic Oscillations in A+B→C SystemsBudroni, Marcello A.; Rossi, Federico; Rongy, LaurenceChemSystemsChem (2022), 4 (1), e202100023CODEN: CSCHCD; ISSN:2570-4206. (Wiley-VCH Verlag GmbH & Co. KGaA)Can simple chem. drive the emergence of self-organised complex behaviors. Addressing this big-picture question crucially impacts the comprehension of fundamental mechanisms at the basis of stationary and dynamical spatio-temporal chem. patterns which represent an integral part of Origin of Life studies and morphogenesis. This is also of paramount importance in cutting-edge approaches for the design and control of bio-inspired self-organised functional materials as well as for understanding how complex biol. networks work. So far, spontaneous chem. self-organization has constituted the realm of Nonlinear Chem. Oscillations, waves, Turing structures have been typically obtained in systems characterised by a complex network of nonlinear reactions activated on appropriate relative timescales. Here we revisit the emergence of oscillatory dynamics in systems characterised by a kinetics as simple and general as a bimol. process, provided that it is actively coupled with transport phenomena, in the absence of any nonlinear or external kinetic feedback. We also present new numerical expts. to substantiate and clarify the minimal ingredients underlying these complex dynamics. The objective of this paper is to discuss chemo-hydrodynamics as a possible mechanism for activating self-organised structures and functional behaviors in contexts characterised by a minimal chem. like prebiotic conditions. In this view, we also highlight the necessity to include convective phenomena in the paradigm of Systems Chem.
- 47Menon, G.; Krishnan, J. Spatial localisation meets biomolecular networks. Nat. Commun. 2021, 12 (1), 5357, DOI: 10.1038/s41467-021-24760-y47https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB3MXitVSju7%252FI&md5=3d9a558aa56e2a8bac080aa27a85c011Spatial localisation meets biomolecular networksMenon, Govind; Krishnan, J.Nature Communications (2021), 12 (1), 5357CODEN: NCAOBW; ISSN:2041-1723. (Nature Research)Spatial organization through localisation/compartmentalisation of species is a ubiquitous but poorly understood feature of cellular biomol. networks. Current technologies in systems and synthetic biol. (spatial proteomics, imaging, synthetic compartmentalisation) necessitate a systematic approach to elucidating the interplay of networks and spatial organization. We develop a systems framework toward this end and focus on the effect of spatial localisation of network components revealing its multiple facets: (i) As a key distinct regulator of network behavior, and an enabler of new network capabilities (ii) As a potent new regulator of pattern formation and self-organization (iii) As an often hidden factor impacting inference of temporal networks from data (iv) As an engineering tool for rewiring networks and network/circuit design. These insights, transparently arising from the most basic considerations of networks and spatial organization, have broad relevance in natural and engineered biol. and in related areas such as cell-free systems, systems chem. and bionanotechnol.
- 48Sharma, C.; Maity, I.; Walther, A. pH-feedback systems to program autonomous self-assembly and material lifecycles. Chem. Commun. 2023, 59, 1125– 1144, DOI: 10.1039/D2CC06402B48https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB3sXpt1amsQ%253D%253D&md5=c81df9a957b093d794e0fb904a0ded10pH-feedback systems to program autonomous self-assembly and material lifecyclesSharma, Charu; Maity, Indrajit; Walther, AndreasChemical Communications (Cambridge, United Kingdom) (2023), 59 (9), 1125-1144CODEN: CHCOFS; ISSN:1359-7345. (Royal Society of Chemistry)A review. PH-responsive systems have gained importance for the development of smart materials and for biomedical applications because they can switch between different states by simple acid/base triggers. However, such equil. systems lack the autonomous behavior that is so ubiquitous in living systems that self-regulate out of equil. As a contribution to the emerging field of autonomous chem. systems, we have developed pH-feedback systems (pH-FS) based on the coupling of acid- and base-producing steps in chem. reaction networks. The resulting autonomous nonlinear pH curves can be coupled with a variety of pH-sensitive building blocks to program the lifecycles of the assocd. transient state at the level of self-assemblies and material systems. In this article, we discuss the different generations of such pH-feedback systems, the principles of their coupling to self-assemblies with lifecycles and highlight emerging concepts for the design of autonomous functional materials. The specificity, robustness, and flexible operation of such pH-FS can also be used to realize chemo-structural and chemo-mech. feedbacks that extend the behavior of such materials systems toward complex and functional life-like systems.
- 49Dúzs, B.; Molnár, I.; Lagzi, I.; Szalai, I. Reaction–diffusion dynamics of pH oscillators in oscillatory forced open spatial reactors. ACS Omega 2021, 6 (50), 34367– 34374, DOI: 10.1021/acsomega.1c0426949https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB3MXislSgu7rI&md5=a6cc93dab9a55f296a18a8c68cbf6ef8Reaction-Diffusion Dynamics of pH Oscillators in Oscillatory Forced Open Spatial ReactorsDuzs, Brigitta; Molnar, Istvan; Lagzi, Istvan; Szalai, IstvanACS Omega (2021), 6 (50), 34367-34374CODEN: ACSODF; ISSN:2470-1343. (American Chemical Society)Studying the effect of coupling and forcing of oscillators is a significant area of interest within nonlinear dynamics and has provided evidence of many interesting phenomena, such as synchronization, beating, oscillatory death, and phase resetting. Many studies have also reported along this line in reaction-diffusion systems, which are preferably explored exptl. by using open reactors. These reactors consist of one or two homogeneous (well-stirred) tanks, which provide the boundary conditions for a spatially distributed part. The spatiotemporal dynamics of this configuration in the presence of temporal oscillations in the homogeneous part has not been systematically investigated. This paper aims to explore numerically the effect of time-periodic boundary conditions on the dynamics of open reactors provided by autonomous and forced oscillations in the well-stirred part. A simple model of pH oscillators can produce various phenomena under these conditions, for example, superposition and modulation of spatiotemporal oscillations and forced bursting. The autonomous oscillatory boundary conditions can be generated by the same kinetic instabilities that result in spatiotemporal oscillations in the spatially distributed part. The forced oscillations are induced by sinusoidal modulation on the inflow concn. of the activator in the tank. The simulations confirmed that this type of forcing is more effective when the modulation period is longer than the residence time of the well-stirred part. The use of time-periodic boundary conditions may open a new perspective in the control and design of spatiotemporal phenomena in open one-side-fed and two-side-fed reactors.
- 50Duzs, B.; Lagzi, I.; Szalai, I. Functional rhythmic chemical systems governed by pH-driven kinetic feedback. ChemSystemsChem 2023, 5, e202200032, DOI: 10.1002/syst.20220003250https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB38XjtFymsLfE&md5=ceef1e19f91a085dcb74f8bcea070d73Functional Rhythmic Chemical Systems Governed by pH-Driven Kinetic FeedbackDuzs, Brigitta; Lagzi, Istvan; Szalai, IstvanChemSystemsChem (2023), 5 (2), e202200032CODEN: CSCHCD; ISSN:2570-4206. (Wiley-VCH Verlag GmbH & Co. KGaA)Hydrogen ion autocatalytic reactions, esp. in combination with an appropriate neg. feedback process, show a wide range of dynamical phenomena, like clock behavior, bistability, oscillations, waves, and stationary patterns. The temporal or spatial variation of pH caused by these reactions is often significant enough to control the actual state (geometry, conformation, reactivity) or drive the mech. motion of coupled pH-sensitive physico-chem. systems. These autonomous operating systems provide nonlinear chem.'s most reliable applications, where the hydrogen ion autocatalytic reactions act as engines. This review briefly summarizes the nonlinear dynamics of these reactions and the different approaches developed to properly couple the pH-sensitive units (e. g., pH-sensitive equil., gels, mol. machines, colloids). We also emphasize the feedback of the coupled processes on the dynamics of the hydrogen ion autocatalytic reactions since the way of coupling is a crit. operational issue.
- 51Bubanja, I. N.; Bánsági, T.; Taylor, A. F. Kinetics of the urea–urease clock reaction with urease immobilized in hydrogel beads. Reac. Kinet. Mech. Catal. 2018, 123 (1), 177– 185, DOI: 10.1007/s11144-017-1296-6There is no corresponding record for this reference.
- 52Miele, Y.; Jones, S. J.; Rossi, F.; Beales, P. A.; Taylor, A. F. Collective behavior of urease pH clocks in nano- and microvesicles controlled by fast ammonia transport. J. Phys. Chem. Lett. 2022, 13 (8), 1979– 1984, DOI: 10.1021/acs.jpclett.2c0006952https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB38Xktl2qurY%253D&md5=14a36537aa46cd55bd13745860a4bc8dCollective Behavior of Urease pH Clocks in Nano- and Microvesicles Controlled by Fast Ammonia TransportMiele, Ylenia; Jones, Stephen J.; Rossi, Federico; Beales, Paul A.; Taylor, Annette F.Journal of Physical Chemistry Letters (2022), 13 (8), 1979-1984CODEN: JPCLCD; ISSN:1948-7185. (American Chemical Society)The transmission of chem. signals via an extracellular soln. plays a vital role in collective behavior in cellular biol. systems and may be exploited in applications of lipid vesicles such as drug delivery. Here, we investigated chem. communication in synthetic micro- and nanovesicles contg. urease in a soln. of urea and acid. We combined expts. with simulations to demonstrate that the fast transport of ammonia to the external soln. governs the pH-time profile and synchronizes the timing of the pH clock reaction in a heterogeneous population of vesicles. This study shows how the rate of prodn. and emission of a small basic product controls pH changes in active vesicles with a distribution of sizes and enzyme amts., which may be useful in bioreactor or healthcare applications.
- 53Markovic, V. M.; Bánsági, T., Jr.; McKenzie, D.; Mai, A.; Pojman, J. A.; Taylor, A. F. Influence of reaction-induced convection on quorum sensing in enzyme-loaded agarose beads. Chaos 2019, 29 (3), 033130 DOI: 10.1063/1.508929553https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC1MXlvVaht7Y%253D&md5=253ed3102986bffa416647741cc8542bInfluence of reaction-induced convection on quorum sensing in enzyme-loaded agarose beadsMarkovic, Vladimir M.; Bansagi, Tamas; McKenzie, Dennel; Mai, Anthony; Pojman, John A.; Taylor, Annette F.Chaos (2019), 29 (3), 033130/1-033130/8CODEN: CHAOEH; ISSN:1054-1500. (American Institute of Physics)In theory, groups of enzyme-loaded particles producing an acid or base may show complex behavior including dynamical quorum sensing, the appearance of synchronized oscillations above a crit. no. or d. of particles. Here, expts. were performed with the enzyme urease loaded into mm-sized agarose beads and placed in a soln. of urea, resulting in an increase in pH. This behavior was found to be dependent upon the no. of beads present in the array; however, reaction-induced convection occurred and plumes of high pH developed that extended to the walls of the reactor. The convection resulted in the motion of the mm-sized particles and conversion of the soln. to high pH. Simulations in a simple model of the beads demonstrated the suppression of dynamical quorum sensing in the presence of flow. (c) 2019 American Institute of Physics.
- 54Muzika, F.; RuZicka, M.; Schreiberova, L.; Schreiber, I. Oscillations of pH in the urea-urease system in a membrane reactor. Phys. Chem. Chem. Phys. 2019, 21 (17), 8619– 8622, DOI: 10.1039/C9CP00630C54https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC1MXmtlyksbY%253D&md5=a78ec2e5982160a8ec8ca2a83a397ab1Oscillations of pH in the urea-urease system in a membrane reactorMuzika, Frantisek; Ruzicka, Matej; Schreiberova, Lenka; Schreiber, IgorPhysical Chemistry Chemical Physics (2019), 21 (17), 8619-8622CODEN: PPCPFQ; ISSN:1463-9076. (Royal Society of Chemistry)Herein, we present direct exptl. evidence of pH oscillatory dynamics in the urea-urease enzymic reaction conducted in a continuous reactor-membrane-reservoir system. Our results are consistent with earlier model predictions requiring differential transport of H+ and substrate. We report oscillations with periods in hundreds of seconds and the amplitude of ∼0.1 pH units.
- 55Muzika, F.; Bánsági, T., Jr.; Schreiber, I.; Schreiberova, L.; Taylor, A. F. A bistable switch in pH in urease-loaded alginate beads. Chem. Commun. 2014, 50 (76), 11107– 11109, DOI: 10.1039/C4CC03936J55https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2cXht1ylsLfM&md5=ab6e71b5ceabd086472dc0da60a86b16A bistable switch in pH in urease-loaded alginate beadsMuzika, F.; Bansagi, T.; Schreiber, I.; Schreiberova, L.; Taylor, A. F.Chemical Communications (Cambridge, United Kingdom) (2014), 50 (76), 11107-11109CODEN: CHCOFS; ISSN:1359-7345. (Royal Society of Chemistry)A bistable switch from a low pH (unreacted "off") state to a high pH (reacted "on") state was obtained in enzyme-loaded gel beads in response to supra-threshold substrate concns.
- 56Hu, G.; Pojman, J. A.; Scott, S. K.; Wrobel, M. M.; Taylor, A. F. Base-catalyzed feedback in the urea-urease reaction. J. Phys. Chem. B 2010, 114 (44), 14059– 14063, DOI: 10.1021/jp106532d56https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3cXht12lt7nI&md5=03f423f0c3cf6e8af398a2b90feaf934Base-Catalyzed Feedback in the Urea-Urease ReactionHu, Gang; Pojman, John A.; Scott, Stephen K.; Wrobel, Magdelena M.; Taylor, Annette F.Journal of Physical Chemistry B (2010), 114 (44), 14059-14063CODEN: JPCBFK; ISSN:1520-6106. (American Chemical Society)The bell-shaped rate-pH curve coupled to prodn. of base in the urea-urease reaction was utilized to give feedback-driven behavior: an acid-to-base pH clock (a kinetic switch), bistability and hysteresis between an acid/base state when the initial pH was adjusted by a strong acid, and aperiodic pH oscillations when the initial pH was adjusted by a weak acid in an open reactor. A simple model of the reaction reproduced most of the exptl. results and provided insight into the role of self-buffering in the dynamics. This reaction suggests new possibilities in the development of biocompatible feedback to couple to pH-sensitive processes for bioinspired applications in medicine, engineering, or materials science.
- 57Straube, A. V.; Winkelmann, S.; Schütte, C.; Höfling, F. Stochastic pH oscillations in a model of the urea-urease reaction confined to lipid vesicles. J. Phys. Chem. Lett. 2021, 12 (40), 9888– 9893, DOI: 10.1021/acs.jpclett.1c0301657https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB3MXitFKgs7vL&md5=302f4240f6ef6671cea1a311d812818bStochastic pH Oscillations in a Model of the Urea-Urease Reaction Confined to Lipid VesiclesStraube, Arthur V.; Winkelmann, Stefanie; Schuette, Christof; Hoefling, FelixJournal of Physical Chemistry Letters (2021), 12 (40), 9888-9893CODEN: JPCLCD; ISSN:1948-7185. (American Chemical Society)The urea-urease clock reaction is a pH switch from acid to basic that can turn into a pH oscillator if it occurs inside a suitable open reactor. The authors numerically study the confinement of the reaction to lipid vesicles, which permit the exchange with an external reservoir by differential transport, enabling the recovery of the pH level and yielding a const. supply of urea mols. For microscopically small vesicles, the discreteness of the no. of mols. requires a stochastic treatment of the reaction dynamics. The authors' anal. shows that intrinsic noise induces a significant statistical variation of the oscillation period, which increases as the vesicles become smaller. The mean period, however, is remarkably robust for vesicle sizes down to ∼200 nm, but the periodicity of the rhythm is gradually destroyed for smaller vesicles. The obsd. oscillations are explained as a canard-like limit cycle that differs from the wide class of conventional feedback oscillators.
- 58Bánsági, T.; Taylor, A. F. Switches induced by quorum sensing in a model of enzyme-loaded microparticles. J. R. Soc. Interface. 2018, 15, 20170945, DOI: 10.1098/rsif.2017.094558https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC1cXitFyksrjM&md5=7879b8b536206bf09b4f3be0510234a9Switches induced by quorum sensing in a model of enzyme-loaded microparticlesBansagi, Tamas, Jr.; Taylor, Annette F.Journal of the Royal Society, Interface (2018), 15 (140), 20170945/1-20170945/10CODEN: JRSICU; ISSN:1742-5662. (Royal Society)Quorum sensing refers to the ability of bacteria and other single-celled organisms to respond to changes in cell d. or no. with population- wide changes in behavior. Here, simulations were performed to investigate quorum sensing in groups of diffusively coupled enzyme microparticles using a well-characterized autocatalytic reaction which raises the pH of the medium: hydrolysis of urea by urease. The enzyme urease is found in both plants and microorganisms, and has been widely exploited in engineering processes. We demonstrate how increases in group size can be used to achieve a sigmoidal switch in pH at high enzyme loading, oscillations in pH at intermediate enzyme loading and a bistable, hysteretic switch at low enzyme loading. Thus, quorum sensing can be exploited to obtain different types of response in the same system, depending on the enzyme concn. The implications for microorganisms in colonies are discussed, and the results could help in the design of synthetic quorum sensing for biotechnol. applications such as drug delivery.
- 59Heinen, L.; Heuser, T.; Steinschulte, A.; Walther, A. Antagonistic enzymes in a biocatalytic pH feedback system program autonomous DNA hydrogel life cycles. Nano Lett. 2017, 17 (8), 4989– 4995, DOI: 10.1021/acs.nanolett.7b0216559https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2sXhtVOntr7J&md5=7e4447431de1ec8deba85a2979c81bfaAntagonistic Enzymes in a Biocatalytic pH Feedback System Program Autonomous DNA Hydrogel Life CyclesHeinen, Laura; Heuser, Thomas; Steinschulte, Alexander; Walther, AndreasNano Letters (2017), 17 (8), 4989-4995CODEN: NALEFD; ISSN:1530-6984. (American Chemical Society)Enzymes regulate complex functions and active behavior in natural systems and have shown increasing prospect for developing self-regulating soft matter systems. Striving for advanced autonomous hydrogel materials with fully programmable, self-regulated life cycles, we combine two enzymes with an antagonistic pH-modulating effect in a feedback-controlled biocatalytic reaction network (BRN) and couple it to pH-responsive DNA hydrogels to realize hydrogel systems with distinct preprogrammable lag times and lifetimes in closed systems. The BRN enables precise and orthogonal internal temporal control of the "ON" and "OFF" switching times of the temporary gel state by modulation of programmable, non-linear pH changes. The timescales are tunable by variation of the enzyme concns. and addnl. buffer substances. The resulting material system operates in full autonomy after injection of the chem. fuels driving the BRN. The concept may open new applications inherent to DNA hydrogels, for instance, autonomous shape memory behavior for soft robotics. We further foresee general applicability to achieve autonomous life cycles in other pH switchable systems.
- 60Fan, X.; Walther, A. Autonomous transient pH flips shaped by layered compartmentalization of antagonistic enzymatic reactions. Angew. Chem., Int. Ed. 2021, 60, 3619– 3624, DOI: 10.1002/anie.20200954260https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB3cXis1agtbnO&md5=83cc1ce50746ba7ef36516d4d684b92bAutonomous transient pH flips shaped by layered compartmentalization of antagonistic enzymatic reactionsFan, Xinlong; Walther, AndreasAngewandte Chemie, International Edition (2021), 60 (7), 3619-3624CODEN: ACIEF5; ISSN:1433-7851. (Wiley-VCH Verlag GmbH & Co. KGaA)Transient signaling orchestrates complex spatiotemporal behavior in living organisms via (bio)chem. reaction networks (CRNs). Compartmentalization of signal processing is an important aspect for controlling such networks. However, artificial CRNs mostly focus on homogeneous solns. to program autonomous self-assembling systems, which limits their accessible behavior and tuneability. Here, we introduce layered compartments housing antagonistic pH-modulating enzymes and demonstrate that transient pH signals in a supernatant soln. can be programmed based on spatial delays. This overcomes limitations of activity mismatches of antagonistic enzymes in soln. and allows to flexibly program acidic and alk. pH lifecycles beyond the possibilities of homogeneous solns. Lag time, lifetime, and the pH min. and maxima can be precisely programmed by adjusting spatial and kinetic conditions. We integrate these spatially controlled pH flips with switchable peptides, furnishing time-programmed self-assemblies and hydrogel material system.
- 61Wang, X.; Moreno, S.; Boye, S.; Wen, P.; Zhang, K.; Formanek, P.; Lederer, A.; Voit, B.; Appelhans, D. Feedback-induced and oscillating pH regulation of a binary enzyme–polymersomes. System. Chem. Mater. 2021, 33 (17), 6692– 6700, DOI: 10.1021/acs.chemmater.1c00897There is no corresponding record for this reference.
- 62Dúzs, B.; Lagzi, I.; Szalai, I. Functional rhythmic chemical systems governed by pH-Driven kinetic feedback. ChemSystemsChem 2023, 5 (2), e202200032, DOI: 10.1002/syst.20220003262https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB38XjtFymsLfE&md5=ceef1e19f91a085dcb74f8bcea070d73Functional Rhythmic Chemical Systems Governed by pH-Driven Kinetic FeedbackDuzs, Brigitta; Lagzi, Istvan; Szalai, IstvanChemSystemsChem (2023), 5 (2), e202200032CODEN: CSCHCD; ISSN:2570-4206. (Wiley-VCH Verlag GmbH & Co. KGaA)Hydrogen ion autocatalytic reactions, esp. in combination with an appropriate neg. feedback process, show a wide range of dynamical phenomena, like clock behavior, bistability, oscillations, waves, and stationary patterns. The temporal or spatial variation of pH caused by these reactions is often significant enough to control the actual state (geometry, conformation, reactivity) or drive the mech. motion of coupled pH-sensitive physico-chem. systems. These autonomous operating systems provide nonlinear chem.'s most reliable applications, where the hydrogen ion autocatalytic reactions act as engines. This review briefly summarizes the nonlinear dynamics of these reactions and the different approaches developed to properly couple the pH-sensitive units (e. g., pH-sensitive equil., gels, mol. machines, colloids). We also emphasize the feedback of the coupled processes on the dynamics of the hydrogen ion autocatalytic reactions since the way of coupling is a crit. operational issue.
- 63Jee, E.; Bánsági, T., Jr.; Taylor, A. F.; Pojman, J. A. Temporal control of gelation and polymerization fronts driven by an autocatalytic enzyme reaction. Angew. Chem., Int. Ed. 2016, 55 (6), 2127– 2131, DOI: 10.1002/anie.20151060463https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC28XktlSmsA%253D%253D&md5=2ed1fdf6d04860d0dbf9745a6bb289dbTemporal Control of Gelation and Polymerization Fronts Driven by an Autocatalytic Enzyme ReactionJee, Elizabeth; Bansagi, Tamas Jr.; Taylor, Annette F.; Pojman, John A.Angewandte Chemie, International Edition (2016), 55 (6), 2127-2131CODEN: ACIEF5; ISSN:1433-7851. (Wiley-VCH Verlag GmbH & Co. KGaA)Chem. systems that remain kinetically dormant until activated have numerous applications in materials science. Herein we present a method for the control of gelation that exploits an inbuilt switch: the increase in pH after an induction period in the urease-catalyzed hydrolysis of urea was used to trigger the base-catalyzed Michael addn. of a water-sol. trithiol to a polyethylene glycol diacrylate. The time to gelation (minutes to hours) was either preset through the initial concns. or the reaction was initiated locally by a base, thus resulting in polymn. fronts that converted the mixt. from a liq. into a gel (ca. 0.1 mm min-1). The rate of hydrolytic degrdn. of the hydrogel depended on the initial concns., thus resulting in a gel lifetime of hours to months. In this way, temporal programming of gelation was possible under mild conditions by using the output of an autocatalytic enzyme reaction to drive both the polymn. and subsequent degrdn. of a hydrogel.
- 64Mai, A. Q.; Bánsági, T., Jr.; Taylor, A. F.; Pojman, J. A., Sr. Reaction-diffusion hydrogels from urease enzyme particles for patterned coatings. Commun. Chem. 2021, 4 (1), 101, DOI: 10.1038/s42004-021-00538-764https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB3MXitF2gur3I&md5=af0f5c0f3dcbe53e166935d27ac84dcdReaction-diffusion hydrogels from urease enzyme particles for patterned coatingsMai, Anthony Q.; Bansagi Jr., Tamas; Taylor, Annette F.; Pojman Sr., John A.Communications Chemistry (2021), 4 (1), 101CODEN: CCOHCT; ISSN:2399-3669. (Nature Research)Abstr.: The reaction and diffusion of small mols. is used to initiate the formation of protective polymeric layers, or biofilms, that attach cells to surfaces. Here, inspired by biofilm formation, we present a general method for the growth of hydrogels from urease enzyme-particles by combining prodn. of ammonia with a pH-regulated polymn. reaction in soln. We show through expts. and simulations how the propagating basic front and thiol-acrylate polymn. were continuously maintained by the localized urease reaction in the presence of urea, resulting in hydrogel layers around the enzyme particles at surfaces, interfaces or in motion. The hydrogels adhere the enzyme-particles to surfaces and have a tunable growth rate of the order of 10 μm min-1 that depends on the size and spatial distribution of particles. This approach can be exploited to create enzyme-hydrogels or chem. patterned coatings for applications in biocatalytic flow reactors.
- 65Maity, I.; Sharma, C.; Lossada, F.; Walther, A. Feedback and communication in active hydrogel spheres with pH fronts: facile approaches to grow soft hydrogel structures. Angew. Chem., Int. Ed. 2021, 60 (41), 22537– 22546, DOI: 10.1002/anie.20210973565https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB3MXhvFGmsbvO&md5=fc6e8605c1188dfbdcd3e9571568898bFeedback and Communication in Active Hydrogel Spheres with pH Fronts: Facile Approaches to Grow Soft Hydrogel StructuresMaity, Indrajit; Sharma, Charu; Lossada, Francisco; Walther, AndreasAngewandte Chemie, International Edition (2021), 60 (41), 22537-22546CODEN: ACIEF5; ISSN:1433-7851. (Wiley-VCH Verlag GmbH & Co. KGaA)Compartmentalized reaction networks regulating signal processing, communication and pattern formation are central to living systems. Towards achieving life-like materials, the authors compartmentalized urea-urease and more complex urea-urease/ester-esterase pH-feedback reaction networks into hydrogel spheres and study how fuel-driven pH fronts can be sent out from these spheres and regulated by internal reaction networks. Membrane characteristics are installed by covering urease spheres with responsive hydrogel shells. The authors then encapsulate the two networks (urea-urease and ester-esterase) sep. into different hydrogel spheres to devise communication, pattern formation and attraction. Moreover, these pH fronts and patterns can be used for self-growing hydrogels, and for developing complex geometries from non-injectable hydrogels without 3D printing tools. This study opens possibilities for compartmentalized feedback reactions and their use in next generation materials fabrication.
- 66Rauner, N.; Buenger, L.; Schuller, S.; Tiller, J. C. Post-polymerization of urease-induced calcified, polymer hydrogels. Macromol. Rapid Commun. 2015, 36 (2), 224– 230, DOI: 10.1002/marc.20140042666https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2MXosFyksg%253D%253D&md5=f7c7d33c8fffcdc22bc61188ce70bbc8Post-Polymerization of Urease-Induced Calcified, Polymer HydrogelsRauner, Nicolas; Buenger, Lea; Schuller, Stefanie; Tiller, Joerg C.Macromolecular Rapid Communications (2015), 36 (2), 224-230CODEN: MRCOE3; ISSN:1022-1336. (Wiley-VCH Verlag GmbH & Co. KGaA)Urease-induced calcification is an innovative method to artificially produce highly filled CaCO3-based composite materials by intrinsic mineralization of hydrogels. The mech. properties of these hybrid materials based on poly(2-hydroxyethylacrylate) cross-linked by triethylene glycol dimethacrylate are poor. Increasing the degree of calcification to up to 94 wt% improves the Young's moduli (YM) of the materials from some 40 MPa to more than 300 MPa. The introduction of calcium carbonate affine groups to the hydrogel matrix by copolymg. acrylic acid and [2-(methacryloyloxy) ethyl]trimethylammonium chloride, resp., does not increase the stiffness of the composites. A Young's modulus of more than 1 GPa is achieved by post-polymn. (PP) of the calcified hydrogels, which proves that the size of the contact area between the matrix and calcium carbonate crystals is the most crucial parameter for controlling the stiffness of hybrid materials. Switching from low Tg to high Tg hydrogel matrixes (based on poly(N,N-di-Me acrylamide)) results in a YM of up to 3.5 GPa after PP.
- 67Semenov, S. N.; Wong, A. S.; van der Made, R. M.; Postma, S. G.; Groen, J.; van Roekel, H. W.; de Greef, T. F.; Huck, W. T. Rational design of functional and tunable oscillating enzymatic networks. Nat. Chem. 2015, 7 (2), 160– 165, DOI: 10.1038/nchem.214267https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2MXmtFSgsQ%253D%253D&md5=e5010fdd61b6a845eb70077cfebb232fRational design of functional and tunable oscillating enzymatic networksSemenov, Sergey N.; Wong, Albert S. Y.; van der Made, R. Martijn; Postma, Sjoerd G. J.; Groen, Joost; van Roekel, Hendrik W. H.; de Greef, Tom F. A.; Huck, Wilhelm T. S.Nature Chemistry (2015), 7 (2), 160-165CODEN: NCAHBB; ISSN:1755-4330. (Nature Publishing Group)Life is sustained by complex systems operating far from equil. and consisting of a multitude of enzymic reaction networks. The operating principles of biol.'s regulatory networks are known, but the in vitro assembly of out-of-equil. enzymic reaction networks proved challenging, limiting the development of synthetic systems showing autonomous behavior. Here, the authors present a strategy for the rational design of programmable functional reaction networks that exhibit dynamic behavior. A network built around autoactivation and delayed neg. feedback of the enzyme trypsin is capable of producing sustained oscillating concns. of active trypsin for over 65 h. Other functions, such as amplification, analog-to-digital conversion and periodic control over equil. systems, were obtained by linking multiple network modules in microfluidic flow reactors. The methodol. developed here provides a general framework to construct dissipative, tunable and robust (bio)chem. reaction networks.
- 68Maguire, O. R.; Wong, A. S. Y.; Westerdiep, J. H.; Huck, W. T. S. Early warning signals in chemical reaction networks. Chem. Commun. 2020, 56 (26), 3725– 3728, DOI: 10.1039/D0CC01010C68https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB3cXjvFKiu7c%253D&md5=5bef9631196a02efb799d83b92408a71Early warning signals in chemical reaction networksMaguire, Oliver R.; Wong, Albert S. Y.; Westerdiep, Jan Harm; Huck, Wilhelm T. S.Chemical Communications (Cambridge, United Kingdom) (2020), 56 (26), 3725-3728CODEN: CHCOFS; ISSN:1359-7345. (Royal Society of Chemistry)Complex systems such as ecosystems, the climate and stock markets produce emergent behavior which is capable of undergoing dramatic change when pushed beyond a tipping point. Such complex systems display Early Warning Signals in their behavior when they are close to a tipping point. Here we show that a complex chem. reaction network can also display early warning signals when it is in close proximity to the boundary between oscillatory and steady state concn. behaviors. We identify early warning signals using both an active sensing method, based on the recovery time of an oscillatory response after a perturbation in temp., and a passive sensing method, based upon a change in the shape of the oscillations. The presence of the early warning signals indicates that complex, dissipative chem. networks can intrinsically sense their proximity to a boundary between behaviors.
- 69Maguire, O. R.; Wong, A. S. Y.; Baltussen, M. G.; van Duppen, P.; Pogodaev, A. A.; Huck, W. T. S. Dynamic environments as a tool to preserve desired output in a chemical reaction network. Chem. Eur. J. 2020, 26, 1676– 1682, DOI: 10.1002/chem.20190472569https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB3cXht1Khu7o%253D&md5=eb697b3888e013a653c608bf30c55677Dynamic environments as tool to preserve desired output in chemical reaction networkMaguire, Oliver R.; Wong, Albert S. Y.; Baltussen, Mathieu G.; van Duppen, Peer; Pogodaev, Aleksandr A.; Huck, Wilhelm T. S.Chemistry - A European Journal (2020), 26 (7), 1676-1682CODEN: CEUJED; ISSN:0947-6539. (Wiley-VCH Verlag GmbH & Co. KGaA)Current efforts to design functional mol. systems have overlooked the importance of coupling out-of-equil. behavior with changes in the environment. Here, the authors use an oscillating reaction network and demonstrate that the application of environmental forcing, in the form of periodic changes in temp. and in the inflow of the concn. of one of the network components, removes the dependency of the periodicity of this network on temp. or flow rates and enforces a stable periodicity across a wide range of conditions. Coupling a system to a dynamic environment can thus be used as a simple tool to regulate the output of a network. In addn., the authors show that coupling can also induce an increase in behavioral complexity to include quasi-periodic oscillations.
- 70Helwig, B.; van Sluijs, B.; Pogodaev, A. A.; Postma, S. G. J.; Huck, W. T. S. Bottom-up construction of an adaptive enzymatic reaction network. Angew. Chem., Int. Ed. 2018, 57 (43), 14065– 14069, DOI: 10.1002/anie.20180694470https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC1cXhvVejs7%252FI&md5=376c5ca98743d8247e0116a3309693e1Bottom-Up Construction of an Adaptive Enzymatic Reaction NetworkHelwig, Britta; van Sluijs, Bob; Pogodaev, Aleksandr A.; Postma, Sjoerd G. J.; Huck, Wilhelm T. S.Angewandte Chemie, International Edition (2018), 57 (43), 14065-14069CODEN: ACIEF5; ISSN:1433-7851. (Wiley-VCH Verlag GmbH & Co. KGaA)The reprodn. of emergent behaviors in nature using reaction networks is an important objective in synthetic biol. and systems chem. Herein, the first exptl. realization of an enzymic reaction network capable of an adaptive response is reported. The design is based on the dual activity of trypsin (Tr), which activates chymotrypsin (Cr) while at the same time generating a fluorescent output from a fluorogenic substrate. Once activated, chymotrypsin counteracts the trypsin output by competing for the fluorogenic substrate and producing a non-fluorescent output. It is demonstrated that this network produces a transient fluorescent output under out-of-equil. conditions while the input signal persists. Importantly, in agreement with math. simulations, we show that optimization of the pulse-like response is an inherent trade-off between max. amplitude and lowest residual fluorescence.
- 71Semenov, S. N.; Markvoort, A. J.; de Greef, T. F.; Huck, W. T. Threshold sensing through a synthetic enzymatic reaction-diffusion network. Angew. Chem., Int. Ed. 2014, 53 (31), 8066– 8069, DOI: 10.1002/anie.20140232771https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2cXlsFyisrw%253D&md5=57191864d1fcaa1a82b52db1977c29d4Threshold Sensing Through a Synthetic Enzymatic Reaction-Diffusion NetworkSemenov, Sergey N.; Markvoort, Albert J.; de Greef, Tom F. A.; Huck, Wilhelm T. S.Angewandte Chemie, International Edition (2014), 53 (31), 8066-8069CODEN: ACIEF5; ISSN:1433-7851. (Wiley-VCH Verlag GmbH & Co. KGaA)A wet stamping method to precisely control concns. of enzymes and inhibitors in place and time inside layered gels is reported. By combining enzymic reactions such as autocatalysis and inhibition with spatial delivery of components through soft lithog. techniques, a biochem. reaction network capable of recognizing the spatial distribution of an enzyme was constructed. The exptl. method can be used to assess fundamental principles of spatiotemporal order formation in chem. reaction networks.
- 72Kriukov, D. V.; Koyuncu, A. H.; Wong, A. S. Y. History dependence in a chemical reaction network enables dynamic switching. Small 2022, 18 (16), 2107523, DOI: 10.1002/smll.20210752372https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB38Xms1Omu7c%253D&md5=fc91fbb1e0b1af424562d6dda6e51c76History Dependence in a Chemical Reaction Network Enables Dynamic SwitchingKriukov, Dmitrii V.; Koyuncu, A. Hazal; Wong, Albert S. Y.Small (2022), 18 (16), 2107523CODEN: SMALBC; ISSN:1613-6810. (Wiley-VCH Verlag GmbH & Co. KGaA)This work describes an enzymic autocatalytic network capable of dynamic switching under out-of-equil. conditions. The network, wherein a mol. fuel (trypsinogen) and an inhibitor (soybean trypsin inhibitor) compete for a catalyst (trypsin), is kept from reaching equil. using a continuous flow stirred tank reactor. A so-called 'linear inhibition sweep' is developed (i.e., a mol. analog of linear sweep voltammetry) to intentionally perturb the competition between autocatalysis and inhibition, and used to demonstrate that a simple mol. system, comprising only three components, is already capable of a variety of essential neuromorphic behaviors (hysteresis, synchronization, resonance, and adaptation). This research provides the first steps in the development of a strategy that uses the principles in systems chem. to transform chem. reaction networks into platforms capable of neural network computing.
- 73Pogodaev, A. A.; Fernández Regueiro, C. L.; Jakštaitė, M.; Hollander, M. J.; Huck, W. T. S. Modular design of small enzymatic reaction networks based on reversible and cleavable inhibitors. Angew. Chem., Int. Ed. 2019, 58 (41), 14539– 14543, DOI: 10.1002/anie.20190799573https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC1MXhslWkurnM&md5=8e800d63a72c0a71f00f1ecf02c54b04Modular Design of Small Enzymatic Reaction Networks Based on Reversible and Cleavable InhibitorsPogodaev, Aleksandr A.; Fernandez Regueiro, Cristina Lia; Jakstaite, Migle; Hollander, Marijn J.; Huck, Wilhelm T. S.Angewandte Chemie, International Edition (2019), 58 (41), 14539-14543CODEN: ACIEF5; ISSN:1433-7851. (Wiley-VCH Verlag GmbH & Co. KGaA)Systems chem. aims to mimic the functional behavior of living systems by constructing chem. reaction networks with well-defined dynamic properties. Enzymes can play a key role in such networks, but there is currently no general and scalable route to the design and construction of enzymic reaction networks. Here, we introduce reversible, cleavable peptide inhibitors that can link proteolytic enzymic activity into simple network motifs. As a proof-of-principle, we show auto-activation topologies producing sigmoidal responses in enzymic activity, explore cross-talk in minimal systems, design a simple enzymic cascade, and introduce non-inhibiting phosphorylated peptides that can be activated using a phosphatase.
- 74Yamazaki, I.; Yokota, K.; Nakajima, R. Oscillatory oxidations of reduced pyridine nucleotide by peroxidase. Biochem. Biophys. Res. Commun. 1965, 21 (6), 582– 6, DOI: 10.1016/0006-291X(65)90525-574https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaF28XjslehtA%253D%253D&md5=944b16272e50aff3e58d1c39ae519aebOscillatory oxidations of reduced pyridine nucleotide by peroxidaseYamazaki, I.; Yokota, K.; Nakajima, R.Biochemical and Biophysical Research Communications (1965), 21 (6), 582-6CODEN: BBRCA9; ISSN:0006-291X.Observation of periodic reactions catalyzed by peroxidase in the presence of reduced pyridine nucleotide under aerobic conditions is described. Addn. of glucose-6-phosphate dehydrogenase to a soln. contg. NADP, Mn2+, glucose 6-phosphate, and peroxidase results in a transient redn. of NADP, followed by a few cyclic responses of oxidn. and redn. until all of the O is consumed. A rapid oxidn. of NADP suddenly occurs at a certain reaction time which corresponds to the termination of Compound III accumulation. Addn. of NADH to an aerobic soln. of peroxidase results in a rapid appearance of Compound III. The rate of concomitant consumption of O overcomes that of the O supply and the O concentration of the soln. decreases, followed by the decompn. of Compound III. The formation of Compound III is mainly due to the reaction of peroxidase with the perhydroxyl radical which causes the chain reaction of the NADH oxidn. The sudden decay of Compound III in the presence of low O concns. is likely due to the redn. of Compound III by intermediates, such as the NAD radical and ferrous peroxidase.
- 75Folke Olsen, L. Complex dynamics in an unexplored simple model of the peroxidase-oxidase reaction. Chaos 2023, 33 (2), 023102 DOI: 10.1063/5.012909575https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB3sXisF2ru7Y%253D&md5=881e99d3a1fba1340d8b1276a9220ea5Complex dynamics in an unexplored simple model of the peroxidase-oxidase reactionFolke Olsen, LarsChaos (2023), 33 (2), 023102CODEN: CHAOEH; ISSN:1054-1500. (American Institute of Physics)A previously overlooked version of the so-called Olsen model of the peroxidase-oxidase reaction has been studied numerically using 2D isospike stability and max. Lyapunov exponent diagrams and reveals a rich variety of dynamic behaviors not obsd. before. The model has a complex bifurcation structure involving mixed-mode and bursting oscillations as well as quasiperiodic and chaotic dynamics. In addn., multiple periodic and non-periodic attractors coexist for the same parameters. For some parameter values, the model also reveals formation of mosaic patterns of complex dynamic states. The complex dynamic behaviors exhibited by this model are compared to those of another version of the same model, which has been studied in more detail. The two models show similarities, but also notable differences between them, e.g., the organization of mixed-mode oscillations in parameter space and the relative abundance of quasiperiodic and chaotic oscillations. In both models, domains with chaotic dynamics contain apparently disorganized subdomains of periodic attractors with dinoflagellate-like structures, while the domains with mainly quasiperiodic behavior contain subdomains with periodic attractors organized as regular filamentous structures. These periodic attractors seem to be organized according to Stern-Brocot arithmetics. Finally, it appears that toroidal (quasiperiodic) attractors develop into first wrinkled and then fractal tori before they break down to chaotic attractors. (c) 2023 American Institute of Physics.
- 76Olsen, L. F.; Lunding, A. Chaos in the peroxidase-oxidase oscillator. Chaos 2021, 31 (1), 013119 DOI: 10.1063/5.002225176https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB3MXhtFGnsb%252FP&md5=040d7f99e066ac615c9bb5dc635c0993Chaos in the peroxidase-oxidase oscillatorOlsen, Lars F.; Lunding, AnitaChaos (2021), 31 (1), 013119CODEN: CHAOEH; ISSN:1054-1500. (American Institute of Physics)The peroxidase-oxidase (PO) reaction involves the oxidn. of reduced NAD by mol. oxygen. When both reactants are supplied continuously to a reaction mixt. contg. the enzyme and a phenolic compd., the reaction will exhibit oscillatory behavior. In fact, the reaction exhibits a zoo of dynamical behaviors ranging from simple periodic oscillations to period-doubled and mixed mode oscillations to quasiperiodicity and chaos. The routes to chaos involve period-doubling, period-adding, and torus bifurcations. The dynamic behaviors in the exptl. system can be simulated by detailed semiquant. models. Previous models of the reaction have omitted the phenolic compd. from the reaction scheme. In the current paper, we present new exptl. results with the oscillating PO reaction that add to our understanding of its rich dynamics, and we describe a new variant of a previous model, which includes the chem. of the phenol in the reaction mechanism. This new model can simulate most of the exptl. behaviors of the exptl. system including the new observations presented here. For example, the model reproduces the two main routes to chaos obsd. in expts.: (i) a period-doubling scenario, which takes place at low pH, and a period-adding scenario involving mixed mode oscillations (MMOs), which occurs at high pH. Our simulations suggest alternative explanations for the pH-sensitivity of the dynamics. We show that the MMO domains are sepd. by narrow parameter regions of chaotic behavior or quasiperiodicity. These regions start as tongues of secondary quasiperiodicity and develop into strange attractors through torus breakdown. (c) 2021 American Institute of Physics.
- 77Scheeline, A.; Olson, D. L.; Williksen, E. P.; Horras, G. A.; Klein, M. L.; Larter, R. The peroxidase–oxidase oscillator and its constituent chemistries. Chem. Rev. 1997, 97 (3), 739– 756, DOI: 10.1021/cr960081a77https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaK2sXisF2murg%253D&md5=ee8592a17b3b80df9865655a040f4e7bThe Peroxidase-Oxidase Oscillator and Its Constituent ChemistriesScheeline, Alexander; Olson, Dean L.; Williksen, Erik P.; Horras, Gregg A.; Klein, Margaret L.; Larter, RaimaChemical Reviews (Washington, D. C.) (1997), 97 (3), 739-756CODEN: CHREAY; ISSN:0009-2665. (American Chemical Society)A review with 165 refs. on the chem. and biochem. of peroxidase concg. on aspect relevant to periodic and chaotic oscillations.
- 78Zhang, Y.; Tsitkov, S.; Hess, H. Complex dynamics in a two-enzyme reaction network with substrate competition. Nat. Catal. 2018, 1 (4), 276– 281, DOI: 10.1038/s41929-018-0053-1There is no corresponding record for this reference.
- 79Gyevi-Nagy, L.; Lantos, E.; Gehér-Herczegh, T.; Tóth, A.; Bagyinka, C.; Horváth, D. Reaction fronts of the autocatalytic hydrogenase reaction. J. Chem. Phys. 2018, 148 (16), 165103, DOI: 10.1063/1.502235979https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC1cXosl2htL4%253D&md5=5b7e7b1a5325910a0eb4ea79b2627f91Reaction fronts of the autocatalytic hydrogenase reactionGyevi-Nagy, Laszlo; Lantos, Emese; Geher-Herczegh, Tunde; Toth, Agota; Bagyinka, Csaba; Horvath, DezsoJournal of Chemical Physics (2018), 148 (16), 165103/1-165103/8CODEN: JCPSA6; ISSN:0021-9606. (American Institute of Physics)We have built a model to describe the hydrogenase catalyzed, autocatalytic, reversible hydrogen oxidn. reaction where one of the enzyme forms is the autocatalyst. The model not only reproduces the exptl. obsd. front properties, but also explains the found hydrogen ion dependence. Furthermore, by linear stability anal., two different front types are found in good agreement with the expts. (c) 2018 American Institute of Physics.
- 80Bagyinka, C.; Pankotai-Bodó, G.; Branca, R. M. M.; Debreczeny, M. Oscillating hydrogenase reaction. Int. J. Hydrogen Energy 2014, 39 (32), 18551– 18555, DOI: 10.1016/j.ijhydene.2014.02.01580https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2cXjsVWhsLg%253D&md5=79655c55851cbb0e6f5d40627e5c7abaOscillating hydrogenase reactionBagyinka, Csaba; Pankotai-Bodo, Gabriella; Branca, Rui M. M.; Debreczeny, MonikaInternational Journal of Hydrogen Energy (2014), 39 (32), 18551-18555CODEN: IJHEDX; ISSN:0360-3199. (Elsevier Ltd.)The hydrogenase-catalyzed oxidn. of H2 includes an autocatalytic step in the reaction cycle. The reaction also exhibits different pH dependence in the H2 oxidn. and in the proton redn. directions. This is not only due to the pH titrn. of the amino acid side-chains as protons are also either the substrates or the products of the reaction. Utilizing the autocatalytic nature of the hydrogenase reaction and the multiple roles of protons therein, together with appropriate limitation of the substrate (gaseous H2) supply, oscillations can be induced in the system. The reaction oscillates both in space and in time, and can last for days with decreasing frequency until reaching chem. equil. Of all biol. oscillating systems described so far, this one is the simplest in that it has the fewest biol. components.
- 81Claaßen, C.; Gerlach, T.; Rother, D. Stimulus-responsive regulation of enzyme activity for one-step and multi-step syntheses. Adv. Synth. Catal. 2019, 361 (11), 2387– 2401, DOI: 10.1002/adsc.20190016981https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC1MXntleru74%253D&md5=aa201db447575541f52c94d6d9f6c3d7Stimulus-Responsive Regulation of Enzyme Activity for One-Step and Multi-Step SynthesesClaassen, Christiane; Gerlach, Tim; Rother, DoerteAdvanced Synthesis & Catalysis (2019), 361 (11), 2387-2401CODEN: ASCAF7; ISSN:1615-4150. (Wiley-VCH Verlag GmbH & Co. KGaA)A review. Multi-step biocatalytic reactions have gained increasing importance in recent years because the combination of different enzymes enables the synthesis of a broad variety of industrially relevant products. However, the more enzymes combined, the more crucial it is to avoid cross-reactivity in these cascade reactions and thus achieve high product yields and high purities. The selective control of enzyme activity, i.e., remote on-/off-switching of enzymes, might be a suitable tool to avoid the formation of unwanted byproducts in multi-enzyme reactions. This review compiles a range of methods that are known to modulate enzyme activity in a stimulus-responsive manner. It focuses predominantly on in vitro systems and is subdivided into reversible and irreversible enzyme activity control. Furthermore, a discussion section provides indications as to which factors should be considered when designing and choosing activity control systems for biocatalysis. Finally, an outlook is given regarding the future prospects of the field.
- 82Chen, Z.; Zhao, Y.; Liu, Y. Advanced strategies of enzyme activity regulation for biomedical applications. ChemBioChem 2022, 23 (21), e202200358, DOI: 10.1002/cbic.20220035882https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB38XitV2ksb3O&md5=a6e13b3b38222804c88636870c7dd7ecAdvanced Strategies of Enzyme Activity Regulation for Biomedical ApplicationsChen, Zihan; Zhao, Yu; Liu, YangChemBioChem (2022), 23 (21), e202200358CODEN: CBCHFX; ISSN:1439-4227. (Wiley-VCH Verlag GmbH & Co. KGaA)A review. Enzymes are important macromol. biocatalysts that accelerate chem. and biochem. reactions in living organisms. Most human diseases are related to alterations in enzyme activity. Moreover, enzymes are potential therapeutic tools for treating different diseases, such as cancer, infections, and cardiovascular and cerebrovascular diseases. Precise remote enzyme activity regulation provides new opportunities to combat diseases. This review summarizes recent advances in the field of enzyme activity regulation, including reversible and irreversible regulation. It also discusses the mechanisms and approaches for on-demand control of these activities. Furthermore, a range of stimulus-responsive inhibitors, polymers, and nanoparticles for regulating enzyme activity and their prospective biomedical applications are summarized. Finally, the current challenges and future perspectives on enzyme activity regulation are discussed.
- 83Hoorens, M. W. H.; Szymanski, W. Reversible, spatial and temporal control over protein activity using light. Trends Biochem. Sci. 2018, 43 (8), 567– 575, DOI: 10.1016/j.tibs.2018.05.00483https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC1cXhtFSru7vI&md5=6802043365a055b6a166db2f493fb391Reversible, Spatial and Temporal Control over Protein Activity Using LightHoorens, Mark W. H.; Szymanski, WiktorTrends in Biochemical Sciences (2018), 43 (8), 567-575CODEN: TBSCDB; ISSN:0968-0004. (Elsevier Ltd.)A review. In biomedical sciences, the function of a protein of interest is investigated by altering its net activity and assessing the consequences for the cell or organism. To change the activity of a protein, a wide variety of chem. and genetic tools have been developed. The drawback of most of these tools is that they do not allow for reversible, spatial and temporal control. Here, we describe selected developments in photopharmacol. that aim at establishing such control over protein activity through bioactive mols. with photo-controlled potency. We also discuss why such control is desired and what challenges still need to be overcome for photopharmacol. to reach its maturity as a chem. biol. research tool.
- 84Kneuttinger, A. C. A guide to designing photocontrol in proteins: methods, strategies and applications. Biol. Chem. 2022, 403 (5–6), 573– 613, DOI: 10.1515/hsz-2021-041784https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB38XhtVCmtLzF&md5=f11065ba65174acc0ee78139a82f764dA guide to designing photocontrol in proteins: methods, strategies and applicationsKneuttinger, Andrea C.Biological Chemistry (2022), 403 (5-6), 573-613CODEN: BICHF3; ISSN:1431-6730. (Walter de Gruyter GmbH)A review. Light is essential for various biochem. processes in all domains of life. In its presence certain proteins inside a cell are excited, which either stimulates or inhibits subsequent cellular processes. The artificial photocontrol of specifically proteins is of growing interest for the investigation of scientific questions on the organismal, cellular and mol. level as well as for the development of medicinal drugs or biocatalytic tools. For the targeted design of photocontrol in proteins, three major methods have been developed over the last decades, which employ either chem. engineering of small-mol. photosensitive effectors (photopharmacol.), incorporation of photoactive non-canonical amino acids by genetic code expansion (photoxenoprotein engineering), or fusion with photoreactive biol. modules (hybrid protein optogenetics). This review compares the different methods as well as their strategies and current applications for the light-regulation of proteins and provides background information useful for the implementation of each technique.
- 85Volarić, J.; Szymanski, W.; Simeth, N. A.; Feringa, B. L. Molecular photoswitches in aqueous environments. Chem. Soc. Rev. 2021, 50 (22), 12377– 12449, DOI: 10.1039/D0CS00547A85https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB3MXitFCgtbvK&md5=4fd8e15de13066519d17b50d2fe7fde0Molecular photoswitches in aqueous environmentsVolaric, Jana; Szymanski, Wiktor; Simeth, Nadja A.; Feringa, Ben L.Chemical Society Reviews (2021), 50 (22), 12377-12449CODEN: CSRVBR; ISSN:0306-0012. (Royal Society of Chemistry)A review. Mol. photoswitches enable dynamic control of processes with high spatiotemporal precision, using light as external stimulus, and hence are ideal tools for different research areas spanning from chem. biol. to smart materials. Photoswitches are typically org. mols. that feature extended arom. systems to make them responsive to (visible) light. However, this renders them inherently lipophilic, while water-soly. is of crucial importance to apply photoswitchable org. mols. in biol. systems, like in the rapidly emerging field of photopharmacol. Several strategies for solubilizing org. mols. in water are known, but there are not yet clear rules for applying them to photoswitchable mols. Importantly, rendering photoswitches water-sol. has a serious impact on both their photophys. and biol. properties, which must be taken into consideration when designing new systems. Altogether, these aspects pose considerable challenges for successfully applying mol. photoswitches in aq. systems, and in particular in biol. relevant media. In this , we focus on fully water-sol. photoswitches, such as those used in biol. environments, in both in vitro and in vivo studies. We discuss the design principles and prospects for water-sol. photoswitches to inspire and enable their future applications.
- 86Pogodaev, A. A.; Lap, T. T.; Huck, W. T. S. The dynamics of an oscillating enzymatic reaction network is crucially determined by side reactions. ChemSystemsChem 2021, 3, e2000033, DOI: 10.1002/syst.20200003386https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB3MXovFylsbk%253D&md5=242d0992326356525f85f0960ece3499The Dynamics of an Oscillating Enzymatic Reaction Network is Crucially Determined by Side ReactionsPogodaev, Aleksandr A.; Lap, Tijs T.; Huck, Wilhelm T. S.ChemSystemsChem (2021), 3 (1), e2000033CODEN: CSCHCD; ISSN:2570-4206. (Wiley-VCH Verlag GmbH & Co. KGaA)Synthetic complex chem. systems are often subject to perturbations in reaction conditions. To ensure robust functioning of these systems in real-world applications, a better understanding is required of how resilience to perturbations could be included in the design of these systems. In order to develop such an understanding we need a deeper insight into how chem. systems respond to perturbations. Here, we study the effect of spiking concns. in an oscillating enzymic reaction network. We identify that a different magnitude of a perturbation triggers two distinctive responses: obtaining sustained oscillations and causing the loss of the amplitude. We rationalise our findings based on non-linear dynamics and identify that non-essential side reactions crucially tailor the obsd. behavior.
- 87Crespi, S.; Simeth, N. A.; König, B. Heteroaryl azo dyes as molecular photoswitches. Nat. Rev. Chem. 2019, 3, 133– 146, DOI: 10.1038/s41570-019-0074-687https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC1MXnsFCltL0%253D&md5=b8fd12702cc1d5c5ad102de3b6ce6378Heteroaryl azo dyes as molecular photoswitchesCrespi, Stefano; Simeth, Nadja A.; Koenig, BurkhardNature Reviews Chemistry (2019), 3 (3), 133-146CODEN: NRCAF7; ISSN:2397-3358. (Nature Research)A review. We have known of azobenzene for over 150 years, the past 80 of which have seen the study and application of its photochromism. Azobenzene derivs. are now considered archetypical mol. switches, and their stability and reliability make them amenable to many fields of modern chem., materials science, biol. and photopharmacol. When developing a photoswitch for a given application, a common approach is to tune the properties of an azobenzene. It is also possible to instead use heteroaryl azo dyes - motifs that are less popular even though their diversity offers distinct features. Despite the first discoveries of switching behavior in heteroaryl azos and azobenzenes being coincident, the former have only recently begun to attract attention. This Review describes how the versatile and multifaceted characteristics of these scaffolds make them serious alternatives to azobenzene derivs. in mol. photoactuation. Heteroaryl azo photoswitches arguably deserve more consideration, and our survey of these systems includes challenges to their successful deployment.
- 88Teders, M.; Pogodaev, A. A.; Bojanov, G.; Huck, W. T. S. Reversible photoswitchable inhibitors generate ultrasensitivity in out-of-equilibrium enzymatic reactions. J. Am. Chem. Soc. 2021, 143 (15), 5709– 5716, DOI: 10.1021/jacs.0c1295688https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB3MXosVWntLo%253D&md5=4aa47d83f4357ed58cd6585db025bb59Reversible photoswitchable inhibitors generate ultrasensitivity in out-of-equil. enzymic reactionsTeders, Michael; Pogodaev, Aleksandr A.; Bojanov, Glenn; Huck, Wilhelm T. S.Journal of the American Chemical Society (2021), 143 (15), 5709-5716CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)Ultrasensitivity is a ubiquitous emergent property of biochem. reaction networks. The design and construction of synthetic reaction networks exhibiting ultrasensitivity has been challenging, but would greatly expand the potential properties of life-like materials. Herein, we exploit a general and modular strategy to reversibly regulate the activity of enzymes using light and show how ultrasensitivity arises in simple out-of-equil. enzymic systems upon incorporation of reversible photoswitchable inhibitors (PIs). Utilizing a chromophore/warhead strategy, PIs of the protease α-chymotrypsin were synthesized, which led to the discovery of inhibitors with large differences in inhibition consts. (Ki) for the different photoisomers. A microfluidic flow setup was used to study enzymic reactions under out-of-equil. conditions by continuous addn. and removal of reagents. Upon irradn. of the continuously stirred tank reactor with different light pulse sequences, i.e., varying the pulse duration or frequency of UV and blue light irradn., reversible switching between photoisomers resulted in ultrasensitive responses in enzymic activity as well as frequency filtering of input signals. This general and modular strategy enables reversible and tunable control over the kinetic rates of individual enzyme-catalyzed reactions and makes a programmable linkage of enzymes to a wide range of network topologies feasible.
- 89Teders, M.; Murray, N. R.; Huck, W. T. S. Reversible photoswitchable inhibitors enable wavelength-selective regulation of out-of-equilibrium bi-enzymatic systems. ChemSystemsChem 2021, 3 (6), e2100020, DOI: 10.1002/syst.20210002089https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB3MXisFSjsrrF&md5=2266f480fd59765360dab98b60932eb3Reversible Photoswitchable Inhibitors Enable Wavelength-Selective Regulation of Out-of-Equilibrium Bi-enzymatic SystemsTeders, Michael; Murray, Nicholas R.; Huck, Wilhelm T. S.ChemSystemsChem (2021), 3 (6), e2100020CODEN: CSCHCD; ISSN:2570-4206. (Wiley-VCH Verlag GmbH & Co. KGaA)The construction of synthetic enzymic reaction networks can provide new insights into the design principles of living systems. However, the programmable connection of enzymes into a wide range of network topologies has been challenging due to the lack of a general strategy enabling a reversible activity regulation of individual network enzymes. Here, we exploit a general and modular strategy based on the external regulation of enzymes using light and photoswitchable inhibitors (PIs) that enables the bottom-up construction and control of enzymic systems studied under out-of-equil. conditions. Upon synthesis and incorporation of potent photoswitchable trypsin inhibitors (Tr-PIs), the output of several functional enzymic systems could be photoregulated using 390/460 nm light as a trigger signal. In addn., the wavelength-selective control over the activity of two enzymes within a functional bi-enzymic system was achieved using a suitable combination of two PIs.
- 90Li, K.; Liu, M. D.; Huang, Q. X.; Liu, C. J.; Zhang, X. Z. Nanoplatforms with donor-acceptor Stenhouse adduct molecular switch for enzymatic reactions remotely controlled with near-infrared light. Sci. China Mater. 2023, 66, 375– 384, DOI: 10.1007/s40843-022-2107-090https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB38XhvVGkur3L&md5=b5f8248b37bd48da19c39abc5f802662Nanoplatforms with donor-acceptor Stenhouse adduct molecular switch for enzymatic reactions remotely controlled with near-infrared lightLi, Ke; Liu, Miao-Deng; Huang, Qian-Xiao; Liu, Chuan-Jun; Zhang, Xian-ZhengScience China Materials (2023), 66 (1), 375-384CODEN: SCMCDB; ISSN:2095-8226. (Science China Press)Enzymes have been widely used in biomedical applications owing to their excellent biocatalysis functions. However, the precise control of enzymic reactions remains a challenge in vivo. Here, a nanoplatform functionalized with upconversion nanoparticles (UCNPs) as the core and a donor-acceptor Stenhouse adduct as a mol. switch (MS) was designed for near-IR (NIR)-controlled enzymic reactions. Under NIR irradn., the UCNPs could emit bright green light to isomerize the MS and then transform the permeability of the MS-gated polymer layer, allowing small mols. to penetrate the polymer layer. Consequently, the contact between the enzyme and substrate could be regulated to control the enzymic reaction remotely. As the photoisomerism of MS is reversible and the enzyme activity is not changed, the enzyme reactor based on this nanoplatform is also reversible, which can achieve precise temporal and spatial control of the enzyme reaction. Glucose oxidase (GOX), lactate oxidase (LOX), urate oxidase (UOX), and luciferase were used to assess the controllability of the enzymic reactions. Both the substrates in and out of the nanoplatform were satisfactorily controlled, indicating that the gated nanoplatform equipped with valve-like characteristics could realize remotely controlled enzymic reactions upon NIR irradn.
- 91Hindley, J. W.; Elani, Y.; McGilvery, C. M.; Ali, S.; Bevan, C. L.; Law, R. V.; Ces, O. Light-triggered enzymatic reactions in nested vesicle reactors. Nat. Commun. 2018, 9 (1), 1093, DOI: 10.1038/s41467-018-03491-791https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A280%3ADC%252BC1MnhslKisg%253D%253D&md5=96a3060c2d2721132e189f186947f5c3Light-triggered enzymatic reactions in nested vesicle reactorsHindley James W; Elani Yuval; Law Robert V; Ces Oscar; Hindley James W; Elani Yuval; Law Robert V; Ces Oscar; McGilvery Catriona M; Ali Simak; Bevan Charlotte LNature communications (2018), 9 (1), 1093 ISSN:.Cell-sized vesicles have tremendous potential both as miniaturised pL reaction vessels and in bottom-up synthetic biology as chassis for artificial cells. In both these areas the introduction of light-responsive modules affords increased functionality, for example, to initiate enzymatic reactions in the vesicle interior with spatiotemporal control. Here we report a system composed of nested vesicles where the inner compartments act as phototransducers, responding to ultraviolet irradiation through diacetylene polymerisation-induced pore formation to initiate enzymatic reactions. The controlled release and hydrolysis of a fluorogenic β-galactosidase substrate in the external compartment is demonstrated, where the rate of reaction can be modulated by varying ultraviolet exposure time. Such cell-like nested microreactor structures could be utilised in fields from biocatalysis through to drug delivery.
- 92Babii, O.; Afonin, S.; Diel, C.; Huhn, M.; Dommermuth, J.; Schober, T.; Koniev, S.; Hrebonkin, A.; Nesterov-Mueller, A.; Komarov, I. V. Diarylethene-based photoswitchable inhibitors of serine proteases. Angew. Chem., Int. Ed. 2021, 60 (40), 21789– 21794, DOI: 10.1002/anie.20210884792https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB3MXhvFShu7zP&md5=204d0959bc8b05f71e2b016406e4554cDiarylethene-Based Photoswitchable Inhibitors of Serine ProteasesBabii, Oleg; Afonin, Sergii; Diel, Christian; Huhn, Marcel; Dommermuth, Jennifer; Schober, Tim; Koniev, Serhii; Hrebonkin, Andrii; Nesterov-Mueller, Alexander; Komarov, Igor V.; Ulrich, Anne S.Angewandte Chemie, International Edition (2021), 60 (40), 21789-21794CODEN: ACIEF5; ISSN:1433-7851. (Wiley-VCH Verlag GmbH & Co. KGaA)A bicyclic peptide scaffold was chem. adapted to generate diarylethene-based photoswitchable inhibitors of serine protease Bos taurus trypsin 1 (T1). Starting from a prototype mol.-sunflower trypsin inhibitor-1 (SFTI-1)-the authors obtained light-controllable inhibitors of T1 with Ki in the low nanomolar range, whose activity could be modulated over 20-fold by irradn. The inhibitory potency as well as resistance to proteolytic degrdn. were systematically studied on a series of 17 SFTI-1 analogs. The hydrogen bond network that stabilizes the structure of inhibitors and possibly the enzyme-inhibitor binding dynamics were affected by isomerization of the photoswitch. The feasibility of manipulating enzyme activity in time and space was demonstrated by controlled digestion of gelatin-based hydrogel and an antimicrobial peptide BP100-RW. Finally, the authors' design principles of diarylethene photoswitches apply also for the development of other serine protease inhibitors.
- 93Rifaie-Graham, O.; Yeow, J.; Najer, A.; Wang, R.; Sun, R.; Zhou, K.; Dell, T. N.; Adrianus, C.; Thanapongpibul, C.; Chami, M. Photoswitchable gating of non-equilibrium enzymatic feedback in chemically communicating polymersome nanoreactors. Nat. Chem. 2023, 15 (1), 110– 118, DOI: 10.1038/s41557-022-01062-493https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB38XivVajs7bL&md5=b01721d704940a001212e55306e28618Photoswitchable gating of non-equilibrium enzymatic feedback in chemically communicating polymersome nanoreactorsRifaie-Graham, Omar; Yeow, Jonathan; Najer, Adrian; Wang, Richard; Sun, Rujie; Zhou, Kun; Dell, Tristan N.; Adrianus, Christopher; Thanapongpibul, Chalaisorn; Chami, Mohamed; Mann, Stephen; de Alaniz, Javier Read; Stevens, Molly M.Nature Chemistry (2023), 15 (1), 110-118CODEN: NCAHBB; ISSN:1755-4330. (Nature Portfolio)The circadian rhythm generates out-of-equil. metabolite oscillations that are controlled by feedback loops under light/dark cycles. Here we describe a non-equil. nanosystem comprising a binary population of enzyme-contg. polymersomes capable of light-gated chem. communication, controllable feedback and coupling to macroscopic oscillations. The populations consist of esterase-contg. polymersomes functionalized with photo-responsive donor-acceptor Stenhouse adducts (DASA) and light-insensitive semipermeable urease-loaded polymersomes. The DASA-polymersome membrane becomes permeable under green light, switching on esterase activity and decreasing the pH, which in turn initiates the prodn. of alkali in the urease-contg. population. A pH-sensitive pigment that absorbs green light when protonated provides a neg. feedback loop for deactivating the DASA-polymersomes. Simultaneously, increased alkali prodn. deprotonates the pigment, reactivating esterase activity by opening the membrane gate. We utilize light-mediated fluctuations of pH to perform non-equil. communication between the nanoreactors and use the feedback loops to induce work as chemomech. swelling/deswelling oscillations in a crosslinked hydrogel. We envision possible applications in artificial organelles, protocells and soft robotics.
- 94Bisswanger, H. Enzyme assays. Perspectives in Science 2014, 1 (1–6), 41– 55, DOI: 10.1016/j.pisc.2014.02.005There is no corresponding record for this reference.
- 95Zhang, Y.; Wang, Q.; Hess, H. Increasing enzyme cascade throughput by pH-engineering the microenvironment of individual enzymes. ACS Catal. 2017, 7 (3), 2047– 2051, DOI: 10.1021/acscatal.6b0343195https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2sXisVOkurk%253D&md5=50fb5a9647fbe533d55dff99c707712fIncreasing enzyme cascade throughput by pH-engineering the microenvironment of individual enzymesZhang, Yifei; Wang, Qin; Hess, HenryACS Catalysis (2017), 7 (3), 2047-2051CODEN: ACCACS; ISSN:2155-5435. (American Chemical Society)The throughput of enzyme cascade reactions could be increased if the environmental conditions could be optimized for each enzyme in the cascade individually. Here, we describe the engineering of the microenvironment of a hemoprotein, cytochrome c (cyt c), which is active under acidic conditions, with respect to pH, so that it operates optimally together with a the enzyme, D-amino acid oxidase, which is active under alk. conditions. Conjugation of cyt c with a neg. charged polyelectrolyte [poly(methacrylic acid)] lowered the local pH at the cyt c active site and enabled a 10-fold enhancement in cascade throughput.
- 96Fan, X.; Walther, A. pH feedback lifecycles programmed by enzymatic logic gates using common foods as fuels. Angew. Chem., Int. Ed. 2021, 60, 11398, DOI: 10.1002/anie.20201700396https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB3MXotlGktrs%253D&md5=eec6c760a9a40322bed51e3b5b458040pH Feedback Lifecycles Programmed by Enzymatic Logic Gates Using Common Foods as FuelsFan, Xinlong; Walther, AndreasAngewandte Chemie, International Edition (2021), 60 (20), 11398-11405CODEN: ACIEF5; ISSN:1433-7851. (Wiley-VCH Verlag GmbH & Co. KGaA)Artificial temporal signaling systems, which mimic living out-of-equil. conditions, have made large progress. However, systems programmed by enzymic reaction networks in multicomponent and unknown environments, and using biocompatible components remain a challenge. Herein, we demonstrate an approach to program temporal pH signals by enzymic logic gates. They are realized by an enzymic disaccharide-to-monosaccharide-to-sugar acid reaction cascade catalyzed by two metabolic chains: invertase-glucose oxidase and β-galactosidase-glucose oxidase, resp. Lifetimes of the transient pH signal can be programmed from less than 15 min to more than 1 day. We study enzymic kinetics of the reaction cascades and reveal the underlying regulatory mechanisms. Operating with all-food grade chems. and coupling to self-regulating hydrogel, our system is quite robust to work in a complicated medium with unknown components and in a biocompatible fashion.
- 97Che, H.; Cao, S.; van Hest, J. C. M. Feedback-induced temporal control of ″breathing″ polymersomes to create self-adaptive nanoreactors. J. Am. Chem. Soc. 2018, 140 (16), 5356– 5359, DOI: 10.1021/jacs.8b0238797https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC1cXntVCmurk%253D&md5=178dddacb91d1c063f0d6104e216d2e6Feedback-Induced Temporal Control of "Breathing" Polymersomes To Create Self-Adaptive NanoreactorsChe, Hailong; Cao, Shoupeng; van Hest, Jan C. M.Journal of the American Chemical Society (2018), 140 (16), 5356-5359CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)Here the authors present the development of self-regulated "breathing" polymersome nanoreactors that show temporally programmable biocatalysis induced by a chem. fuel. pH-sensitive polymersomes loaded with horseradish peroxidase (HRP) and urease were developed. Addn. of an acidic urea soln. ("fuel") endowed the polymersomes with a transient size increase and permeability enhancement, driving a temporal "ON" state of the HRP enzymic catalysis; subsequent depletion of fuel led to shrinking of the polymersomes, resulting in the catalytic "OFF" state. Moreover, the nonequil. nanoreactors could be reinitiated several cycles as long as fuel was supplied. This feedback-induced temporal control of catalytic activity in polymersome nanoreactors provides a platform for functional nonequil. systems as well as for artificial organelles with precisely controlled adaptivity.
- 98Wells, P. K.; Smutok, O.; Melman, A.; Katz, E. Switchable biocatalytic reactions controlled by interfacial pH changes produced by orthogonal biocatalytic processes. ACS Appl. Mater. Interfaces 2021, 13 (29), 33830– 33839, DOI: 10.1021/acsami.1c0739398https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB3MXhsFCltrvK&md5=35c6c8a040f735ffddb70ceb0fe21816Switchable Biocatalytic Reactions Controlled by Interfacial pH Changes Produced by Orthogonal Biocatalytic ProcessesWells, Paulina K.; Smutok, Oleh; Melman, Artem; Katz, EvgenyACS Applied Materials & Interfaces (2021), 13 (29), 33830-33839CODEN: AAMICK; ISSN:1944-8244. (American Chemical Society)Enzymes immobilized on a nano-structured surface were used to switch the activity of one enzyme by a local pH change produced by another enzyme. Immobilized amyloglucosidase (AMG) and trypsin were studied as examples of the pH-dependent switchable "target enzymes." The reactions catalyzed by co-immobilized urease or esterase were increasing or decreasing the local pH, resp., thus operating as "actuator enzymes." Both kinds of the enzymes, producing local pH changes and changing biocatalytic activity with the pH variation, were orthogonal in terms of the biocatalytic reactions; however, their operation was coupled with the local pH produced near the surface with the immobilized enzymes. The "target enzymes" (AMG and trypsin) were changed reversibly between the active and inactive states by applying input signals (urea or ester, substrates for the urease or esterase operating as the "actuator enzymes") and washing them out with a new portion of the background soln. The developed approach can potentially lead to switchable operation of several enzymes, while some of them are inhibited when the others are activated upon receiving external signals processed by the "actuator enzymes." More complex systems with branched biocatalytic cascades can be controlled by orthogonal biocatalytic reactions activating selected pathways and changing the final output.
- 99Wang, C.; Fischer, A.; Ehrlich, A.; Nahmias, Y.; Willner, I. Biocatalytic reversible control of the stiffness of DNA-modified responsive hydrogels: applications in shape-memory, self-healing and autonomous controlled release of insulin. Chem. Sci. 2020, 11 (17), 4516– 4524, DOI: 10.1039/D0SC01319F99https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB3cXmvFOitrc%253D&md5=8da648a257fc758d89335776e8164d59Biocatalytic reversible control of the stiffness of DNA-modified responsive hydrogels: applications in shape-memory, self-healing and autonomous controlled release of insulinWang, Chen; Fischer, Amit; Ehrlich, Avner; Nahmias, Yaakov; Willner, ItamarChemical Science (2020), 11 (17), 4516-4524CODEN: CSHCCN; ISSN:2041-6520. (Royal Society of Chemistry)The enzymes glucose oxidase (GOx), acetylcholine esterase (AchE) and urease that drive biocatalytic transformations to alter pH, are integrated into pH-responsive DNA-based hydrogels. A two-enzyme-loaded hydrogel composed of GOx/urease or AchE/urease and a three-enzyme-loaded hydrogel composed of GOx/AchE/urease are presented. The biocatalytic transformations within the hydrogels lead to the dictated reconfiguration of nucleic acid bridges and the switchable control over the stiffness of the resp. hydrogels. The switchable stiffness features are used to develop biocatalytically guided shape-memory and self-healing matrixes. In addn., loading of GOx/insulin in a pH-responsive DNA-based hydrogel yields a glucose-triggered matrix for the controlled release of insulin, acting as an artificial pancreas. The release of insulin is controlled by the concns. of glucose, hence, the biocatalytic insulin-loaded hydrogel provides an interesting sense-and-treat carrier for controlling diabetes.
- 100Zhang, Y.; Nie, N.; Wang, H.; Tong, Z.; Xing, H.; Zhang, Y. Smart enzyme catalysts capable of self-separation by sensing the reaction extent. Biosens. Bioelectron. 2023, 239, 115585, DOI: 10.1016/j.bios.2023.115585100https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB3sXhslWns73P&md5=3e0c482c8735cb453cfa2580577caf5eSmart enzyme catalysts capable of self-separation by sensing the reaction extentZhang, Yinchen; Nie, Ning; Wang, Haoran; Tong, Ziyi; Xing, Hao; Zhang, YifeiBiosensors & Bioelectronics (2023), 239 (), 115585CODEN: BBIOE4; ISSN:0956-5663. (Elsevier B.V.)A smart biocatalyst should dissolve homogeneously for catalysis and recover spontaneously at the end of the reaction. In this study, we present a strategy for prepg. self-pptg. enzyme catalysts by exploiting reaction-induced pH decreases, which connect the reaction extent to the catalyst aggregation state. Using poly(methacrylic acid)-functionalized gold nanoparticles as carriers, we construct smart catalysts with three model systems, including the glucose oxidase (GOx)-catalase (CAT) cascade, the alc. dehydrogenase (ADH)-glucose dehydrogenase (GDH) cascade, and a combination of two lipases. All smart catalysts can self-sep. with a nearly 100% recovery efficiency when a certain conversion threshold is reached. The threshold can be adjusted depending on the reaction demand and buffer capacity. By monitoring the optical signals caused by the dissoln./pptn. of smart catalysts, we propose a prototypic automation system that may enable unsupervised batch/fed-batch bioprocessing.
- 101Jain, M.; Ravoo, B. J. Fuel-driven and enzyme-regulated redox-responsive supramolecular hydrogels. Angew. Chem., Int. Ed. 2021, 60 (38), 21062– 21068, DOI: 10.1002/anie.202107917101https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB3MXhsl2ltb7F&md5=644dd8f13ce610d6bbbdd4018b3e5a5cFuel-Driven and Enzyme-Regulated Redox-Responsive Supramolecular HydrogelsJain, Mehak; Ravoo, Bart JanAngewandte Chemie, International Edition (2021), 60 (38), 21062-21068CODEN: ACIEF5; ISSN:1433-7851. (Wiley-VCH Verlag GmbH & Co. KGaA)Chem. reaction networks (CRN) embedded in hydrogels can transform responsive materials into complex self-regulating materials that generate feedback to counter the effect of external stimuli. This study presents hydrogels contg. the β-cyclodextrin (CD) and ferrocene (Fc) host-guest pair as supramol. crosslinks where redox-responsive behavior is driven by the enzyme-fuel couples horse radish peroxidase (HRP)-H2O2 and glucose oxidase (GOx)-D-glucose. The hydrogel can be tuned from a responsive to a self-regulating supramol. system by varying the concn. of added redn. fuel D-glucose. The onset of self-regulating behavior is due to formation of oxidn. fuel in the hydrogel by a cofactor intermediate GOx[FADH2]. UV/Vis spectroscopy, rheol., and kinetic modeling were employed to understand the emergence of out-of-equil. behavior and reveal the programmable neg. feedback response of the hydrogel, including the adaptation of its elastic modulus and its potential as a glucose sensor.
- 102Eixelsberger, T.; Nidetzky, B. Enzymatic redox cascade for one-pot synthesis of uridine 5′-diphosphate xylose from uridine 5′-diphosphate glucose. Adv. Synth. Catal. 2014, 356 (17), 3575– 3584, DOI: 10.1002/adsc.201400766102https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2cXhvVKlsLvO&md5=fcd9da668f65eddf73f1bc5bb9886e73Enzymatic Redox Cascade for One-Pot Synthesis of Uridine 5'-Diphosphate Xylose from Uridine 5'-Diphosphate GlucoseEixelsberger, Thomas; Nidetzky, BerndAdvanced Synthesis & Catalysis (2014), 356 (17), 3575-3584CODEN: ASCAF7; ISSN:1615-4150. (Wiley-VCH Verlag GmbH & Co. KGaA)Synthetic ways towards UDP (UDP)-xylose are scarce and not well established, although this compd. plays an important role in the glycobiol. of various organisms and cell types. We show here how UDP-glucose 6-dehydrogenase (hUGDH) and UDP-xylose synthase 1 (hUXS) from Homo sapiens can be used for the efficient prodn. of pure UDP-α-xylose from UDP-glucose. In a mimic of the natural biosynthetic route, UDP-glucose is converted to UDP-glucuronic acid by hUGDH, followed by subsequent formation of UDP-xylose by hUXS. The NAD (NAD+) required in the hUGDH reaction is continuously regenerated in a three-step chemo-enzymic cascade. In the first step, reduced NAD+ (NADH) is recycled by xylose reductase from Candida tenuis via redn. of 9,10-phenanthrenequinone (PQ). Radical chem. re-oxidn. of this mediator in the second step reduces mol. oxygen to hydrogen peroxide (H2O2) that is cleaved by bovine liver catalase in the last step. A comprehensive anal. of the coupled chemo-enzymic reactions revealed pronounced inhibition of hUGDH by NADH and UDP-xylose as well as an adequate oxygen supply for PQ re-oxidn. as major bottlenecks of effective performance of the overall multi-step reaction system. Net oxidn. of UDP-glucose to UDP-xylose by hydrogen peroxide (H2O2) could thus be achieved when using an in situ oxygen supply through periodic external feed of H2O2 during the reaction. Engineering of the interrelated reaction parameters finally enabled prodn. of 19.5 mM (10.5 g L-1) UDP-α-xylose. After two-step chromatog. purifn. the compd. was obtained in high purity (>98%) and good overall yield (46%). The results provide a strong case for application of multi-step redox cascades in the synthesis of nucleotide sugar products.
- 103Kuk, S. K.; Singh, R. K.; Nam, D. H.; Singh, R.; Lee, J. K.; Park, C. B. Photoelectrochemical reduction of carbon dioxide to methanol through a highly efficient enzyme cascade. Angew. Chem., Int. Ed. 2017, 56 (14), 3827– 3832, DOI: 10.1002/anie.201611379103https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2sXhs1ems7o%253D&md5=db856189622518381382e2fd5d0b43b6Photoelectrochemical Reduction of Carbon Dioxide to Methanol through a Highly Efficient Enzyme CascadeKuk, Su Keun; Singh, Raushan K.; Nam, Dong Heon; Singh, Ranjitha; Lee, Jung-Kul; Park, Chan BeumAngewandte Chemie, International Edition (2017), 56 (14), 3827-3832CODEN: ACIEF5; ISSN:1433-7851. (Wiley-VCH Verlag GmbH & Co. KGaA)Natural photosynthesis is an effective route for the clean and sustainable conversion of CO2 into high-energy chems. Inspired by the natural process, a tandem photoelectrochem. (PEC) cell with an integrated enzyme-cascade (TPIEC) system was designed, which transfers photogenerated electrons to a multienzyme cascade for the biocatalyzed redn. of CO2 to methanol. A hematite photoanode and a bismuth ferrite photocathode were applied to fabricate the iron oxide based tandem PEC cell for visible-light-assisted regeneration of the nicotinamide cofactor (NADH). The cell utilized water as an electron donor and spontaneously regenerated NADH. To complete the TPIEC system, a superior three-dehydrogenase cascade system was employed in the cathodic part of the PEC cell. Under applied bias, the TPIEC system achieved a high methanol conversion output of 220 μm h-1, 1280 μmol g-1 h-1 using readily available solar energy and water.
- 104Mallawarachchi, S.; Gejji, V.; Sierra, L. S.; Wang, H.; Fernando, S. Electrical field reversibly modulates enzyme kinetics of hexokinase entrapped in an electro-responsive hydrogel. ACS Appl. Bio Mater. 2019, 2 (12), 5676– 5686, DOI: 10.1021/acsabm.9b00748104https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC1MXitVOht7rO&md5=349857855cebc50da63f91fdc2e2f765Electrical Field Reversibly Modulates Enzyme Kinetics of Hexokinase Entrapped in an Electro-Responsive HydrogelMallawarachchi, Samavath; Gejji, Varun; Sierra, Laura Soto; Wang, Haoqi; Fernando, SandunACS Applied Bio Materials (2019), 2 (12), 5676-5686CODEN: AABMCB; ISSN:2576-6422. (American Chemical Society)In this paper, the potential use of electro-responsive poly(acrylic acid) (PAA) gels as reversible enzyme activity regulators is analyzed. This was evaluated by measuring the glucose conversion by hexokinase embedded PAA hydrogels under external elec. stimuli. Hexokinase phys. entrapped within PAA gels showed a significant increase in activity under an elec. stimulus as compared to in the absence of a stimulus. Kinetic studies revealed that the change in reaction rate could be attributed to the change of Vmax under a stimulus, while Km was unaffected by the stimulus, which suggested that the increase in reaction rate under an elec. stimulus was due to increased accessibility of the active site. Optimum stimuli-responsive behavior that resulted in max. conversion under a stimulus and min. conversion in the absence of a stimulus was obtained at 5.5 pH and 30 °C. The significant difference between the pH optima for the entrapped enzyme and the pure enzyme can be attributed to the acidic nature of the polymeric matrix. Higher cross-linker concns. resulted in a redn. of both enzyme release and glucose conversion, and a reasonable trade-off between conversion and release could be obtained at 5% cross-linker concn. Application of a stepwise elec. stimulus revealed that the entrapped enzymes could sustain responsive properties over multiple cycles of elec. switching. Entrapped hexokinase also showed much better reusability compared to pure hexokinase, a combined result of higher enzyme retention and increased stability. No significant impact of the polymer on the interaction between enzyme and glucose was obsd. Thus, this system enables electro-responsive modulation of enzyme activity without any redn. in enzyme activity. The studies revealed that conjugation of electro-responsive polymers to enzymes has the potential to reversibly modulate enzymic reactions via the application of external elec. stimuli, which is promising for bioprocessing and enzymic sepn. applications.
- 105Morello, G.; Megarity, C. F.; Armstrong, F. A. The power of electrified nanoconfinement for energising, controlling and observing long enzyme cascades. Nat. Commun. 2021, 12 (1), 340, DOI: 10.1038/s41467-020-20403-w105https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB3MXhsVelsrk%253D&md5=144f16dcc5a3103a0ecddb9dabb20714The power of electrified nanoconfinement for energising, controlling and observing long enzyme cascadesMorello, Giorgio; Megarity, Clare F.; Armstrong, Fraser A.Nature Communications (2021), 12 (1), 340CODEN: NCAOBW; ISSN:2041-1723. (Nature Research)Abstr.: Multistep enzyme-catalyzed cascade reactions are highly efficient in nature due to the confinement and concn. of the enzymes within nanocompartments. In this way, rates are exceptionally high, and loss of intermediates minimised. Similarly, extended enzyme cascades trapped and crowded within the nanoconfined environment of a porous conducting metal oxide electrode material form the basis of a powerful way to study and exploit myriad complex biocatalytic reactions and pathways. One of the confined enzymes, ferredoxin-NADP+ reductase, serves as a transducer, rapidly and reversibly recycling nicotinamide cofactors electrochem. for immediate delivery to the next enzyme along the chain, thereby making it possible to energize, control and observe extended cascade reactions driven in either direction depending on the electrode potential that is applied. Here we show as proof of concept the synthesis of aspartic acid from pyruvic acid or its reverse oxidative decarboxylation/deamination, involving five nanoconfined enzymes.
- 106Milton, R. D.; Cai, R.; Abdellaoui, S.; Leech, D.; De Lacey, A. L.; Pita, M.; Minteer, S. D. Bioelectrochemical Haber-Bosch process: an ammonia-producing H2/N2 fuel cell. Angew. Chem., Int. Ed. 2017, 56 (10), 2680– 2683, DOI: 10.1002/anie.201612500106https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2sXitVKnurg%253D&md5=fe1ceeac4a36827692ba7997994321c3Bioelectrochemical Haber-Bosch Process: An Ammonia-Producing H2/N2 Fuel CellMilton, Ross D.; Cai, Rong; Abdellaoui, Sofiene; Leech, Donal; De Lacey, Antonio L.; Pita, Marcos; Minteer, Shelley D.Angewandte Chemie, International Edition (2017), 56 (10), 2680-2683CODEN: ACIEF5; ISSN:1433-7851. (Wiley-VCH Verlag GmbH & Co. KGaA)Nitrogenases are the only enzymes known to reduce mol. nitrogen (N2) to ammonia (NH3). By using Me viologen (N,N'-dimethyl-4,4'-bipyridinium) to shuttle electrons to nitrogenase, N2 redn. to NH3 can be mediated at an electrode surface. The coupling of this nitrogenase cathode with a bioanode that utilizes the enzyme hydrogenase to oxidize mol. hydrogen (H2) results in an enzymic fuel cell (EFC) that is able to produce NH3 from H2 and N2 while simultaneously producing an elec. current. To demonstrate this, a charge of 60 mC was passed across H2 /N2 EFCs, which resulted in the formation of 286 nmol NH3 mg-1 MoFe protein, corresponding to a Faradaic efficiency of 26.4 %.
- 107Cao, Y.; Wang, Y. Temperature-mediated regulation of enzymatic activity. ChemCatChem. 2016, 8 (17), 2740– 2747, DOI: 10.1002/cctc.201600406107https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC28XhtFersb7I&md5=3fa509ec15c4fdab3c62abebd2bc6bf7Temperature-Mediated Regulation of Enzymatic ActivityCao, Yuanyuan; Wang, YapeiChemCatChem (2016), 8 (17), 2740-2747CODEN: CHEMK3; ISSN:1867-3880. (Wiley-VCH Verlag GmbH & Co. KGaA)A review. Controllable nanobiocatalysis using enzymes has evolved to play an important role in the specific and efficient prepn. of bioproducts. Temp. change is one promising tool owing to the dependence of enzyme catalysis on temp. Some examples of regulation of enzymic activity through direct or indirect heating methods with the assistance of thermoresponsive or thermal harvesting materials are summarized in this concept article. Several fascinating applications including remote control of enzyme and/or substrate release, on-off enzyme reactions triggered by temp. variation to sep. products from enzymes and remote acceleration of enzyme reactions even at room temp. have been highlighted. All this provides a new perspective for establishing on-demand enzyme catalysis to further their applications in biol. engineering, chem. industry, and scientific research fields.
- 108Zhang, S.; Wang, C.; Chang, H.; Zhang, Q.; Cheng, Y. Off-on switching of enzyme activity by near-infrared light-induced photothermal phase transition of nanohybrids. Sci. Adv. 2019, 5, eaaw4252, DOI: 10.1126/sciadv.aaw4252There is no corresponding record for this reference.
- 109Gobbo, P.; Patil, A. J.; Li, M.; Harniman, R.; Briscoe, W. H.; Mann, S. Programmed assembly of synthetic protocells into thermoresponsive prototissues. Nat. Mater. 2018, 17 (12), 1145– 1153, DOI: 10.1038/s41563-018-0183-5109https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC1cXhvV2rurfM&md5=f18bafe4760311f97789519a09e1fcf6Programmed assembly of synthetic protocells into thermoresponsive prototissuesGobbo, Pierangelo; Patil, Avinash J.; Li, Mei; Harniman, Robert; Briscoe, Wuge H.; Mann, StephenNature Materials (2018), 17 (12), 1145-1153CODEN: NMAACR; ISSN:1476-1122. (Nature Research)Although several new types of synthetic cell-like entities are now available, their structural integration into spatially interlinked prototissues that communicate and display coordinated functions remains a considerable challenge. Here the authors describe the programmed assembly of synthetic prototissue constructs based on the bio-orthogonal adhesion of a spatially confined binary community of protein-polymer protocells, termed proteinosomes. The thermoresponsive properties of the interlinked proteinosomes were used collectively to generate prototissue spheroids capable of reversible contractions that can be enzymically modulated and exploited for mechanochem. transduction. Overall, the authors' methodol. opens up a route to the fabrication of artificial tissue-like materials capable of collective behaviors, and addresses important emerging challenges in bottom-up synthetic biol. and bioinspired engineering.
- 110Szekeres, K.; Bollella, P.; Kim, Y.; Minko, S.; Melman, A.; Katz, E. Magneto-controlled enzyme activity with locally produced pH changes. J. Phys. Chem. Lett. 2021, 12 (10), 2523– 2527, DOI: 10.1021/acs.jpclett.1c00036110https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB3MXlslWgtro%253D&md5=a0a2d91316b29753fa48826093def8b6Magneto-Controlled Enzyme Activity with Locally Produced pH ChangesSzekeres, Krisztina; Bollella, Paolo; Kim, Yongwook; Minko, Sergiy; Melman, Artem; Katz, EvgenyJournal of Physical Chemistry Letters (2021), 12 (10), 2523-2527CODEN: JPCLCD; ISSN:1948-7185. (American Chemical Society)Biocatalytic activity of amyloglucosidase (AMG), immobilized on superparamagnetic nanoparticles, is dynamically and reversibly activated or inhibited by applying a magnetic field. The magnetic field triggers aggregation/deaggregation of magnetic particles that are also functionalized with urease or esterase enzymes. These enzymes produce a local pH change in the vicinity of the particles changing the AMG activity.
- 111Dhasaiyan, P.; Ghosh, T.; Lee, H. G.; Lee, Y.; Hwang, I.; Mukhopadhyay, R. D.; Park, K. M.; Shin, S.; Kang, I. S.; Kim, K. Cascade reaction networks within audible sound induced transient domains in a solution. Nat. Commun. 2022, 13 (1), 2372, DOI: 10.1038/s41467-022-30124-x111https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB38XhtFyisrbJ&md5=f9d49f81b13de09e13f615b0b5d3f163Cascade reaction networks within audible sound induced transient domains in a solutionDhasaiyan, Prabhu; Ghosh, Tanwistha; Lee, Hong-Guen; Lee, Yeonsang; Hwang, Ilha; Mukhopadhyay, Rahul Dev; Park, Kyeng Min; Shin, Seungwon; Kang, In Seok; Kim, KimoonNature Communications (2022), 13 (1), 2372CODEN: NCAOBW; ISSN:2041-1723. (Nature Portfolio)Spatiotemporal control of chem. cascade reactions within compartmentalized domains is one of the difficult challenges to achieve. To implement such control, scientists have been working on the development of various artificial compartmentalized systems such as liposomes, vesicles, polymersomes, etc. Although a considerable amt. of progress has been made in this direction, one still needs to develop alternative strategies for controlling cascade reaction networks within spatiotemporally controlled domains in a soln., which remains a non-trivial issue. Herein, we present the utilization of audible sound induced liq. vibrations for the generation of transient domains in an aq. medium, which can be used for the control of cascade chem. reactions in a spatiotemporal fashion. This approach gives us access to highly reproducible spatiotemporal chem. gradients and patterns, in situ growth and aggregation of gold nanoparticles at predetd. locations or domains formed in a soln. Our strategy also gives us access to nanoparticle patterned hydrogels and their applications for region specific cell growth.
- 112Küchler, A.; Yoshimoto, M.; Luginbühl, S.; Mavelli, F.; Walde, P. Enzymatic reactions in confined environments. Nat. Nanotechnol. 2016, 11 (5), 409– 420, DOI: 10.1038/nnano.2016.54112https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC28XnsVWruro%253D&md5=f476bcda9da7f4fccdb11cb2cd11ccd2Enzymatic reactions in confined environmentsKuchler, Andreas; Yoshimoto, Makoto; Luginbuhl, Sandra; Mavelli, Fabio; Walde, PeterNature Nanotechnology (2016), 11 (5), 409-420CODEN: NNAABX; ISSN:1748-3387. (Nature Publishing Group)A review. Within each biol. cell, surface- and vol.-confined enzymes control a highly complex network of chem. reactions. These reactions are efficient, timely, and spatially defined. Efforts to transfer such appealing features to in vitro systems have led to several successful examples of chem. reactions catalyzed by isolated and immobilized enzymes. In most cases, these enzymes are either bound or adsorbed to an insol. support, phys. trapped in a macromol. network, or encapsulated within compartments. Advanced applications of enzymic cascade reactions with immobilized enzymes include enzymic fuel cells and enzymic nanoreactors, both for in vitro and possible in vivo applications. Here, the authors discuss some of the general principles of enzymic reactions confined on surfaces, at interfaces, and inside small vols. The authors also highlight the similarities and differences between the in vivo and in vitro cases and attempt to critically evaluate some of the necessary future steps to improve the fundamental understanding of these systems.
- 113Hwang, E. T.; Lee, S. Multienzymatic Cascade Reactions Enzyme complex by immobilization. ACS Catal. 2019, 9 (5), 4402– 4425, DOI: 10.1021/acscatal.8b04921113https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC1MXmvVemsL4%253D&md5=57580b50c6cd302510726c71da408601Multienzymatic cascade reactions via enzyme complex by immobilizationHwang, Ee Taek; Lee, SeonbyulACS Catalysis (2019), 9 (5), 4402-4425CODEN: ACCACS; ISSN:2155-5435. (American Chemical Society)A review. Multienzymic cascade reactions are a most important technol. to succeed in industrial process development, such as synthesis of pharmaceutical, cosmetic, and nutritional compds. Different strategies to construct multienzyme structures have been widely reported. Enzymes complexes are designed by three types of routes: (i) fusion proteins, (ii) enzyme scaffolds, or (iii) immobilization. As a result, enzyme complexes can enhance cascade enzymic activity through substrate channeling. In particular, recent advances in materials science have led to syntheses of various materials applicable for enzyme immobilization. This review discusses different cases for assembling multienzyme complexes via random co-immobilization, compartmentalization, and positional co-immobilization. The advantages of using immobilized multienzymes include not only improved cascade enzymic activity via substrate channeling but also enhanced enzyme stability and ease of recovery for reuse. In this review, we also consider the latest studies of different model enzyme reactions immobilized on various support materials, as multienzyme systems allow for economical product synthesis through bioprocesses.
- 114Ji, Q.; Wang, B.; Tan, J.; Zhu, L.; Li, L. Immobilized multienzymatic systems for catalysis of cascade reactions. Process Biochemistry 2016, 51 (9), 1193– 1203, DOI: 10.1016/j.procbio.2016.06.004114https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC28XhtVOntLjE&md5=769953a90f9288de812d6159383ea053Immobilized multienzymatic systems for catalysis of cascade reactionsJi, Qingzhi; Wang, Bochu; Tan, Jun; Zhu, Liancai; Li, LiuyingProcess Biochemistry (Oxford, United Kingdom) (2016), 51 (9), 1193-1203CODEN: PBCHE5; ISSN:1359-5113. (Elsevier Ltd.)A review. Cascade reactions catalyzed by immobilized multienzymic systems are an emerging technol., making catalytic strategies more sophisticated and effective. Thus, immobilized multienzymic systems that exploit the chemo-, regio-, and stereoselectivity of biocatalysts have been developed. Recently, a variety of bioinspired immobilized multienzymic systems was fabricated. In this review, immobilized multienzymic systems were divided into three types: stepwise immobilized enzymes, mixed immobilized enzymes, or co-immobilized enzymes. General considerations such as coupling pH, coupling temp., proportion of each enzyme, and cascade bottleneck anal. for the enzymic cascade reaction were presented. For greater insight, we analyzed the effects of substrate channeling, mass transport limitation, synergistic mechanisms, and promotion of cofactor regeneration on immobilized multienzymic systems. Some recent novel examples of immobilized multienzymic systems were summarized. The aim of this review is to promote and broaden the application of immobilized multienzymic systems in biocatalysis and other related fields.
- 115Kazenwadel, F.; Franzreb, M.; Rapp, B. E. Synthetic enzyme supercomplexes: co-immobilization of enzyme cascades. Anal. Methods 2015, 7 (10), 4030– 4037, DOI: 10.1039/C5AY00453E115https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2MXmvFartbY%253D&md5=d1e8c020ccd7c3ef41f9b4a42a5916faSynthetic enzyme supercomplexes: co-immobilization of enzyme cascadesKazenwadel, F.; Franzreb, M.; Rapp, B. E.Analytical Methods (2015), 7 (10), 4030-4037CODEN: AMNEGX; ISSN:1759-9679. (Royal Society of Chemistry)A review. A sustainable alternative to traditional chem. synthesis is the use of enzymes as biocatalysts. Using enzymes, different advantages such as mild reaction conditions and high turnover rates are combined. However, the approach of using sol. enzymes suffers from the fact that enzymes have to be sepd. from the product post-synthesis and can be inactivated by this process. Therefore, enzymes are often immobilized to solid carriers to enable easy sepn. from the product as well as stabilization of the enzyme structure. In order to mimic the metabolic pathways of living cells and thus to create more complex bioproducts in a cell-free manner, a series of consecutive reactions can be realized by applying whole enzyme cascades. As enzymes from different host organisms can be combined, this offers enormous opportunities for creating advanced metabolic pathways that do not occur in nature. When immobilizing these enzyme cascades in a co-localized pattern a further advantage emerges: as the product of the previous enzyme is directly transferred to its co-immobilized subsequent catalyst, the overall performance of the cascade can be enhanced. Furthermore when enzymes are in close proximity to each other, the generation of byproducts is reduced and obstructive effects like product inhibition and unfavorable kinetics can be disabled. This review provides an overview of the current state of the art in the application of enzyme cascades in immobilized forms. Furthermore it focuses on different immobilization techniques for structured immobilizates and the use of enzyme cascade in specially designed (microfluidic) reactor devices.
- 116Xu, K.; Chen, X.; Zheng, R.; Zheng, Y. Immobilization of multi-enzymes on support materials for efficient biocatalysis. Front. Bioeng. Biotechnol. 2020, 8, 660, DOI: 10.3389/fbioe.2020.00660116https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A280%3ADC%252BB38jlsFGmsw%253D%253D&md5=9429722fa4f60a98dc174b0dc75690e9Immobilization of Multi-Enzymes on Support Materials for Efficient BiocatalysisXu Kongliang; Chen Xuexiao; Zheng Renchao; Zheng Yuguo; Xu Kongliang; Chen Xuexiao; Zheng Renchao; Zheng YuguoFrontiers in bioengineering and biotechnology (2020), 8 (), 660 ISSN:2296-4185.Multi-enzyme biocatalysis is an important technology to produce many valuable chemicals in the industry. Different strategies for the construction of multi-enzyme systems have been reported. In particular, immobilization of multi-enzymes on the support materials has been proved to be one of the most efficient approaches, which can increase the enzymatic activity via substrate channeling and improve the stability and reusability of enzymes. A general overview of the characteristics of support materials and their corresponding attachment techniques used for multi-enzyme immobilization will be provided here. This review will focus on the materials-based techniques for multi-enzyme immobilization, which aims to present the recent advances and future prospects in the area of multi-enzyme biocatalysis based on support immobilization.
- 117Bié, J.; Sepodes, B.; Fernandes, P. C. B.; Ribeiro, M. H. L. Enzyme immobilization and co-immobilization: main framework, advances and some applications. Processes 2022, 10 (3), 494, DOI: 10.3390/pr10030494117https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB38XhtVSltr%252FL&md5=3138ce7588d05413bd16d4ea4514ac36Enzyme Immobilization and Co-Immobilization: Main Framework, Advances and Some ApplicationsBie, Joaquim; Sepodes, Bruno; Fernandes, Pedro C. B.; Ribeiro, Maria H. L.Processes (2022), 10 (3), 494CODEN: PROCCO; ISSN:2227-9717. (MDPI AG)Enzymes are outstanding (bio)catalysts, not solely on account of their ability to increase reaction rates by up to several orders of magnitude but also for the high degree of substrate specificity, regiospecificity and stereospecificity. The use and development of enzymes as robust biocatalysts is one of the main challenges in biotechnol. However, despite the high specificities and turnover of enzymes, there are also drawbacks. At the industrial level, these drawbacks are typically overcome by resorting to immobilized enzymes to enhance stability. Immobilization of biocatalysts allows their reuse, increases stability, facilitates process control, eases product recovery, and enhances product yield and quality. This is esp. important for expensive enzymes, for those obtained in low fermn. yield and with relatively low activity. This review provides an integrated perspective on (multi)enzyme immobilization that abridges a crit. evaluation of immobilization methods and carriers, biocatalyst metrics, impact of key carrier features on biocatalyst performance, trends towards miniaturization and detailed illustrative examples that are representative of biocatalytic applications promoting sustainability.
- 118Benitez-Mateos, A. I.; Roura Padrosa, D.; Paradisi, F. Multistep enzyme cascades as a route towards green and sustainable pharmaceutical syntheses. Nat. Chem. 2022, 14 (5), 489– 499, DOI: 10.1038/s41557-022-00931-2118https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB38Xht1Wis7rE&md5=926da86ead0775bd6729686f9f1afc4bMultistep enzyme cascades as a route towards green and sustainable pharmaceutical synthesesBenitez-Mateos, Ana I.; Roura Padrosa, David; Paradisi, FrancescaNature Chemistry (2022), 14 (5), 489-499CODEN: NCAHBB; ISSN:1755-4330. (Nature Portfolio)A review. Enzyme cascades are a powerful technol. to develop environmentally friendly and cost-effective synthetic processes to manuf. drugs, as they couple different biotransformations in sequential reactions to synthesize the product. These biocatalytic tools can address two key parameters for the pharmaceutical industry: an improved selectivity of synthetic reactions and a redn. of potential hazards by using biocompatible catalysts, which can be produced from sustainable sources, which are biodegradable and, generally, non-toxic. Here we outline a broad variety of enzyme cascades used either in vivo (whole cells) or in vitro (purified enzymes) to specifically target pharmaceutically relevant mols., from simple building blocks to complex drugs. We also discuss the advantages and requirements of multistep enzyme cascades and their combination with chem. catalysts through a series of reported examples. Finally, we examine the efficiency of enzyme cascades and how they can be further improved by enzyme engineering, process intensification in flow reactors and/or enzyme immobilization to meet all the industrial requirements.
- 119Liang, J.; Liang, K. Multi-enzyme cascade reactions in metal-organic frameworks. Chem. Rec. 2020, 20 (10), 1100– 1116, DOI: 10.1002/tcr.202000067119https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB3cXhsVClt7fF&md5=c3527de6b56da9e62ac78172ce1c6057Multi-enzyme Cascade Reactions in Metal-Organic FrameworksLiang, Jieying; Liang, KangChemical Record (2020), 20 (10), 1100-1116CODEN: CRHEAK; ISSN:1528-0691. (Wiley-VCH Verlag GmbH & Co. KGaA)Multi-enzyme cascade reactions are indispensable in biotechnol. and many industrial (bio)chem. processes. However, most natural enzymes have poor stability and reusability, and tend to inactivate in toxic media or high temp., which significantly limit their broader applications. Metal-org. frameworks (MOFs) are promising candidates for enzymes immobilization to produce nanocomposite structures that not only could shield the enzymes from harsh environments, but also facilitate selective diffusion of substrates and intermediates to the reactive site via their tailorable and ordered pore network. Multi-enzyme cascade reactions in MOFs have recently attracted considerable attention. This Personal Account discusses the different strategies for multi-enzyme-MOF interfaces and their cutting-edge applications from biosensing and catalytic nanomedicine to artificial/hybrid cells. At last, we provide a crit. evaluation and future prospects to outline future research directions.
- 120Tsitkov, S.; Hess, H. Design principles for a compartmentalized enzyme cascade reaction. ACS Catal. 2019, 9, 2432– 2439, DOI: 10.1021/acscatal.8b04419120https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC1MXitlChtLs%253D&md5=9e6ee51aa63f5a4311db731647ce276aDesign principles for a compartmentalized enzyme cascade reactionTsitkov, Stanislav; Hess, HenryACS Catalysis (2019), 9 (3), 2432-2439CODEN: ACCACS; ISSN:2155-5435. (American Chemical Society)Compartmentalization of enzyme cascade reactions can both create a safe space for volatile reaction intermediates, where they are protected from cellular degrdn. mechanisms, and a quarantine for toxic intermediates, where they are prevented from impeding cellular function. The quant. understanding of biol. compartments and the design of bioengineered compartments and synthetic cells would be facilitated by a general and concise model. Most existing models for studying compartmentalized cascades focus on a specific biol. system and are highly detailed. In this study, we develop a simple model for a compartmentalized two-enzyme cascade reaction and analyze it in the well-mixed, steady-state regime. An immediate and intuitive result is that the fundamental parameter governing compartment cascade throughput is the resistance to diffusion of substrate and intermediate mols. across the compartment boundary. We then use this model to develop a design process for a compartmentalized cascade, where intermediate loss is minimized while maintaining a desired product outflux. Finally, the model also reveals that there is a crit. threshold at which compartmentalization provides benefits over the free-soln. reaction. Our model not only provides many insights into the design of compartmentalized cascade reactions, but also captures the essential physics of the problem, as it can replicate the results of more complex models.
- 121Oh, W.; Jeong, D.; Park, J.-W. An Artificial compartmentalized biocatalytic cascade system constructed with enzyme-caged reticulate nanoporous membranes. Adv. Mater. Interfaces 2023, 10, 2300185, DOI: 10.1002/admi.202300185121https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB3sXhtVClt7jI&md5=61941208ae34d0a151e41830a1332838An Artificial Compartmentalized Biocatalytic Cascade System Constructed With Enzyme-Caged Reticulate Nanoporous MembranesOh, Wangsuk; Jeong, Dawoon; Park, Ji-WoongAdvanced Materials Interfaces (2023), 10 (17), 2300185CODEN: AMIDD2; ISSN:2196-7350. (Wiley-VCH Verlag GmbH & Co. KGaA)Compartmentalization is a ubiquitous feature of life, with a membrane interface that regulates mol. transport and exhibits bioactivities. An artificial enzyme-integrated membrane cascade system can mimic such interactive properties across different length scales for in vitro application. Here, it is shown that a reticulated nanoporous framework membrane with nano-caged enzymes presents the first modular macroscale platform for the compartmentalized biocatalytic system interfaced with an external medium. Catalase (CAT)-integrated polyurea membrane scaffold is prepd. by a simple pressurization procedure for a model cyclic cascade of glucose oxidase/catalase (GOx/CAT). The bicontinuous nanoporous membrane readily constructs a compartmentalized system interfacing the glucose/GOx soln. and the external environment and mediates glucose oxidn. by a cascade reaction under anaerobic or aerobic conditions. Furthermore, the membrane compartment exhibits multiple bioactive capabilities and barrier properties, including hydrogen peroxide detoxification, in situ oxygen generation, and protease exclusion. This work suggests a new strategy toward a robust modular biocatalytic membranous interface to mediate biochem. reaction cascades occurring in a compartment interfaced to an external environment, promising for potential applications in biohybrid devices embedded with tissues and cells.
- 122Diamanti, E.; Andrés-Sanz, D.; Orrego, A. H.; Carregal-Romero, S.; López-Gallego, F. Surpassing substrate–enzyme competition by compartmentalization. ACS Catal. 2023, 13 (17), 11441– 11454, DOI: 10.1021/acscatal.3c01965122https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB3sXhs1ygtb%252FO&md5=ac45f276ec4ceb2c664ef5c9988e8327Surpassing Substrate-Enzyme Competition by CompartmentalizationDiamanti, Eleftheria; Andres-Sanz, Daniel; Orrego, Alejandro H.; Carregal-Romero, Susana; Lopez-Gallego, FernandoACS Catalysis (2023), 13 (17), 11441-11454CODEN: ACCACS; ISSN:2155-5435. (American Chemical Society)Enzyme compartmentalization is one of the main strategies exploited by nature to create phys. sepd. chem. environments that allow simultaneous enzyme reactions within the cell metabolic networks. However, designing nanostructured architectures that mimic cellular compartments remains a challenge when two competing enzymes must work simultaneously over the same substrate. Herein, we develop a method to fabricate soft hybrids that phys. sep. two oxidoreductases that compete for NADH with greatly different kinetics. The less competitive enzyme is encapsulated into polymeric capsules capable of recruiting NADH, which are then assembled on porous agarose microbeads where the most competitive enzyme is immobilized. As a result, this functional hybrid enables the simultaneous action of two competing enzymes in the same reaction media, which would otherwise be impossible in a non-compartmentalized system. We demonstrate that substrate recruitment is a powerful approach to building up enzymic reaction networks with complex dynamics. Moreover, single-particle anal. under operando conditions reveals the impact of enzyme spatial organization on the overall performance of these soft hybrids, underlining the importance of understanding the functional variability within compartmentalized systems. Finally, integrating this compartmentalized system into a model cell-free biosynthetic cascade, we transform vinyl acetate into (S)-β-hydroxybutyrate with a 2 times higher titer than the non-compartmentalized free system. The proposed strategy can be generalized to produce compartmentalized cell-free biosynthetic pathways and multienzyme cascades where enzyme competition is an issue.
- 123Aumiller, W. M., Jr.; Uchida, M.; Douglas, T. Protein cage assembly across multiple length scales. Chem. Soc. Rev. 2018, 47, 3433– 3469, DOI: 10.1039/C7CS00818J123https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC1cXjs1OjsL0%253D&md5=da2a83077dd878cab68e8334e78c974cProtein cage assembly across multiple length scalesAumiller, William M., Jr.; Uchida, Masaki; Douglas, TrevorChemical Society Reviews (2018), 47 (10), 3433-3469CODEN: CSRVBR; ISSN:0306-0012. (Royal Society of Chemistry)Within the materials science community, proteins with cage-like architectures are being developed as versatile nanoscale platforms for use in protein nanotechnol. Much effort has been focused on the functionalization of protein cages with biol. and non-biol. moieties to bring about new properties of not only individual protein cages, but collective bulk-scale assemblies of protein cages. In this review, we report on the current understanding of protein cage assembly, both of the cages themselves from individual subunits, and the assembly of the individual protein cages into higher order structures. We start by discussing the key properties of natural protein cages (for example: size, shape and structure) followed by a review of some of the mechanisms of protein cage assembly and the factors that influence it. We then explore the current approaches for functionalizing protein cages, on the interior or exterior surfaces of the capsids. Lastly, we explore the emerging area of higher order assemblies created from individual protein cages and their potential for new and exciting collective properties.
- 124Jaekel, A.; Stegemann, P.; Saccà, B. Manipulating enzymes properties with DNA nanostructures. Molecules 2019, 24 (20), 3694, DOI: 10.3390/molecules24203694124https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC1MXit1yisb3P&md5=d3a16abadf59421439624c5f4f6360c4Manipulating enzymes properties with DNA nanostructuresJaekel, Andreas; Stegemann, Pierre; Sacca, BarbaraMolecules (2019), 24 (20), 3694CODEN: MOLEFW; ISSN:1420-3049. (MDPI AG)A review. Nucleic acids and proteins are two major classes of biopolymers in living systems. Whereas nucleic acids are characterized by robust mol. recognition properties, essential for the reliable storage and transmission of the genetic information, the variability of structures displayed by proteins and their adaptability to the environment make them ideal functional materials. One of the major goals of DNA nanotechnol.-and indeed its initial motivation-is to bridge these two worlds in a rational fashion. Combining the predictable base-pairing rule of DNA with chem. conjugation strategies and modern protein engineering methods has enabled the realization of complex DNA-protein architectures with programmable structural features and intriguing functionalities. In this review, we will focus on a special class of biohybrid structures, characterized by one or many enzyme mols. linked to a DNA scaffold with nanometer-scale precision. After an initial survey of the most important methods for coupling DNA oligomers to proteins, we will report the strategies adopted until now for organizing these conjugates in a predictable spatial arrangement. The major focus of this review will be on the consequences of such manipulations on the binding and kinetic properties of single enzymes and enzyme complexes: an interesting aspect of artificial DNA-enzyme hybrids, often reported in the literature, however, not yet entirely understood and whose full comprehension may open the way to new opportunities in protein science.
- 125Jiao, Y.; Shang, Y.; Li, N.; Ding, B. DNA-based enzymatic systems and their applications. iScience 2022, 25, 104018, DOI: 10.1016/j.isci.2022.104018125https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB38XhsVOmsbbE&md5=4b88367fa3b27d7f14575f2345c4c014DNA-based enzymatic systems and their applicationsJiao, Yunfei; Shang, Yingxu; Li, Na; Ding, BaoquaniScience (2022), 25 (4), 104018CODEN: ISCICE; ISSN:2589-0042. (Elsevier B.V.)A review. DNA strands with unique secondary structures can catalyze various chem. reactions and mimic natural enzymes with the assistance of cofactors, which have attracted much research attention. At the same time, the emerging DNA nanotechnol. provides an efficient platform to organize functional components of the enzymic systems and regulate their catalytic performances. In this review, we summarize the recent progress of DNA-based enzymic systems. First, DNAzymes (Dzs) are introduced, and their versatile utilities are summarized. Then, G-quadruplex/hemin (G4/hemin) Dzs with unique oxidase/peroxidase-mimicking activities and representative examples where these Dzs served as biosensors are explicitly elaborated. Next, the DNA-based enzymic cascade systems fabricated by the structural DNA nanotechnol. are depicted. In addn., the applications of catalytic DNA nanostructures in biosensing and biomedicine are included. At last, the challenges and the perspectives of the DNA-based enzymic systems for practical applications are also discussed.
- 126Engelen, W.; Janssen, B. M. G.; Merkx, M. DNA-based control of protein activity. Chem. Commun. 2016, 52, 3598– 3610, DOI: 10.1039/C5CC09853J126https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC28XhtF2isrg%253D&md5=2205b7f35ee3a448b4a7b09acfa3abd7DNA-based control of protein activityEngelen, W.; Janssen, B. M. G.; Merkx, M.Chemical Communications (Cambridge, United Kingdom) (2016), 52 (18), 3598-3610CODEN: CHCOFS; ISSN:1359-7345. (Royal Society of Chemistry)A review. DNA has emerged as a highly versatile construction material for nanometer-sized structures and sophisticated mol. machines and circuits. The successful application of nucleic acid based systems greatly relies on their ability to autonomously sense and act on their environment. In this feature article, the development of DNA-based strategies to dynamically control protein activity via oligonucleotide triggers is discussed. Depending on the desired application, protein activity can be controlled by directly conjugating them to an oligonucleotide handle, or expressing them as a fusion protein with DNA binding motifs. To control proteins without modifying them chem. or genetically, multivalent ligands and aptamers that reversibly inhibit their function provide valuable tools to regulate proteins in a noncovalent manner. The goal of this feature article is to give an overview of strategies developed to control protein activity via oligonucleotide-based triggers, as well as hurdles yet to be taken to obtain fully autonomous systems that interrogate, process and act on their environments by DNA-based protein control.
- 127Fu, J.; Wang, Z.; Liang, X. H.; Oh, S. W.; St. Iago-McRae, E.; Zhang, T. DNA-scaffolded proximity assembly and confinement of multienzyme reactions. Top. Curr. Chem. 2020, 378, 38, DOI: 10.1007/s41061-020-0299-3127https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB3cXmsVert7k%253D&md5=2ee0e83645bdd98cffc3f83314bb4e00DNA-Scaffolded Proximity Assembly and Confinement of Multienzyme ReactionsFu, Jinglin; Wang, Zhicheng; Liang, Xiao Hua; Oh, Sung Won; St. Iago-McRae, Ezry; Zhang, TingTopics in Current Chemistry (2020), 378 (3), 38CODEN: TPCCAQ; ISSN:2364-8961. (Springer International Publishing AG)A review. Cellular functions rely on a series of organized and regulated multienzyme cascade reactions. The catalytic efficiencies of these cascades depend on the precise spatial organization of the constituent enzymes, which is optimized to facilitate substrate transport and regulate activities. Mimicry of this organization in a non-living, artificial system would be very useful in a broad range of applications-with impacts on both the scientific community and society at large. Self-assembled DNA nanostructures are promising applications to organize biomol. components into prescribed, multidimensional patterns. In this review, we focus on recent progress in the field of DNA-scaffolded assembly and confinement of multienzyme reactions. DNA self-assembly is exploited to build spatially organized multienzyme cascades with control over their relative distance, substrate diffusion paths, compartmentalization and activity actuation. The combination of addressable DNA assembly and multienzyme cascades can deliver breakthroughs toward the engineering of novel synthetic and biomimetic reactors.
- 128Kröll, S.; Niemeyer, C. M. Nucleic acid-based enzyme cascades─current trends and future perspectives. Angew. Chem., Int. Ed. 2024, 63, e202314452, DOI: 10.1002/anie.202314452There is no corresponding record for this reference.
- 129Liang, J.; Liang, K. Multi-enzyme cascade reactions in metal-organic frameworks. Chem. Rec. 2020, 20, 1100, DOI: 10.1002/tcr.202000067129https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB3cXhsVClt7fF&md5=c3527de6b56da9e62ac78172ce1c6057Multi-enzyme Cascade Reactions in Metal-Organic FrameworksLiang, Jieying; Liang, KangChemical Record (2020), 20 (10), 1100-1116CODEN: CRHEAK; ISSN:1528-0691. (Wiley-VCH Verlag GmbH & Co. KGaA)Multi-enzyme cascade reactions are indispensable in biotechnol. and many industrial (bio)chem. processes. However, most natural enzymes have poor stability and reusability, and tend to inactivate in toxic media or high temp., which significantly limit their broader applications. Metal-org. frameworks (MOFs) are promising candidates for enzymes immobilization to produce nanocomposite structures that not only could shield the enzymes from harsh environments, but also facilitate selective diffusion of substrates and intermediates to the reactive site via their tailorable and ordered pore network. Multi-enzyme cascade reactions in MOFs have recently attracted considerable attention. This Personal Account discusses the different strategies for multi-enzyme-MOF interfaces and their cutting-edge applications from biosensing and catalytic nanomedicine to artificial/hybrid cells. At last, we provide a crit. evaluation and future prospects to outline future research directions.
- 130Dhakshinamoorthy, A.; Asiri, A. M.; Garcia, H. Integration of metal organic frameworks with enzymes as multifunctional solids for cascade catalysis. Dalton Trans. 2020, 49, 11059– 11072, DOI: 10.1039/D0DT02045A130https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB3cXht1ClsrbN&md5=63dc33498161488f91cb98a222f2781fIntegration of metal organic frameworks with enzymes as multifunctional solids for cascade catalysisDhakshinamoorthy, Amarajothi; Asiri, Abdullah M.; Garcia, HermenegildoDalton Transactions (2020), 49 (32), 11059-11072CODEN: DTARAF; ISSN:1477-9226. (Royal Society of Chemistry)A review. Enzymes exhibit a large degree of compatibility with metal-org. frameworks (MOFs) which allows the development of multicomponent catalysts consisting of enzymes adsorbed or occluded by MOFs. The combination of enzymes and MOFs in a multicomponent catalyst can be used to promote cascade reactions in which two or more individual reactions are performed in a single step. Cascade reactions take place due to the cooperation of active sites present on the MOF with the enzyme. A survey of the available data establishes that often an enzyme undergoes stabilization by assocn. with a MOF and the system exhibits notable recyclability. In addn., the existence of synergism is obsd. as a consequence of the close proximity of all the required active sites in the multicomponent catalyst. After an introductory section describing the specific features and properties of enzyme-MOF assemblies, the main part of the present review focuses on the description of the cascade reactions that have been reported with com. enzymes assocd. with MOFs, paying special attention to the advantages derived from the multicomponent catalyst. Related to the catalytic activity to metabolize glucose, generating reactive oxygen species (ROS) and decreasing the soln. pH, an independent section describes the recent use of enzyme-MOF catalysts in cancer therapy. The last paragraphs summarize the current state of the art and provide our view on future developments in this field.
- 131Wang, X.; Lan, P. C.; Ma, S. Metal–organic frameworks for enzyme immobilization: beyond host matrix materials. ACS Cent. Sci. 2020, 6 (9), 1497– 1506, DOI: 10.1021/acscentsci.0c00687131https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB3cXhs1OrsLrI&md5=8b6eba4e66b83d884976908803e31e12Metal-Organic Frameworks for Enzyme Immobilization: Beyond Host Matrix MaterialsWang, Xiaoliang; Lan, Pui Ching; Ma, ShengqianACS Central Science (2020), 6 (9), 1497-1506CODEN: ACSCII; ISSN:2374-7951. (American Chemical Society)A review. Enzyme immobilization in metal-org. frameworks (MOFs) as a promising strategy is attracting the interest of scientists from different disciplines with the expansion of MOFs' development. Different from other traditional host materials, their unique strengths of high surface areas, large yet adjustable pore sizes, functionalizable pore walls, and diverse architectures make MOFs an ideal platform to study hosted enzymes, which is crit. to the industrial and com. process. In addn. to the protective function of MOFs, the extensive roles of MOFs in the enzyme immobilization are being well-explored by making full use of their remarkable properties like well-defined structure, high porosity, and tunable functionality. Such development shifts the focus from the exploration of immobilization strategies toward functionalization. Meanwhile, this would undoubtedly contribute to a better understanding of enzymes in regards to the structural transformation after being hosted in a confinement environment, particularly to the orientation and conformation change as well as the interplay between enzyme and matrix MOFs. In this Outlook, the authors target a comprehensive review of the role diversities of the host matrix MOF based on the current enzyme immobilization research, along with proposing an outlook toward the future development of this field, including the representatives of potential techniques and methodologies being capable of studying the hosted enzymes. Current research of role/function diversities of host matrix MOFs is reviewed, noting the importance of understanding enzyme structural alternation and enzyme-MOF interaction after immobilization.
- 132Lian, X.; Fang, Y.; Joseph, E.; Wang, Q.; Li, J.; Banerjee, S.; Lollar, C.; Wang, X.; Zhou, H.-C. Enzyme–MOF (metal–organic framework) composites. Chem. Soc. Rev. 2017, 46, 3386– 3401, DOI: 10.1039/C7CS00058H132https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2sXmslOku7s%253D&md5=329ea8161e9200a133c76ba386fc70a7Enzyme-MOF (metal-organic framework) compositesLian, Xizhen; Fang, Yu; Joseph, Elizabeth; Wang, Qi; Li, Jialuo; Banerjee, Sayan; Lollar, Christina; Wang, Xuan; Zhou, Hong-CaiChemical Society Reviews (2017), 46 (11), 3386-3401CODEN: CSRVBR; ISSN:0306-0012. (Royal Society of Chemistry)A review. The ex vivo application of enzymes in various processes, esp. via enzyme immobilization techniques, has been extensively studied in recent years in order to enhance the recyclability of enzymes, to minimize enzyme contamination in the product, and to explore novel horizons for enzymes in biomedical applications. Possessing remarkable amenability in structural design of the frameworks as well as almost unparalelled surface tunability, Metal-Org. Frameworks (MOFs) have been gaining popularity as candidates for enzyme immobilization platforms. Many MOF-enzyme composites have achieved unprecedented results, far outperforming free enzymes in many aspects. This review summarizes recent developments of MOF-enzyme composites with special emphasis on preparative techniques and the synergistic effects of enzymes and MOFs. The applications of MOF-enzyme composites, primarily in transferation, catalysis and sensing, are presented as well. The enhancement of enzymic activity of the composites over free enzymes in biol. incompatible conditions is emphasized in many cases.
- 133Liang, W.; Wied, P.; Carraro, F.; Sumby, C. J.; Nidetzky, B.; Tsung, C.-K.; Falcaro, P.; Doonan, C. J. Metal–organic framework-based enzyme biocomposites. Chem. Rev. 2021, 121 (3), 1077– 1129, DOI: 10.1021/acs.chemrev.0c01029133https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB3MXpvFylsQ%253D%253D&md5=22a04b209c88b1c69ee3e73f8aeba6c3Metal-Organic Framework-Based Enzyme BiocompositesLiang, Weibin; Wied, Peter; Carraro, Francesco; Sumby, Christopher J.; Nidetzky, Bernd; Tsung, Chia-Kuang; Falcaro, Paolo; Doonan, Christian J.Chemical Reviews (Washington, DC, United States) (2021), 121 (3), 1077-1129CODEN: CHREAY; ISSN:0009-2665. (American Chemical Society)A review. Because of their efficiency, selectivity, and environmental sustainability, there are significant opportunities for enzymes in chem. synthesis and biotechnol. However, as the three-dimensional active structure of enzymes is predominantly maintained by weaker noncovalent interactions, thermal, pH, and chem. stressors can modify or eliminate activity. Metal-org. frameworks (MOFs), which are extended porous network materials assembled by a bottom-up building block approach from metal-based nodes and org. linkers, can be used to afford protection to enzymes. The self-assembled structures of MOFs can be used to encase an enzyme in a process called encapsulation when the MOF is synthesized in the presence of the biomol. Alternatively, enzymes can be infiltrated into mesoporous MOF structures or surface bound via covalent or noncovalent processes. Integration of MOF materials and enzymes in this way affords protection and allows the enzyme to maintain activity in challenge conditions (e.g., denaturing agents, elevated temp., non-native pH, and org. solvents). In addn. to forming simple enzyme/MOF biocomposites, other materials can be introduced to the composites to improve recovery or facilitate advanced applications in sensing and fuel cell technol. This review canvasses enzyme protection via encapsulation, pore infiltration, and surface adsorption and summarizes strategies to form multicomponent composites. Also, given that enzyme/MOF biocomposites straddle materials chem. and enzymol., this review provides an assessment of the characterization methodologies used for MOF-immobilized enzymes and identifies some key parameters to facilitate development of the field.
- 134Hu, J.; Zhang, G.; Liu, S. Enzyme-responsive polymeric assemblies, nanoparticles and hydrogels. Chem. Soc. Rev. 2012, 41 (18), 5933– 5949, DOI: 10.1039/c2cs35103j134https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC38Xht1aqsbrF&md5=d18156de75b1053ac9e63aadfa9661f5Enzyme-responsive polymeric assemblies, nanoparticles and hydrogelsHu, Jinming; Zhang, Guoqing; Liu, ShiyongChemical Society Reviews (2012), 41 (18), 5933-5949CODEN: CSRVBR; ISSN:0306-0012. (Royal Society of Chemistry)A review. Being responsive and adaptive to external stimuli is an intrinsic feature characteristic of all living organisms and soft matter. Specifically, responsive polymers can exhibit reversible or irreversible changes in chem. structures and/or phys. properties in response to a specific signal input such as pH, temp., ionic strength, light irradn., mech. force, elec. and magnetic fields, and analyte of interest (e.g., ions, bioactive mols., etc.) or an integration of them. The past decade has evidenced tremendous growth in the fundamental research of responsive polymers, and accordingly, diverse applications in fields ranging from drug or gene nanocarriers, imaging, diagnostics, smart actuators, adaptive coatings, to self-healing materials were explored and suggested. Among a variety of external stimuli that were utilized for the design of novel responsive polymers, enzymes have recently emerged to be a promising triggering motif. Enzyme-catalyzed reactions are highly selective and efficient toward specific substrates under mild conditions. They are involved in all biol. and metabolic processes, serving as the prime protagonists in the chem. of living organisms at a mol. level. The integration of enzyme-catalyzed reactions with responsive polymers can further broaden the design flexibility and scope of applications by endowing the latter with enhanced triggering specificity and selectivity. In this tutorial review, the authors describe recent developments concerning enzyme-responsive polymeric assemblies, nanoparticles, and hydrogels by highlighting this research area with selected literature reports. Three different types of systems, namely, enzyme-triggered self-assembly and aggregation of synthetic polymers, enzyme-driven disintegration and structural reorganization of polymeric assemblies and nanoparticles, and enzyme-triggered sol-to-gel and gel-to-sol transitions, are described. Their promising applications in drug controlled release, biocatalysis, imaging, sensing, and diagnostics are also discussed.
- 135Li, P.; Zhong, Y.; Wang, X.; Hao, J. Enzyme-regulated healable polymeric hydrogels. ACS Cent. Sci. 2020, 6 (9), 1507– 1522, DOI: 10.1021/acscentsci.0c00768135https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB3cXhsFWnt73F&md5=1fcff98ac8ba657eece9f626d7f4ebc7Enzyme-Regulated Healable Polymeric HydrogelsLi, Panpan; Zhong, Yuanbo; Wang, Xu; Hao, JingchengACS Central Science (2020), 6 (9), 1507-1522CODEN: ACSCII; ISSN:2374-7951. (American Chemical Society)A review. The enzyme-regulated healable polymeric hydrogels are a kind of emerging soft material capable of repairing the structural defects and recovering the hydrogel properties, wherein their fabrication, self-healing, or degrdn. is mediated by enzymic reactions. Despite achievements that have been made in controllable crosslinking and de-crosslinking of hydrogels by utilizing enzyme-catalyzed reactions in the past few years, this substrate-specific strategy for regulating healable polymeric hydrogels remains in its infancy, because both the intelligence and practicality of current man-made enzyme-regulated healable materials are far below the levels of living organisms. A systematic summary of current achievements and a reasonable prospect at this point can play pos. roles for the future development in this field. This Outlook focuses on the emerging and rapidly developing research area of bioinspired enzyme-regulated self-healing polymeric hydrogel systems. The enzymic fabrication and degrdn. of healable polymeric hydrogels, as well as the enzymically regulated self-healing of polymeric hydrogels, are reviewed. The functions and applications of the enzyme-regulated healable polymeric hydrogels are discussed. A systematic summary of current achievements in the area of enzyme-regulated healable polymeric hydrogels is given by detailing their fabrication, self-healing, degrdn., and applications.
- 136Amir, R. J.; Zhong, S.; Pochan, D. J.; Hawker, C. J. Enzymatically triggered self-assembly of block copolymers. J. Am. Chem. Soc. 2009, 131 (39), 13949– 13951, DOI: 10.1021/ja9060917136https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD1MXhtFWqtLnO&md5=4bd33ad61070f9c98a4359ea77b02664Enzymatically Triggered Self-Assembly of Block CopolymersAmir, Roey J.; Zhong, Sheng; Pochan, Darrin J.; Hawker, Craig J.Journal of the American Chemical Society (2009), 131 (39), 13949-13951CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)The polymn. of vinyl monomers with cleavable enzymic substrates has been shown to lead to water-sol. double-hydrophilic block copolymers which, upon enzymic activation of the diblock copolymers, become amphiphilic and undergo self-assembly into colloidal nanostructures. The ability to change the chem. and phys. characteristics of polymeric materials by an enzymic reaction opens the way for novel and exciting applications such as enzymic-triggered activation of surfaces and formation of nanostructures in vivo in a highly controlled manner.
- 137Kobayashi, S.; Uyama, H.; Kimura, S. Enzymatic polymerization. Chem. Rev. 2001, 101 (12), 3793– 3818, DOI: 10.1021/cr990121l137https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD3MXotVOht7Y%253D&md5=6bdfb08b5a7bea461570df0bae3547f4Enzymatic polymerizationKobayashi, Shiro; Uyama, Hiroshi; Kimura, ShunsakuChemical Reviews (Washington, D. C.) (2001), 101 (12), 3793-3818CODEN: CHREAY; ISSN:0009-2665. (American Chemical Society)A review with refs. on recent advances in enzymic polymn. Enzymes discussed included peroxidases, laccases, glycosyltransferases, acyltransferases, glycosidases, lipases, and proteases.
- 138Klemperer, R. G.; Shannon, M. R.; Ross Anderson, J. L.; Perriman, A. W. Bienzymatic generation of interpenetrating polymer networked engineered living materials with shape changing Pproperties. Adv. Mater. Technol. 2023, 8, 2300626, DOI: 10.1002/admt.202300626138https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB3sXhsVKltLfL&md5=3fb13931d0d744a3167696ab4af0b185Bienzymatic Generation of Interpenetrating Polymer Networked Engineered Living Materials with Shape Changing PropertiesKlemperer, R. George; Shannon, Mark R.; Ross Anderson, J. L.; Perriman, Adam W.Advanced Materials Technologies (Weinheim, Germany) (2023), 8 (18), 2300626CODEN: AMTDCM; ISSN:2365-709X. (Wiley-VCH Verlag GmbH & Co. KGaA)The synthesis of a porous shape-changing interpenetrating network (IPN) bioink for the fabrication of large-scale (cm) reversibly thermosensitive structures is described. The poly(N-isopropylacrylamide) (PNIPAm) IPN is generated in situ within an ionically crosslinked alginate hydrogel at room temp. and under aerobic conditions using a horseradish peroxidase (HRP)/glucose oxidase (GOx) bienzymic initiation system. Mech. testing assessment of the IPN hydrogels confirm mech. reinforcement via covalent single network interdigitation. Furthermore, the thermosensitive bioink can be used to print biohybrid reactors contg. genetically engineered phosphotriesterase-expressing E. coli capable of hydrolyzing toxic organophosphorus compds. Herein, increasing the bioink pore size using the contractile-thermosensitive response of the IPN improves the temp.-dependent theor. mass-transfer-limited enzyme catalyzed reaction rate, providing a plausible route to externally regulated enzymic catalysis within bioprinted structures.
- 139Mao, Y.; Su, T.; Wu, Q.; Liao, C.; Wang, Q. Dual enzymatic formation of hybrid hydrogels with supramolecular-polymeric networks. Chem. Commun. 2014, 50 (92), 14429– 14432, DOI: 10.1039/C4CC06472K139https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2cXhsFyrurzO&md5=65fed273b072c904d9ac9f48cb433722Dual enzymatic formation of hybrid hydrogels with supramolecular-polymeric networksMao, Yanjie; Su, Teng; Wu, Qing; Liao, Chuanan; Wang, QigangChemical Communications (Cambridge, United Kingdom) (2014), 50 (92), 14429-14432CODEN: CHCOFS; ISSN:1359-7345. (Royal Society of Chemistry)This communication describes a mild construction of hybrid hydrogels with supramol.-polymeric networks via a dual enzymic reaction.
- 140Wei, Q.; Xu, M.; Liao, C.; Wu, Q.; Liu, M.; Zhang, Y.; Wu, C.; Cheng, L.; Wang, Q. Printable hybrid hydrogel by dual enzymatic polymerization with superactivity. Chem. Sci. 2016, 7 (4), 2748– 2752, DOI: 10.1039/C5SC02234G140https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC28Xhtl2lsw%253D%253D&md5=d9cbcf00eddb407ca0a7943980ef46aaPrintable hybrid hydrogel by dual enzymatic polymerization with superactivityWei, Qingcong; Xu, Mengchi; Liao, Chuanan; Wu, Qing; Liu, Mingyu; Zhang, Ye; Wu, Chengtie; Cheng, Liming; Wang, QigangChemical Science (2016), 7 (4), 2748-2752CODEN: CSHCCN; ISSN:2041-6520. (Royal Society of Chemistry)A new approach has been developed to fabricate tough hybrid hydrogels by employing dual enzyme-mediated redox initiation to achieve post-self-assembly crosslinking polymn. The resulting hydrogel combines the merits of supramol. hydrogels with polymeric hydrogels to achieve higher mech. strength and porous networks. Designed 3D constructs were fabricated via in situ 3D printing. The in situ immobilized GOx/HRP in Gel II exhibited superactivity compared to free enzymes, which might be attributed to the synergistic effect of co-localized GOx and HRP minimizing the distances for mass transport between the gel and the bulk soln. This mech. strong hybrid hydrogel maintained high reusability and thermal stability as well. In addn., in situ 3D cell culture was demonstrated, thus indicating that this biodegradable hybrid hydrogel is biocompatible with cells. The subsequent 3D cell printing further indicates that the hybrid hydrogel is a promising scaffold for bio-related applications such as biocatalysis and tissue engineering.
- 141Yang, Z.; Liang, G.; Wang, L.; Xu, B. Using a kinase/phosphatase switch to regulate a supramolecular hydrogel and forming the supramolecular hydrogel in vivo. J. Am. Chem. Soc. 2006, 128 (9), 3038– 3043, DOI: 10.1021/ja057412y141https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD28Xht1Cjs70%253D&md5=326363d3b9d418c808c8749b2e6592c0Using a Kinase/Phosphatase Switch to Regulate a Supramolecular Hydrogel and Forming the Supramolecular Hydrogel in VivoYang, Zhimou; Liang, Gaolin; Wang, Ling; Xu, BingJournal of the American Chemical Society (2006), 128 (9), 3038-3043CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)We have designed and synthesized a new hydrogelator Nap-FFGEY (1), which forms a supramol. hydrogel. A kinase/phosphatase switch is used to control the phosphorylation and dephosphorylation of the hydrogelator and to regulate the formation of supramol. hydrogels. Adding a kinase to the hydrogel induces a gel-sol phase transition in the presence of adenosine triphosphates (ATP) because the tyrosine residue is converted into tyrosine phosphate by the kinase to give a more hydrophilic mol. of Nap-FFGEY-P(O)(OH)2 (2); treating the resulting soln. with a phosphatase transforms 2 back to 1 and restores the hydrogel. Electron micrographs of the hydrogels indicate that 1 self-assembles into nanofibers. S.c. injection of 2 in mice shows that 80.5±1.2% of 2 turns into 1 and results in the formation of the supramol. hydrogel of 1 in vivo. This simple biomimetic approach for regulating the states of supramol. hydrogels promises a new way to design and construct biomaterials.
- 142Knipe, J. M.; Chen, F.; Peppas, N. A. Enzymatic biodegradation of hydrogels for protein delivery targeted to the small intestine. Biomacromolecules 2015, 16 (3), 962– 972, DOI: 10.1021/bm501871a142https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2MXisF2gurk%253D&md5=377318e0f9e887be253719e9b3bf98bfEnzymatic Biodegradation of Hydrogels for Protein Delivery Targeted to the Small IntestineKnipe, Jennifer M.; Chen, Frances; Peppas, Nicholas A.Biomacromolecules (2015), 16 (3), 962-972CODEN: BOMAF6; ISSN:1525-7797. (American Chemical Society)Multiresponsive poly(methacrylic acid-co-N-vinylpyrrolidone) hydrogels were synthesized with biodegradable oligopeptide crosslinks. The oligopeptide crosslinks were incorporated using EDC-NHS zero-length links between the carboxylic acid groups of the polymer and free primary amines on the peptide. The reaction of the peptide was confirmed by primary amine assay and IR spectroscopy. The microgels exhibited pH-responsive swelling as well as enzyme-catalyzed degrdn. targeted by trypsin present in the small intestine, as demonstrated upon incubation with gastrointestinal fluids from rats. Relative turbidity was used to evaluate enzyme-catalyzed degrdn. as a function of time, and initial trypsin concn. controlled both the degrdn. mechanism as well as the extent of degrdn. Trypsin activity was effectively extinguished by incubation at 70 °C, and both the microgels and degrdn. products posed no cytotoxic effect toward two different cell lines. The microgels demonstrated pH-dependent loading of the protein insulin for oral delivery to the small intestine.
- 143Pappas, C. G.; Sasselli, I. R.; Ulijn, R. V. Biocatalytic pathway selection in transient tripeptide nanostructures. Angew. Chem., Int. Ed. 2015, 54 (28), 8119– 8123, DOI: 10.1002/anie.201500867143https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2MXptFert7s%253D&md5=d57863a160d87e7b9d913c2c0d9ebda0Biocatalytic Pathway Selection in Transient Tripeptide NanostructuresPappas, Charalampos G.; Sasselli, Ivan R.; Ulijn, Rein V.Angewandte Chemie, International Edition (2015), 54 (28), 8119-8123CODEN: ACIEF5; ISSN:1433-7851. (Wiley-VCH Verlag GmbH & Co. KGaA)Structural adaptation in living systems is achieved by competing catalytic pathways that drive assembly and disassembly of mol. components under the influence of chem. fuels. We report on a simple mimic of such a system that displays transient, sequence-dependent formation of supramol. nanostructures based on biocatalytic formation and hydrolysis of self-assembling tripeptides. The systems are catalyzed by α-chymotrypsin and driven by hydrolysis of dipeptide aspartyl-phenylalanine-Me ester (the sweetener aspartame, DF-OMe). We obsd. switch-like pathway selection, with the kinetics and consequent lifetime of transient nanostructures controlled by the peptide sequence. In direct competition, kinetic (rather than thermodn.) component selection is obsd.
- 144Cook, A. B.; Decuzzi, P. Harnessing endogenous stimuli for responsive materials in theranostics. ACS Nano 2021, 15 (2), 2068– 2098, DOI: 10.1021/acsnano.0c09115144https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB3MXjt1Kht70%253D&md5=3502531fce5be079450b300c9a292506Harnessing Endogenous Stimuli for Responsive Materials in TheranosticsCook, Alexander B.; Decuzzi, PaoloACS Nano (2021), 15 (2), 2068-2098CODEN: ANCAC3; ISSN:1936-0851. (American Chemical Society)A review. Materials that respond to endogenous stimuli are being leveraged to enhance spatiotemporal control in a range of biomedical applications from drug delivery to diagnostic tools. The design of materials that undergo morphol. or chem. changes in response to specific biol. cues or pathologies will be an important area of research for improving efficacies of existing therapies and imaging agents, while also being promising for developing personalized theranostic systems. Internal stimuli-responsive systems can be engineered across length scales from nanometers to macroscopic and can respond to endogenous signals such as enzymes, pH, glucose, ATP, hypoxia, redox signals, and nucleic acids by incorporating synthetic bio-inspired moieties or natural building blocks. This Review will summarize response mechanisms and fabrication strategies used in internal stimuli-responsive materials with a focus on drug delivery and imaging for a broad range of pathologies, including cancer, diabetes, vascular disorders, inflammation, and microbial infections. We will also discuss obsd. challenges, future research directions, and clin. translation aspects of these responsive materials.
- 145Ku, T. H.; Chien, M. P.; Thompson, M. P.; Sinkovits, R. S.; Olson, N. H.; Baker, T. S.; Gianneschi, N. C. Controlling and switching the morphology of micellar nanoparticles with enzymes. J. Am. Chem. Soc. 2011, 133 (22), 8392– 8395, DOI: 10.1021/ja2004736145https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3MXkt1ylu7c%253D&md5=d326644c6353068e202c39e927798e8fControlling and Switching the Morphology of Micellar Nanoparticles with EnzymesKu, Ti-Hsuan; Chien, Miao-Ping; Thompson, Matthew P.; Sinkovits, Robert S.; Olson, Norman H.; Baker, Timothy S.; Gianneschi, Nathan C.Journal of the American Chemical Society (2011), 133 (22), 8392-8395CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)Micelles were prepd. from polymer-peptide block copolymer amphiphiles contg. substrates for protein kinase A, protein phosphatase-1, and matrix metalloproteinases 2 and 9. The authors examine reversible switching of the morphol. of these micelles through a phosphorylation-dephosphorylation cycle and study peptide-sequence directed changes in morphol. in response to proteolysis. Furthermore, the exceptional uniformity of these polymer-peptide particles makes them amenable to cryo-TEM reconstruction techniques lending insight into their internal structure.
- 146Postma, S. G.; Vialshin, I. N.; Gerritsen, C. Y.; Bao, M.; Huck, W. T. Preprogramming complex hydrogel responses using enzymatic reaction networks. Angew. Chem., Int. Ed. 2017, 56 (7), 1794– 1798, DOI: 10.1002/anie.201610875146https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2sXlsFCjuw%253D%253D&md5=cf0189e888c27ea1641e5dade88eea86Preprogramming Complex Hydrogel Responses using Enzymatic Reaction NetworksPostma, Sjoerd G. J.; Vialshin, Ilia N.; Gerritsen, Casper Y.; Bao, Min; Huck, Wilhelm T. S.Angewandte Chemie, International Edition (2017), 56 (7), 1794-1798CODEN: ACIEF5; ISSN:1433-7851. (Wiley-VCH Verlag GmbH & Co. KGaA)The creation of adaptive matter is heavily inspired by biol. systems. However, it remains challenging to design complex material responses that are governed by reaction networks, which lie at the heart of cellular complexity. The main reason for this slow progress is the lack of a general strategy to integrate reaction networks with materials. Herein we use a systematic approach to preprogram the response of a hydrogel to a trigger, in this case the enzyme trypsin, which activates a reaction network embedded within the hydrogel. A full characterization of all the kinetic rate consts. in the system enabled the construction of a computational model, which predicted different hydrogel responses depending on the input concn. of the trigger. The results of the simulation are in good agreement with exptl. findings. Our methodol. can be used to design new, adaptive materials of which the properties are governed by reaction networks of arbitrary complexity.
- 147Ikeda, M.; Tanida, T.; Yoshii, T.; Kurotani, K.; Onogi, S.; Urayama, K.; Hamachi, I. Installing logic-gate responses to a variety of biological substances in supramolecular hydrogel-enzyme hybrids. Nat. Chem. 2014, 6 (6), 511– 518, DOI: 10.1038/nchem.1937147https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2cXntlyis7c%253D&md5=4b6d233a620c107f2a4ca3484442e945Installing logic-gate responses to a variety of biological substances in supramolecular hydrogel-enzyme hybridsIkeda, Masato; Tanida, Tatsuya; Yoshii, Tatsuyuki; Kurotani, Kazuya; Onogi, Shoji; Urayama, Kenji; Hamachi, ItaruNature Chemistry (2014), 6 (6), 511-518CODEN: NCAHBB; ISSN:1755-4330. (Nature Publishing Group)Soft materials that exhibit stimuli-responsive behavior under aq. conditions (such as supramol. hydrogels composed of self-assembled nanofibres) have many potential biol. applications. However, designing a macroscopic response to structurally complex biochem. stimuli in these materials still remains a challenge. Here we show that redox-responsive peptide-based hydrogels have the ability to encapsulate enzymes and still retain their activities. Moreover, cooperative coupling of enzymic reactions with the gel response enables us to construct unique stimuli-responsive soft materials capable of sensing a variety of disease-related biomarkers. The programmable gel-sol response (even to biol. samples) is visible to the naked eye. Furthermore, we built Boolean logic gates (OR and AND) into the hydrogel-enzyme hybrid materials, which were able to sense simultaneously plural specific biochems. and execute a controlled drug release in accordance with the logic operation. The intelligent soft materials that we have developed may prove valuable in future medical diagnostics or treatments.
- 148Heuser, T.; Merindol, R.; Loescher, S.; Klaus, A.; Walther, A. Photonic devices out of equilibrium: transient memory, signal propagation, and sensing. Adv. Mater. 2017, 29, 1606842, DOI: 10.1002/adma.201606842There is no corresponding record for this reference.
- 149Hong, Y.; Velegol, D.; Chaturvedi, N.; Sen, A. Biomimetic behavior of synthetic particles: from microscopic randomness to macroscopic control. Phys. Chem. Chem. Phys. 2010, 12 (7), 1423– 1435, DOI: 10.1039/B917741H149https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3cXhsVGrtbw%253D&md5=08a39a9ff8e6fa837c98a023e717d636Biomimetic behavior of synthetic particles: from microscopic randomness to macroscopic controlHong, Yiying; Velegol, Darrell; Chaturvedi, Neetu; Sen, AyusmanPhysical Chemistry Chemical Physics (2010), 12 (7), 1423-1435CODEN: PPCPFQ; ISSN:1463-9076. (Royal Society of Chemistry)Randomness is an inherent property of biol. systems. In contrast, randomness has been mostly avoided in designing synthetic or artificial systems. Particularly, in designing micro/nano-motors, some researchers have successfully used external fields to gain deterministic control over the directionality of the objects, which otherwise move in completely random directions due to Brownian motion. However, a partial control that preserves a certain degree of randomness can be very useful in certain applications of micro/nano-motors. In this Perspective we review the current progress in establishing autonomous motion of micro/nano-particles that possess controlled randomness, provide insight into the phenomena where macroscopic order originates from microscopic disorder and discuss the resemblance between these artificial systems and biol. emergent/collective behaviors.
- 150Sengupta, S.; Dey, K. K.; Muddana, H. S.; Tabouillot, T.; Ibele, M. E.; Butler, P. J.; Sen, A. Enzyme molecules as nanomotors. J. Am. Chem. Soc. 2013, 135 (4), 1406– 1414, DOI: 10.1021/ja3091615150https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3sXms1yitQ%253D%253D&md5=962d69545d196b4117014792625d036fEnzyme Molecules as NanomotorsSengupta, Samudra; Dey, Krishna K.; Muddana, Hari S.; Tabouillot, Tristan; Ibele, Michael E.; Butler, Peter J.; Sen, AyusmanJournal of the American Chemical Society (2013), 135 (4), 1406-1414CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)Using fluorescence correlation spectroscopy, we show that the diffusive movements of catalase enzyme mols. increase in the presence of the substrate, hydrogen peroxide, in a concn.-dependent manner. Employing a microfluidic device to generate a substrate concn. gradient, we show that both catalase and urease enzyme mols. spread toward areas of higher substrate concn., a form of chemotaxis at the mol. scale. Using glucose oxidase and glucose to generate a hydrogen peroxide gradient, we induce the migration of catalase toward glucose oxidase, thereby showing that chem. interconnected enzymes can be drawn together.
- 151Zhao, X.; Gentile, K.; Mohajerani, F.; Sen, A. Powering motion with enzymes. Acc. Chem. Res. 2018, 51 (10), 2373– 2381, DOI: 10.1021/acs.accounts.8b00286151https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC1cXhslyjsrbI&md5=55244a7c7b0a2dc5f1f7fb656fcf2e38Powering Motion with EnzymesZhao, Xi; Gentile, Kayla; Mohajerani, Farzad; Sen, AyusmanAccounts of Chemical Research (2018), 51 (10), 2373-2381CODEN: ACHRE4; ISSN:0001-4842. (American Chemical Society)Enzymes are ubiquitous in living systems. Apart from traditional motor proteins, the function of enzymes was assumed to be confined to the promotion of biochem. reactions. Recent work shows that free swimming enzymes, when catalyzing reactions, generate enough mech. force to cause their own movement, typically obsd. as substrate-concn.-dependent enhanced diffusion. Preliminary indication is that the impulsive force generated per turnover is comparable to the force produced by motor proteins and is within the range to activate biol. adhesion mols. responsible for mechanosensation by cells, making force generation by enzymic catalysis a novel mechanobiol.-relevant event. Furthermore, when exposed to a gradient in substrate concn., enzymes move up the gradient: an example of chemotaxis at the mol. level. The driving force for mol. chemotaxis appears to be the lowering of chem. potential due to thermodynamically favorable enzyme-substrate interactions and we suggest that chemotaxis promotes enzymic catalysis by directing the motion of the catalyst and substrates toward each other. Enzymes that are part of a reaction cascade have been shown to assemble through sequential chemotaxis; each enzyme follows its own specific substrate gradient, which in turn is produced by the preceding enzymic reaction. Thus, sequential chemotaxis in catalytic cascades allows time-dependent, self-assembly of specific catalyst particles. This is an example of how information can arise from chem. gradients, and it is tempting to suggest that similar mechanisms underlie the organization of living systems. On a practical level, chemotaxis can be used to sep. out active catalysts from their less active or inactive counterparts in the presence of their resp. substrates and should, therefore, find wide applicability. When attached to bigger particles, enzyme ensembles act as "engines", imparting motility to the particles and moving them directionally in a substrate gradient. The impulsive force generated by enzyme catalysis can also be transmitted to the surrounding fluid and mol. and colloidal tracers, resulting in convective fluid pumping and enhanced tracer diffusion. Enzyme-powered pumps that transport fluid directionally can be fabricated by anchoring enzymes onto a solid support and supplying the substrate. Thus, enzyme pumps constitute a novel platform that combines sensing and microfluidic pumping into a single self-powered microdevice. Taken in its entirety, force generation by active enzymes has potential applications ranging from nanomachinery, nanoscale assembly, cargo transport, drug delivery, micro- and nanofluidics, and chem./biochem. sensing. We also hypothesize that, in vivo, enzymes may be responsible for the stochastic motion of the cytoplasm, the organization of metabolons and signaling complexes, and the convective transport of fluid in cells. A detailed understanding of how enzymes convert chem. energy to directional mech. force can lead us to the basic principles of fabrication, development, and monitoring of biol. and biomimetic mol. machines.
- 152Zhang, Y.; Hess, H. Enhanced diffusion of catalytically active enzymes. ACS Cen. Sci. 2019, 5 (6), 939– 948, DOI: 10.1021/acscentsci.9b00228152https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC1MXpsVejsb4%253D&md5=2f39b427e23ce42ee54b229000e9d7dfEnhanced Diffusion of Catalytically Active EnzymesZhang, Yifei; Hess, HenryACS Central Science (2019), 5 (6), 939-948CODEN: ACSCII; ISSN:2374-7951. (American Chemical Society)A review. The past decade has seen an increasing no. of investigations into enhanced diffusion of catalytically active enzymes. These studies suggested that enzymes are actively propelled as they catalyze reactions or bind with ligands (e.g., substrates or inhibitors). In this Outlook, we chronol. summarize and discuss the exptl. observations and theor. interpretations and emphasize the potential contradictions in these efforts. We point out that the existing multimeric forms of enzymes or isoenzymes may cause artifacts in measurements and that the conformational changes upon substrate binding are usually not sufficient to give rise to a diffusion enhancement greater than 30%. Therefore, more rigorous expts. and a more comprehensive theory are urgently needed to quant. validate and describe the enhanced enzyme diffusion.
- 153Zhang, Y.; Hess, H. Chemically-powered swimming and diffusion in the microscopic world. Nat. Rev. Chem. 2021, 5, 500– 510, DOI: 10.1038/s41570-021-00281-6153https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB3MXhvVGgs73E&md5=b029942d817871f9955deb37d7f0ab0eChemically-powered swimming and diffusion in the microscopic worldZhang, Yifei; Hess, HenryNature Reviews Chemistry (2021), 5 (7), 500-510CODEN: NRCAF7; ISSN:2397-3358. (Nature Portfolio)A review. The past decade has seen intriguing reports and heated debates concerning the chem.-driven enhanced motion of objects ranging from small mols. to millimeter-size synthetic robots. These objects, in solns. in which chem. reactions were occurring, were obsd. to diffuse (spread non-directionally) or swim (move directionally) at rates exceeding those expected from Brownian motion alone. The debates have focused on whether obsd. enhancement is an exptl. artifact or a real phenomenon. If the latter were true, then we would also need to explain how the chem. energy is converted into mech. work. In this Perspective, we summarize and discuss recent observations and theories of active diffusion and swimming. Notably, the chemomech. coupling and magnitude of diffusion enhancement are strongly size-dependent and should vanish as the size of the swimmers approaches the mol. scale. We evaluate the reliability of common techniques to measure diffusion coeffs. and finish by considering the potential applications and chem. to mech. energy conversion efficiencies of typical nanoswimmers and microswimmers.
- 154Patiño, T.; Arqué, X.; Mestre, R.; Palacios, L.; Sánchez, S. Fundamental aspects of enzyme-powered micro- and nanoswimmers. Acc. Chem. Res. 2018, 51 (11), 2662– 2671, DOI: 10.1021/acs.accounts.8b00288154https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC1cXhvVOgs7bK&md5=ea807f80bdec589b2886c1f5e4640ec9Fundamental Aspects of Enzyme-Powered Micro- and NanoswimmersPatino, Tania; Arque, Xavier; Mestre, Rafael; Palacios, Lucas; Sanchez, SamuelAccounts of Chemical Research (2018), 51 (11), 2662-2671CODEN: ACHRE4; ISSN:0001-4842. (American Chemical Society)A review and discussion with 54 refs. Self-propulsion at the nanoscale constitutes a challenge due to the need for overcoming viscous forces and Brownian motion. Inspired by nature, artificial micro- and nanomachines powered by catalytic reactions have been developed. Due to the toxicity of the most commonly used fuels, enzyme catalysis has emerged as a versatile and biocompatible alternative to generate self-propulsion. Different swimmer sizes, ranging from the nanoscale to the microscale, and geometries, including tubular and spherical shapes, have been explored by our group and others. However, there is still a lack of understanding of the mechanisms underlying enzyme-mediated propulsion. Size, shape, and enzyme distribution, as well as their intrinsic enzymic properties, may play a crucial role in motion dynamics. In this Account, we present the efforts carried out by our group and others on the use of enzymes to power micro- and nanoswimmers. We examine the different structures, materials and enzymes reported so far to fabricate biocatalytic micro- and nanoswimmers with special emphasis on their effect in motion dynamics. We discuss the development of tubular microjets and, in particular the biocompatible propulsion of nanotubular jets, after which our group reported bubble-free enzymically propelled tubular structures with different dynamics depending on the tube length and enzyme localization (inside, outside or both). In the case of swimmers of spherical shape, we highlight the role of asymmetry in enzyme coverage and how it can affect their motion dynamics. Different approaches have been described to generate asym. distribution of enzymes, namely Janus particles, polymeric vesicles and the non-Janus with patch-like enzyme distribution that we recently demonstrated which produce motion as well. We also examine the correlation between enzyme kinetics and active motion. Enzyme activity, and consequently speed, can be modulated by modifying substrate concn. or adding specific inhibitors. Finally, we review the theory of active Brownian motion and how the size of the particles can influence the anal. of the results. Fundamentally, nanoscaled swimmers are more affected by Brownian fluctuations than microsized swimmers and, therefore, their motion is an enhanced diffusion with respect to the passive case. Microswimmers, however, can overcome these fluctuations and show propulsive or ballistic trajectories. We provide some considerations on how to analyze the motion of these swimmers from an exptl. point of view. Despite the rapid progress in enzyme-based micro- and nanoswimmers, deeper understanding of the mechanisms of motion is needed and further efforts should be aimed to study their lifetime, long-term stability and their ability to navigate in complex media.
- 155Wang, L.; Song, S.; van Hest, J.; Abdelmohsen, L.; Huang, X.; Sánchez, S. Biomimicry of cellular motility and communication based on synthetic soft-architectures. Small 2020, 16 (27), 1907680, DOI: 10.1002/smll.201907680155https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB3cXmsVSlsLg%253D&md5=0c324d0e4564083add69a7f4cc66e60dBiomimicry of Cellular Motility and Communication Based on Synthetic Soft-ArchitecturesWang, Lei; Song, Shidong; van Hest, Jan; Abdelmohsen, Loai K. E. A.; Huang, Xin; Sanchez, SamuelSmall (2020), 16 (27), 1907680CODEN: SMALBC; ISSN:1613-6810. (Wiley-VCH Verlag GmbH & Co. KGaA)A review. Cells, sophisticated membrane-bound units that contain the fundamental mols. of life, provide a precious library for inspiration and motivation for both society and academia. Scientists from various disciplines have made great endeavors toward the understanding of the cellular evolution by engineering artificial counterparts (protocells) that mimic or initiate structural or functional cellular aspects. In this regard, several works have discussed possible building blocks, designs, functions, or dynamics that can be applied to achieve this goal. Although great progress has been made, fundamental-yet complex-behaviors such as cellular communication, responsiveness to environmental cues, and motility remain a challenge, yet to be resolved. Herein, recent efforts toward utilizing soft systems for cellular mimicry are summarized-following the main outline of cellular evolution, from basic compartmentalization, and biol. reactions for energy prodn., to motility and communicative behaviors between artificial cell communities or between artificial and natural cell communities. Finally, the current challenges and future perspectives in the field are discussed, hoping to inspire more future research and to help the further advancement of this field.
- 156Hermanová, S.; Pumera, M. Biocatalytic micro- and nanomotors. Chem. Eur. J. 2020, 26, 1108, DOI: 10.1002/chem.202084962There is no corresponding record for this reference.
- 157Muddana, H. S.; Sengupta, S.; Mallouk, T. E.; Sen, A.; Butler, P. J. Substrate catalysis enhances single-enzyme diffusion. J. Am. Chem. Soc. 2010, 132 (7), 2110– 2111, DOI: 10.1021/ja908773a157https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3cXht1SgtLo%253D&md5=2f32d56d7eaa33a5c3f8a59e8aac82e6Substrate Catalysis Enhances Single-Enzyme DiffusionMuddana, Hari S.; Sengupta, Samudra; Mallouk, Thomas E.; Sen, Ayusman; Butler, Peter J.Journal of the American Chemical Society (2010), 132 (7), 2110-2111CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)We show that diffusion of single urease enzyme mols. increases in the presence of urea in a concn.-dependent manner and calc. the force responsible for this increase. Urease diffusion measured using fluorescence correlation spectroscopy increased by 16-28% over buffer controls at urea concns. ranging from 0.001 to 1 M. This increase was significantly attenuated when urease was inhibited with pyrocatechol, demonstrating that the increase in diffusion was the result of enzyme catalysis of urea. Local mol. pH changes as measured using the pH-dependent fluorescence lifetime of SNARF-1 conjugated to urease were not sufficient to explain the increase in diffusion. Thus, a force generated by self-electrophoresis remains the most plausible explanation. This force, evaluated using Brownian dynamics simulations, was 12 pN per reaction turnover. These measurements demonstrate force generation by a single enzyme mol. and lay the foundation for a further understanding of biol. force generation and the development of enzyme-driven nanomotors.
- 158Hortelão, A. C.; García-Jimeno, S.; Cano-Sarabia, M.; Patiño, T.; Maspoch, D.; Sanchez, S. LipoBots: Using liposomal vesicles as protective shell of urease-based nanomotors. Adv. Funct. Mater. 2020, 30 (42), 2002767, DOI: 10.1002/adfm.202002767158https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB3cXhs1Wrs7vK&md5=4c05f90974703c5e86e8c291e0908163LipoBots when using Liposomal Vesicles as Protective Shell of Urease-Based NanomotorsHortelao, Ana C.; Garcia-Jimeno, Sonia; Cano-Sarabia, Mary; Patino, Tania; Maspoch, Daniel; Sanchez, SamuelAdvanced Functional Materials (2020), 30 (42), 2002767CODEN: AFMDC6; ISSN:1616-301X. (Wiley-VCH Verlag GmbH & Co. KGaA)Developing self-powered nanomotors made of biocompatible and functional components is of paramount importance in future biomedical applications. Herein, the functional features of LipoBots (LBs) composed of a liposomal carrier contg. urease enzymes for propulsion, including their protective properties against acidic conditions and their on-demand triggered activation, are reported. Given the functional nature of liposomes, enzymes can be either encapsulated or coated on the surface of the vesicles. The influence of the location of urease on motion dynamics is first studied, finding that the surface-urease LBs undergo self-propulsion whereas the encapsulated-urease LBs do not. However, adding a percolating agent present in the bile salts to the encapsulated-urease LBs triggers active motion. Moreover, it is found that when both types of nanomotors are exposed to a medium of similar pH found in the stomach, the surface-urease LBs lose activity and motion capabilities, while the encapsulated-urease LBs retain activity and mobility. The results for the protection enzyme activity through encapsulation within liposomes and in situ triggering of the motion of LBs upon exposure to bile salts may open new avenues for the use of liposome-based nanomotors in drug delivery, for example, in the gastrointestinal tract, where bile salts are naturally present in the intestine.
- 159Choi, H.; Cho, S. H.; Hahn, S. K. Urease-powered polydopamine nanomotors for intravesical therapy of bladder diseases. ACS Nano 2020, 14 (6), 6683– 6692, DOI: 10.1021/acsnano.9b09726159https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB3cXhtVyis77E&md5=e26e764ddceeccee66212cc4ad32d045Urease-Powered Polydopamine Nanomotors for Intravesical Therapy of Bladder DiseasesChoi, Hyunsik; Cho, Seong Hwi; Hahn, Sei KwangACS Nano (2020), 14 (6), 6683-6692CODEN: ANCAC3; ISSN:1936-0851. (American Chemical Society)Intravesical therapeutic delivery has been extensively investigated for various bladder diseases such as bladder cancer, overactive bladder, urinary incontinence, and interstitial cystitis. However, conventional drug carriers have a low therapeutic delivery efficiency because of the passive diffusion of drug mols. in a bladder and the rapid clearance by periodic urination. Here, we report biocompatible and bioavailable enzyme-powered polymer nanomotors which can deeply penetrate into a mucosa layer of the bladder wall and remain for a long-term period in the bladder. The successful fabrication of nanomotors was confirmed by high-resoln. transmission electron microscopy, energy-dispersive X-ray mapping, zeta-potential anal., Fourier transform IR spectroscopy, and urease activity and nanomotor trajectory analyses. After injection into the bladder, urease-immobilized nanomotors became active, moving around in the bladder by converting urea into carbon dioxide and ammonia. The nanomotors resulted in the facilitated penetration to the mucosa layer of the bladder wall and the prolonged retention in the bladder even after repeated urination. The enhanced penetration and retention of the nanomotors as a drug delivery carrier in the bladder would be successfully harnessed for treating a variety of bladder diseases.
- 160Yang, Z.; Wang, L.; Gao, Z.; Hao, X.; Luo, M.; Yu, Z.; Guan, J. Ultrasmall enzyme-powered janus nanomotor working in blood circulation system. ACS Nano 2023, 17 (6), 6023– 6035, DOI: 10.1021/acsnano.3c00548160https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB3sXkvVKrtbY%253D&md5=f5847c9fc47492c4de9ddb183016deb3Ultrasmall Enzyme-Powered Janus Nanomotor Working in Blood Circulation SystemYang, Zili; Wang, Liangmeng; Gao, Zhixue; Hao, Xiaomeng; Luo, Ming; Yu, Zili; Guan, JianguoACS Nano (2023), 17 (6), 6023-6035CODEN: ANCAC3; ISSN:1936-0851. (American Chemical Society)Injectable chem. powered nanomotors may revolutionize biomedical technologies, but to date, it is a challenge for them to move autonomously in the blood circulation system and they are too large in size to break through the biol. barriers therein. Herein, we report a general scalable colloidal chem. synthesis approach for the fabrication of ultrasmall urease-powered Janus nanomotors (UPJNMs) that have a size (100-30 nm) meeting the requirement to break through the biol. barriers in the blood circulation system and can efficiently move in body fluids with only endogenous urea as fuel. In our protocol, the two hemispheroid surfaces of eccentric Au-polystyrene nanoparticles are stepwise grafted with poly(ethylene glycol) brushes and ureases via selective etching and chem. coupling, resp., forming the UPJNMs. The UPJNMs have lasting powerful mobility with ionic tolerance and pos. chemotaxis, while they are able to be dispersed steadily and self-propelled in real body fluids, as well as demonstrate good biosafety and a long circulation time in the blood circulation system of mice. Thus, the as-prepd. UPJNMs are promising as an active theranostics nanosystem for future biomedical applications.
- 161Toebes, B. J.; Cao, F.; Wilson, D. A. Spatial control over catalyst positioning on biodegradable polymeric nanomotors. Nat. Commun. 2019, 10 (1), 5308, DOI: 10.1038/s41467-019-13288-x161https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A280%3ADC%252BB3Mfit1egsg%253D%253D&md5=430b34dab0e78d12f9becc3da31918f1Spatial control over catalyst positioning on biodegradable polymeric nanomotorsToebes B Jelle; Cao F; Wilson Daniela ANature communications (2019), 10 (1), 5308 ISSN:.Scientists over the world are inspired by biological nanomotors and try to mimic these complex structures. In recent years multiple nanomotors have been created for various fields, such as biomedical applications or environmental remediation, which require a different design both in terms of size and shape, as well as material properties. So far, only relatively simple designs for synthetic nanomotors have been reported. Herein, we report an approach to create biodegradable polymeric nanomotors with a multivalent design. PEG-PDLLA (poly(ethylene glycol)-b-poly(D,L-lactide)) stomatocytes with azide handles were created that were selectively reduced on the outside surface by TCEP (tris(2-carboxyethyl)phosphine) functionalized beads. Thereby, two different functional handles were created, both on the inner and outer surface of the stomatocytes, providing spatial control for catalyst positioning. Enzymes were coupled on the inside of the stomatocyte to induce motion in the presence of fuel, while fluorophores and other molecules can be attached on the outside.
- 162Qiu, B.; Xie, L.; Zeng, J.; Liu, T.; Yan, M.; Zhou, S.; Liang, Q.; Tang, J.; Liang, K.; Kong, B. Interfacially super-assembled asymmetric and H2O2 sensitive multilayer-sandwich magnetic mesoporous silica nanomotors for detecting and removing heavy metal ions. Adv. Funct. Mater. 2021, 31, 2010694, DOI: 10.1002/adfm.202010694162https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB3MXmvVyjtLw%253D&md5=00fa2e105237342feedcdb693c43f840Interfacially Super-Assembled Asymmetric and H2O2 Sensitive Multilayer-Sandwich Magnetic Mesoporous Silica Nanomotors for Detecting and Removing Heavy Metal IonsQiu, Beilei; Xie, Lei; Zeng, Jie; Liu, Tianyi; Yan, Miao; Zhou, Shan; Liang, Qirui; Tang, Jinyao; Liang, Kang; Kong, BiaoAdvanced Functional Materials (2021), 31 (21), 2010694CODEN: AFMDC6; ISSN:1616-301X. (Wiley-VCH Verlag GmbH & Co. KGaA)Asym. hollow and magnetic mesoporous silica nanocomposites have great potential applications due to their unique structural-functional properties. Here, asym. multilayer-sandwich magnetic mesoporous silica nanobottles (MMSNBs) are presented through an interfacial super-assembly strategy. Asym. hollow silica nanobottles (SNBs) are first prepd., and Fe3O4 nanoparticles monolayers and mesoporous silica layers are uniformly super-assembled on the surfaces of SNBs, resp. The high Fe3O4 nanoparticles loading endows MMSNBs with a high magnetization (8.5 emu g-1), while the mesoporous silica layers exhibit high surface area (613.4 m2 g-1) and large pore size (3.6 nm). MMSNBs can be employed as a novel type of enzyme-powered nanomotors by integrating catalase (Cat-MMSNBs), which show an av. speed of 7.59μm s-1 (≈25 body lengths s-1) at 1.5 wt% H2O2. Accordingly, the water quality can be monitored by evaluating the movement speed of Cat-MMSNBs. Moreover, MMSNBs act as a good adsorbent for removing more than 90% of the heavy metal ions with the advantage of the mesoporous structure. In addn., the good magnetic response enables the MMSNBs with precise directional control and is conducive to recycling for repeated operation. This bottom-up interfacial super-assembly construction strategy allows for a new understanding of the rational design and synthesis of multi-functional nanomotors.
- 163Abdelmohsen, L. K.; Nijemeisland, M.; Pawar, G. M.; Janssen, G. J.; Nolte, R. J.; van Hest, J. C.; Wilson, D. A. Dynamic loading and unloading of proteins in polymeric stomatocytes: formation of an enzyme-loaded supramolecular nanomotor. ACS Nano 2016, 10 (2), 2652– 2660, DOI: 10.1021/acsnano.5b07689163https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC28XhtlymsLs%253D&md5=950ae40c6f7c8ae1a6b6d370c5fd71d5Dynamic Loading and Unloading of Proteins in Polymeric Stomatocytes: Formation of an Enzyme-Loaded Supramolecular NanomotorAbdelmohsen, Loai K. E. A.; Nijemeisland, Marlies; Pawar, Gajanan M.; Janssen, Geert-Jan A.; Nolte, Roeland J. M.; van Hest, Jan C. M.; Wilson, Daniela A.ACS Nano (2016), 10 (2), 2652-2660CODEN: ANCAC3; ISSN:1936-0851. (American Chemical Society)Self-powered artificial nanomotors are currently attracting increased interest as mimics of biol. motors but also as potential components of nanomachinery, robotics, and sensing devices. We have recently described the controlled shape transformation of polymersomes into bowl-shaped stomatocytes and the assembly of platinum-driven nanomotors. However, the platinum encapsulation inside the structures was low; only 50% of the structures contained the catalyst and required both high fuel concns. for the propelling of the nanomotors and harsh conditions for the shape transformation. Application of the nanomotors in a biol. setting requires the nanomotors to be efficiently propelled by a naturally available energy source and at biol. relevant concns. Here we report a strategy for enzyme entrapment and nanomotor assembly via controlled and reversible folding of polymersomes into stomatocytes under mild conditions, allowing the encapsulation of the proteins inside the stomach with almost 100% efficiency and retention of activity. The resulting enzyme-driven nanomotors are capable of propelling these structures at low fuel concns. (hydrogen peroxide or glucose) via a one-enzyme or two-enzyme system. The confinement of the enzymes inside the stomach does not hinder their activity and in fact facilitates the transfer of the substrates, while protecting them from the deactivating influences of the media. This is particularly important for future applications of nanomotors in biol. settings esp. for systems where fast autonomous movement occurs at physiol. concns. of fuel.
- 164Nijemeisland, M.; Abdelmohsen, L. K.; Huck, W. T.; Wilson, D. A.; van Hest, J. C. A compartmentalized out-of-equilibrium enzymatic reaction network for sustained autonomous movement. ACS Cent. Sci. 2016, 2 (11), 843– 849, DOI: 10.1021/acscentsci.6b00254164https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC28Xhsl2gtbfP&md5=c4c80a73fb5d6109de97e97bcb43ce09A Compartmentalized Out-of-Equilibrium Enzymatic Reaction Network for Sustained Autonomous MovementNijemeisland, Marlies; Abdelmohsen, Loai K. E. A.; Huck, Wilhelm T. S.; Wilson, Daniela A.; van Hest, Jan C. M.ACS Central Science (2016), 2 (11), 843-849CODEN: ACSCII; ISSN:2374-7951. (American Chemical Society)Every living cell is a compartmentalized out-of-equil. system exquisitely able to convert chem. energy into function. In order to maintain homeostasis, the flux of metabolites is tightly controlled by regulatory enzymic networks. A crucial prerequisite for the development of life-like materials is the construction of synthetic systems with compartmentalized reaction networks that maintain out-of-equil. function. Here, we aim for autonomous movement as an example of the conversion of feedstock mols. into function. The flux of the conversion is regulated by a rationally designed enzymic reaction network with multiple feed forward loops. By compartmentalizing the network into bowl-shaped nanocapsules the output of the network is harvested as kinetic energy. The entire system shows sustained and tunable microscopic motion resulting from the conversion of multiple external substrates. The successful compartmentalization of an out-of-equil. reaction network is a major first step in harnessing the design principles of life for construction of adaptive and internally regulated life-like systems.
- 165Chatterjee, A.; Ghosh, S.; Ghosh, C.; Das, D. Fluorescent microswimmers based on cross-beta amyloid nanotubes and divergent cascade networks. Angew. Chem., Int. Ed. 2022, 61 (29), e202201547, DOI: 10.1002/anie.202201547165https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB38XhsVCgtL%252FJ&md5=d724d4f4ffe85e8ea7e5fcc1e577582eFluorescent Microswimmers Based on Cross-β Amyloid Nanotubes and Divergent Cascade NetworksChatterjee, Ayan; Ghosh, Souvik; Ghosh, Chandranath; Das, DibyenduAngewandte Chemie, International Edition (2022), 61 (29), e202201547CODEN: ACIEF5; ISSN:1433-7851. (Wiley-VCH Verlag GmbH & Co. KGaA)Shaped through millions of years of evolution, the spatial localization of multiple enzymes in living cells employs extensive cascade reactions to enable highly coordinated multimodal functions. Herein, by utilizing a complex divergent cascade, we exploit the catalytic potential as well as templating abilities of streamlined cross-β amyloid nanotubes to yield two orthogonal roles simultaneously. The short peptide based paracryst. nanotube surfaces demonstrated the generation of fluorescence signals within entangled networks loaded with alc. dehydrogenase (ADH). The nanotubular morphologies were further used to generate cascade-driven microscopic motility through surface entrapment of sarcosine oxidase (SOX) and catalase (Cat). Moreover, a divergent cascade network was initiated by upstream catalysis of the substrate mols. through the surface mutation of catalytic moieties. Notably, the resultant downstream products led to the generation of motile fluorescent microswimmers by utilizing the two sets of orthogonal properties and, thus, mimicked the complex cascade-mediated functionalities of extant biol.
- 166Dey, K. K.; Zhao, X.; Tansi, B. M.; Mendez-Ortiz, W. J.; Cordova-Figueroa, U. M.; Golestanian, R.; Sen, A. Micromotors powered by enzyme catalysis. Nano Lett. 2015, 15 (12), 8311– 8315, DOI: 10.1021/acs.nanolett.5b03935166https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2MXhvVOrtrvJ&md5=cfb4f6a75427fa6c4b9ae436c731d197Micromotors Powered by Enzyme CatalysisDey, Krishna K.; Zhao, Xi; Tansi, Benjamin M.; Mendez-Ortiz, Wilfredo J.; Cordova-Figueroa, Ubaldo M.; Golestanian, Ramin; Sen, AyusmanNano Letters (2015), 15 (12), 8311-8315CODEN: NALEFD; ISSN:1530-6984. (American Chemical Society)Active biocompatible systems are of great current interest for their possible applications in drug or antidote delivery at specific locations. Herein, we report the synthesis and study of self-propelled microparticles powered by enzymic reactions and their directed movement in substrate concn. gradient. Polystyrene microparticles were functionalized with the enzymes urease and catalase using a biotin-streptavidin linkage procedure. The motion of the enzyme-coated particles was studied in the presence of the resp. substrates, using optical microscopy and dynamic light scattering anal. The diffusion of the particles was found to increase in a substrate concn. dependent manner. The directed chemotactic movement of these enzyme-powered motors up the substrate gradient was studied using three-inlet microfluidic channel architecture.
- 167Zhao, X.; Palacci, H.; Yadav, V.; Spiering, M. M.; Gilson, M. K.; Butler, P. J.; Hess, H.; Benkovic, S. J.; Sen, A. Substrate-driven chemotactic assembly in an enzyme cascade. Nat. Chem. 2018, 10 (3), 311– 317, DOI: 10.1038/nchem.2905167https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2sXhvF2lsb3F&md5=3c60afd0a0cbcd0a0f3cefb403e1f20bSubstrate-driven chemotactic assembly in an enzyme cascadeZhao, Xi; Palacci, Henri; Yadav, Vinita; Spiering, Michelle M.; Gilson, Michael K.; Butler, Peter J.; Hess, Henry; Benkovic, Stephen J.; Sen, AyusmanNature Chemistry (2018), 10 (3), 311-317CODEN: NCAHBB; ISSN:1755-4330. (Nature Research)Enzymic catalysis is essential to cell survival. In many instances, enzymes that participate in reaction cascades have been shown to assemble into metabolons in response to the presence of the substrate for the first enzyme. However, what triggers metabolon formation has remained an open question. Through a combination of theory and expts., we show that enzymes in a cascade can assemble via chemotaxis. We apply microfluidic and fluorescent spectroscopy techniques to study the coordinated movement of the first four enzymes of the glycolysis cascade: hexokinase, phosphoglucose isomerase, phosphofructokinase and aldolase. We show that each enzyme independently follows its own specific substrate gradient, which in turn is produced by the preceding enzymic reaction. Furthermore, we find that the chemotactic assembly of enzymes occurs even under cytosolic crowding conditions.
- 168Wang, J.; Toebes, B. J.; Plachokova, A. S.; Liu, Q.; Deng, D.; Jansen, J. A.; Yang, F.; Wilson, D. A. Self-propelled PLGA micromotor with chemotactic response to inflammation. Adv. Healthcare Mater. 2020, 9 (7), 1901710, DOI: 10.1002/adhm.201901710168https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB3cXktlGhurk%253D&md5=230e9df65435e02415eb5cb517cfb1ccSelf-Propelled PLGA Micromotor with Chemotactic Response to InflammationWang, Jiamian; Toebes, B. Jelle; Plachokova, Adelina S.; Liu, Qian; Deng, Dongmei; Jansen, John A.; Yang, Fang; Wilson, Daniela A.Advanced Healthcare Materials (2020), 9 (7), 1901710CODEN: AHMDBJ; ISSN:2192-2640. (Wiley-VCH Verlag GmbH & Co. KGaA)Local drug delivery systems have recently been developed for multiple diseases that have the requirements of site-specific actions, prolonged delivery periods, and decreased drug dosage to reduce undesirable side effects. The challenge for such systems is to achieve directional and precise delivery in inaccessible narrow lesions, such as periodontal pockets or root canals in deeper portions of the dentinal tubules. The primary strategy to tackle this challenge is fabricating a smart tracking delivery system. Here, drug-loaded biodegradable micromotors showing self-propelled directional movement along a hydrogen peroxide concn. gradient produced by phorbol esters-stimulated macrophages are reported. The drug-loaded poly(lactic-co-glycolic acid) micromotors with asym. coverage of enzyme (patch-like enzyme distribution) are prepd. by electrospraying and postfunctionalized with catalase via 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide coupling. Doxycycline, a common drug for the treatment of periodontal disease, is selected as a model drug, and the release study by high-performance liq. chromatog. is shown that both the postfunctionalization step and the presence of hydrogen peroxide have no neg. influence on drug release profiles. The movement behavior in the presence of hydrogen peroxide is confirmed by nanoparticle tracking anal. An in vitro model is designed and confirmed the response efficiency and directional control of the micromotors toward phorbol esters-stimulated macrophages.
- 169Joseph, A.; Contini, C.; Cecchin, D.; Nyberg, S.; Ruiz-Perez, L.; Gaitzsch, J.; Fullstone, G.; Tian, X.; Azizi, J.; Preston, J.; Volpe, G.; Battaglia, G. Chemotactic synthetic vesicles: design and applications in blood-brain barrier crossing. Sci. Adv. 2017, 3, e1700362, DOI: 10.1126/sciadv.1700362169https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC1cXntFGrsbY%253D&md5=0234d41cb96af40a7de3af83d36b1359Chemotactic synthetic vesicles: Design and applications in blood-brain barrier crossingJoseph, Adrian; Contini, Claudia; Cecchin, Denis; Nyberg, Sophie; Ruiz-Perez, Lorena; Gaitzsch, Jens; Fullstone, Gavin; Tian, Xiaohe; Azizi, Juzaili; Preston, Jane; Volpe, Giorgio; Battaglia, GiuseppeScience Advances (2017), 3 (8), e1700362/1-e1700362/12CODEN: SACDAF; ISSN:2375-2548. (American Association for the Advancement of Science)In recent years, scientists have created artificialmicroscopic and nanoscopic self-propelling particles, often referred to as nano- or microswimmers, capable of mimicking biol. locomotion and taxis. This active diffusion enables the engineering of complex operations that so far have not been possible at the micro- and nanoscale. One of the most promising tasks is the ability to engineer nanocarriers that can autonomously navigate within tissues and organs, accessing nearly every site of the human body guided by endogenous chem. gradients.We report a fully synthetic, org., nanoscopic system that exhibits attractive chemotaxisdrivenbyenzymic conversion of glucose.Weachieve this by encapsulating glucose oxidase alone or in combination with catalase into nanoscopic and biocompatible asym. polymer vesicles (known as polymersomes). We show that these vesicles self-propel in response to an external gradient of glucose by inducing a slip velocity on their surface, which makes them move in an extremely sensitive way toward higher-concn. regions. We finally demonstrate that the chemotactic behavior of these nanoswimmers, in combination with LRP-1 (low-d. lipoprotein receptor-related protein 1) targeting, enables a fourfold increase in penetration to the brain compared to nonchemotactic systems.
- 170Kumar, B.; Patil, A. J.; Mann, S. Enzyme-powered motility in buoyant organoclay/DNA protocells. Nat. Chem. 2018, 10 (11), 1154– 116, DOI: 10.1038/s41557-018-0119-3170https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC1cXhsFKqtr7K&md5=a18f59a5d0b21e7a790ea182f37ab3c1Enzyme-powered motility in buoyant organoclay/DNA protocellsKumar, B. V. V. S. Pavan; Patil, Avinash J.; Mann, StephenNature Chemistry (2018), 10 (11), 1154-1163CODEN: NCAHBB; ISSN:1755-4330. (Nature Research)Reconstitution and simulation of cellular motility in microcompartmentalized colloidal objects have important implications for microcapsule-based remote sensing, environmentally induced signaling between artificial cell-like entities and programming spatial migration in synthetic protocell consortia. Here the authors describe the design and construction of catalase-contg. organoclay/DNA semipermeable microcapsules, which in the presence of hydrogen peroxide exhibit enzyme-powered oxygen gas bubble-dependent buoyancy. The authors det. the optimum conditions for single and/or multiple bubble generation per microcapsule, monitor the protocell velocities and resilience, and use remote magnetic guidance to establish reversible changes in the buoyancy. Co-encapsulation of catalase and glucose oxidase is exploited to establish a spatiotemporal response to antagonistic bubble generation and depletion to produce protocells capable of sustained oscillatory vertical movement. The motility of the microcapsules can be used for the flotation of macroscopic objects, self-sorting of mixed protocell communities and the delivery of a biocatalyst from an inert to chem. active environment. These results highlight new opportunities to constructing programmable microcompartmentalized colloids with buoyancy-derived motility.
- 171Gao, N.; Li, M.; Tian, L.; Patil, A. J.; Pavan Kumar, B.; Mann, S. Chemical-mediated translocation in protocell-based microactuators. Nat. Chem. 2021, 13 (9), 868– 879, DOI: 10.1038/s41557-021-00728-9171https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB3MXhsVSmsb3L&md5=8db27f942d6097312a2b8f36a6845274Chemical-mediated translocation in protocell-based microactuatorsGao, Ning; Li, Mei; Tian, Liangfei; Patil, Avinash J.; Pavan Kumar, B. V. V. S.; Mann, StephenNature Chemistry (2021), 13 (9), 868-879CODEN: NCAHBB; ISSN:1755-4330. (Nature Portfolio)Artificial cell-like communities participate in diverse modes of chem. interaction but exhibit minimal interfacing with their local environment. Here we develop an interactive microsystem based on the immobilization of a population of enzyme-active semipermeable proteinosomes within a helical hydrogel filament to implement signal-induced movement. We attach large single-polynucleotide/peptide microcapsules at one or both ends of the helical protocell filament to produce free-standing soft microactuators that sense and process chem. signals to perform mech. work. Different modes of translocation are achieved by synergistic or antagonistic enzyme reactions located within the helical connector or inside the attached microcapsule loads. Mounting the microactuators on a ratchet-like surface produces a directional push-pull movement. Our methodol. opens up a route to protocell-based chem. systems capable of utilizing mech. work and provides a step towards the engineering of soft microscale objects with increased levels of operational autonomy.
- 172Wheeldon, I.; Minteer, S. D.; Banta, S.; Barton, S. C.; Atanassov, P.; Sigman, M. Substrate channelling as an approach to cascade reactions. Nat. Chem. 2016, 8 (4), 299– 309, DOI: 10.1038/nchem.2459172https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC28Xks1Cru7Y%253D&md5=5056edbd76a5aa2266906be9e23c579bSubstrate channelling as an approach to cascade reactionsWheeldon, Ian; Minteer, Shelley D.; Banta, Scott; Barton, Scott Calabrese; Atanassov, Plamen; Sigman, MatthewNature Chemistry (2016), 8 (4), 299-309CODEN: NCAHBB; ISSN:1755-4330. (Nature Publishing Group)A review. Millions of years of evolution have produced biol. systems capable of efficient one-pot multi-step catalysis. The underlying mechanisms that facilitate these reaction processes are increasingly providing inspiration in synthetic chem. Substrate channeling, where intermediates between enzymic steps are not in equil. with the bulk soln., enables increased efficiencies and yields in reaction and diffusion processes. Here, we review different mechanisms of substrate channeling found in nature and provide an overview of the anal. methods used to quantify these effects. The incorporation of substrate channeling into synthetic cascades is a rapidly developing concept, and recent examples of the fabrication of cascades with controlled diffusion and flux of intermediates are presented.
- 173Hinzpeter, F.; Gerland, U.; Tostevin, F. Optimal compartmentalization strategies for metabolic microcompartments. Biophys. J. 2017, 112, 767– 779, DOI: 10.1016/j.bpj.2016.11.3194173https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC28XitVOlur3F&md5=5ec8113d5f4060db6906f9c14160aeecOptimal Compartmentalization Strategies for Metabolic MicrocompartmentsHinzpeter, Florian; Gerland, Ulrich; Tostevin, FilipeBiophysical Journal (2017), 112 (4), 767-779CODEN: BIOJAU; ISSN:0006-3495. (Cell Press)Intracellular compartmentalization of cooperating enzymes is a strategy that is frequently used by cells. Segregation of enzymes that catalyze sequential reactions can alleviate challenges such as toxic pathway intermediates, competing metabolic reactions, and slow reaction rates. Inspired by nature, synthetic biologists also seek to encapsulate engineered metabolic pathways within vesicles or proteinaceous shells to enhance the yield of industrially and pharmaceutically useful products. Although enzymic compartments have been extensively studied exptl., a quant. understanding of the underlying design principles is still lacking. Here, the authors study theor. how the size and enzymic compn. of compartments should be chosen so as to maximize the productivity of a model metabolic pathway. Maximizing productivity requires compartments larger than a certain crit. size. The enzyme d. within each compartment should be tuned according to a power-law scaling in the compartment size. The authors explain these observations using an anal. solvable, well-mixed approxn. The authors also study the qual. different compartmentalization strategies that emerge in parameter regimes where this approxn. breaks down. The authors' results suggest that the different sizes and enzyme packings of α- and β-carboxysomes each constitute an optimal compartmentalization strategy given the properties of their resp. protein shells.
- 174Qiao, Y.; Li, M.; Booth, R.; Mann, S. Predatory behaviour in synthetic protocell communities. Nat. Chem. 2017, 9 (2), 110– 119, DOI: 10.1038/nchem.2617174https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC28Xhs1amtbjJ&md5=b9d48e5a1f98b80c2e32b8cb43c768baPredatory behaviour in synthetic protocell communitiesQiao, Yan; Li, Mei; Booth, Richard; Mann, StephenNature Chemistry (2017), 9 (2), 110-119CODEN: NCAHBB; ISSN:1755-4330. (Nature Publishing Group)Recent progress in the chem. construction of colloidal objects comprising integrated biomimetic functions is paving the way towards rudimentary forms of artificial cell-like entities (protocells). Although several new types of protocells are currently available, the design of synthetic protocell communities and investigation of their collective behavior has received little attention. Here we demonstrate an artificial form of predatory behavior in a community of protease-contg. coacervate microdroplets and protein-polymer microcapsules (proteinosomes) that interact via electrostatic binding. The coacervate microdroplets act as killer protocells for the obliteration of the target proteinosome population by protease-induced lysis of the protein-polymer membrane. As a consequence, the proteinosome payload (dextran, single-stranded DNA, platinum nanoparticles) is trafficked into the attached coacervate microdroplets, which are then released as functionally modified killer protocells capable of rekilling. Our results highlight opportunities for the development of interacting artificial protocell communities, and provide a strategy for inducing collective behavior in soft matter microcompartmentalized systems and synthetic protocell consortia.
- 175Qiao, Y.; Li, M.; Qiu, D.; Mann, S. Response-retaliation behavior in synthetic protocell communities. Angew. Chem., Int. Ed. 2019, 58 (49), 17758– 17763, DOI: 10.1002/anie.201909313175https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC1MXitVais77F&md5=6b6ac4f90fc4f496bcfe91f5e17b4d7aResponse-Retaliation Behavior in Synthetic Protocell CommunitiesQiao, Yan; Li, Mei; Qiu, Dong; Mann, StephenAngewandte Chemie, International Edition (2019), 58 (49), 17758-17763CODEN: ACIEF5; ISSN:1433-7851. (Wiley-VCH Verlag GmbH & Co. KGaA)Two different artificial predation strategies are spatially and temporally coupled to generate a simple tit-for-tat mechanism in a ternary protocell network capable of antagonistic enzyme-mediated interactions. The consortium initially consists of protease-sensitive glucose-oxidase-contg. proteinosomes (1), non-interacting pH-sensitive polypeptide/mononucleotide coacervate droplets contg. proteinase K (2), and proteinosome-adhered pH-resistant polymer/polysaccharide coacervate droplets (3). On receiving a glucose signal, secretion of protons from 1 triggers the disassembly of 2 and the released protease is transferred to 3 to initiate a delayed contact-dependent killing of the proteinosomes and cessation of glucose oxidase activity. Our results provide a step towards complex mesoscale dynamics based on programmable response-retaliation behavior in artificial protocell consortia.
- 176Chakraborty, T.; Wegner, S. V. Cell to cell signaling through light in artificial cell communities: glowing predator lures prey. ACS Nano 2021, 15 (6), 9434– 9444, DOI: 10.1021/acsnano.1c01600176https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB3MXhtlCqu77L&md5=0a547d307006fa725cd1e90d72317dbaCell to Cell Signaling through Light in Artificial Cell Communities: Glowing Predator Lures PreyChakraborty, Taniya; Wegner, Seraphine V.ACS Nano (2021), 15 (6), 9434-9444CODEN: ANCAC3; ISSN:1936-0851. (American Chemical Society)Cells commonly communicate with each other through diffusible mols. but nonchem. communication remains elusive. While bioluminescent organisms communicate through light to find prey or attract mates, it is still under debate if signaling through light is possible at the cellular level. Here, we demonstrate that cell to cell signaling through light is possible in artificial cell communities derived from biomimetic vesicles. In our design, artificial sender cells produce an intracellular light signal, which triggers the adhesion to receiver cells. Unlike sol. mols., the light signal propagates fast, independent of diffusion and without the need for a transporter across membranes. To obtain a predator-prey relationship, the luminescence predator cells is loaded with a secondary diffusible poison, which is transferred to the prey cell upon adhesion and leads to its lysis. This design provides a blueprint for light based intercellular communication, which can be used for programing artificial and natural cell communities.
- 177Mason, A. F.; Buddingh, B. C.; Williams, D. S.; van Hest, J. C. M. Hierarchical self-assembly of a copolymer-stabilized coacervate protocell. J. Am. Chem. Soc. 2017, 139 (48), 17309– 17312, DOI: 10.1021/jacs.7b10846177https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2sXhvVSnurzP&md5=30fde604ab0576771bbc76cbefbd7ccfHierarchical Self-Assembly of a Copolymer-Stabilized Coacervate ProtocellMason, Alexander F.; Buddingh, Bastiaan C.; Williams, David S.; van Hest, Jan C. M.Journal of the American Chemical Society (2017), 139 (48), 17309-17312CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)Complex coacervate microdroplets are finding increased utility in synthetic cell applications due to their cytomimetic properties. However, their intrinsic membrane-free nature results in instability that limits their application in protocell research. Herein, the authors present the development of a new protocell model through the spontaneous interfacial self-assembly of copolymer mols. on biopolymer coacervate microdroplets. This hierarchical protocell model not only incorporates the favorable properties of coacervates (such as spontaneous assembly and macromol. condensation) but also assimilates the essential features of a semipermeable copolymeric membrane (such as discretization and stabilization). This was accomplished by engineering an asym., biodegradable triblock copolymer mol. comprising hydrophilic, hydrophobic, and polyanionic components capable of direct coacervate membranization via electrostatic surface anchoring and chain self-assocn. The resulting hierarchical protocell demonstrated striking integrity as a result of membrane formation, successfully stabilizing enzymic cargo against coalescence and fusion in discrete protocellular populations. The semipermeable nature of the copolymeric membrane enabled the incorporation of a simple enzymic cascade, demonstrating chem. communication between discrete populations of neighboring protocells. In this way, the authors pave the way for the development of new synthetic cell constructs.
- 178Wang, X.; Tian, L.; Du, H.; Li, M.; Mu, W.; Drinkwater, B. W.; Han, X.; Mann, S. Chemical communication in spatially organized protocell colonies and protocell/living cell micro-arrays. Chem. Sci. 2019, 10 (41), 9446– 9453, DOI: 10.1039/C9SC04522H178https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC1MXhslyqt7bK&md5=07d8659b3475a5879e1fe01d00d1d227Chemical communication in spatially organized protocell colonies and protocell/living cell micro-arraysWang, Xuejing; Tian, Liangfei; Du, Hang; Li, Mei; Mu, Wei; Drinkwater, Bruce W.; Han, Xiaojun; Mann, StephenChemical Science (2019), 10 (41), 9446-9453CODEN: CSHCCN; ISSN:2041-6520. (Royal Society of Chemistry)Micro-arrays of discrete or hemifused giant unilamellar lipid vesicles (GUVs) with controllable spatial geometries, lattice dimensions, trapped occupancies and compns. are prepd. by acoustic standing wave patterning, and employed as platforms to implement chem. signaling in GUV colonies and protocell/living cell consortia. The methodol. offers an alternative approach to GUV micro-array fabrication and provides new opportunities in protocell research and bottom-up synthetic biol.
- 179Buddingh’, B. C.; Elzinga, J.; van Hest, J. C. M. Intercellular communication between artificial cells by allosteric amplification of a molecular signal. Nat. Commun. 2020, 11 (1), 1652, DOI: 10.1038/s41467-020-15482-8179https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB3cXmsVWitbg%253D&md5=d3384619028f0aac719d9e96dd6f40b7Intercellular communication between artificial cells by allosteric amplification of a molecular signalBuddingh', Bastiaan C.; Elzinga, Janneke; van Hest, Jan C. M.Nature Communications (2020), 11 (1), 1652CODEN: NCAOBW; ISSN:2041-1723. (Nature Research)Multicellular organisms rely on intercellular communication to coordinate the behavior of individual cells, which enables their differentiation and hierarchical organization. Various cell mimics have been developed to establish fundamental engineering principles for the construction of artificial cells displaying cell-like organization, behavior and complexity. However, collective phenomena, although of great importance for a better understanding of life-like behavior, are underexplored. Here, we construct collectives of giant vesicles that can communicate with each other through diffusing chem. signals that are recognized and processed by synthetic enzymic cascades. Similar to biol. cells, the Receiver vesicles can transduce a weak signal originating from Sender vesicles into a strong response by virtue of a signal amplification step, which facilitates the propagation of signals over long distances within the artificial cell consortia. This design advances the development of interconnected artificial cells that can exchange metabolic and positional information to coordinate their higher-order organization.
- 180Ji, Y.; Chakraborty, T.; Wegner, S. V. Self-regulated and bidirectional communication in synthetic cell communities. ACS Nano 2023, 17 (10), 8992– 9002, DOI: 10.1021/acsnano.2c09908180https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB3sXptlyltrY%253D&md5=20a75b01b8ea454dcc31f96dc900a1d1Self-Regulated and Bidirectional Communication in Synthetic Cell CommunitiesJi, Yuhao; Chakraborty, Taniya; Wegner, Seraphine V.ACS Nano (2023), 17 (10), 8992-9002CODEN: ANCAC3; ISSN:1936-0851. (American Chemical Society)Cell-to-cell communication is not limited to a sender releasing a signaling mol. and a receiver perceiving it but is often self-regulated and bidirectional. Yet, in communities of synthetic cells, such features that render communication efficient and adaptive are missing. Here, we report the design and implementation of adaptive two-way signaling with lipid-vesicle-based synthetic cells. The first layer of self-regulation derives from coupling the temporal dynamics of the signal, H2O2, prodn. in the sender to adhesions between sender and receiver cells. This way the receiver stays within the signaling range for the duration sender produces the signal and detaches once the signal fades. Specifically, H2O2 acts as both a forward signal and a regulator of the adhesions by activating photoswitchable proteins at the surface for the duration of the chemiluminescence. The second layer of self-regulation arises when the adhesions render the receiver permeable and trigger the release of a backward signal, resulting in bidirectional exchange. These design rules provide a concept for engineering multicellular systems with adaptive communication.
- 181Taylor, H.; Gao, N.; Mann, S. Chemical communication and protocell-matrix dynamics in segregated colloidosome micro-colonies. Angew. Chem., Int. Ed. 2023, 62 (24), e202300932, DOI: 10.1002/anie.202300932181https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB3sXpslOqsb8%253D&md5=4e838bb2964fb3423458d0acfb4d9a0fChemical Communication and Protocell-Matrix Dynamics in Segregated Colloidosome Micro-ColoniesTaylor, Hannah; Gao, Ning; Mann, StephenAngewandte Chemie, International Edition (2023), 62 (24), e202300932CODEN: ACIEF5; ISSN:1433-7851. (Wiley-VCH Verlag GmbH & Co. KGaA)Despite an emerging catalog of collective behaviors in communities of homogeneously distributed cell-like objects, microscale protocell colonies with spatially segregated populations have received minimal attention. Here, we use microfluidics to fabricate Janus-like calcium alginate hydrogel microspheres with spatially partitioned populations of enzyme-contg. inorg. colloidosomes and investigate their potential as integrated platforms for domain-mediated chem. communication and programmable protocell-matrix dynamics. Diffusive chem. signalling within the segregated communities gives rise to increased initial enzyme kinetics compared with a homogeneous distribution of protocells. We employ competing enzyme-mediated hydrogel crosslinking and decrosslinking reactions in different domains of the partitioned colonies to undertake selective expulsion of a specific protocell population from the community. Our results offer new possibilities for the design and construction of spatially organized cytomimetic consortia capable of endogenous chem. processing and protocell-environment interactivity.
- 182Fusi, G.; Del Giudice, D.; Skarsetz, O.; Di Stefano, S.; Walther, A. Autonomous soft robots empowered by chemical reaction networks. Adv. Mater. 2023, 35 (7), e2209870, DOI: 10.1002/adma.202209870182https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB38XjtFOmur7O&md5=245f47eab56714beed4e2585f71879feAutonomous Soft Robots Empowered by Chemical Reaction NetworksFusi, Giorgio; Del Giudice, Daniele; Skarsetz, Oliver; Di Stefano, Stefano; Walther, AndreasAdvanced Materials (Weinheim, Germany) (2023), 35 (7), 2209870CODEN: ADVMEW; ISSN:0935-9648. (Wiley-VCH Verlag GmbH & Co. KGaA)Hydrogel actuators are important for designing stimuli-sensitive soft robots. They generate mech. motion by exploiting compartmentalized (de)swelling in response to a stimulus. However, classical switching methods, such as manually lowering or increasing the pH, cannot provide more complex autonomous motions. By coupling an autonomously operating pH-flip with programmable lifetimes to a hydrogel system contg. pH-responsive and non-responsive compartments, autoonenomous forward and backward motion as well as more complex tasks, such as interlocking of "puzzle pieces" and collection of objects are realized. All operations are initiated by one simple trigger, and the devices operate in a "fire and forget" mode. More complex self-regulatory behavior is obtained by adding chemo-mechano-chemo feedback mechanisms. Due to its simplicity, this method shows great potential for the autonomous operation of soft grippers and metamaterials.
- 183Elani, Y.; Law, R. V.; Ces, O. Vesicle-based artificial cells as chemical microreactors with spatially segregated reaction pathways. Nat. Commun. 2014, 5, 5305, DOI: 10.1038/ncomms6305183https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2cXitVaksb%252FM&md5=9552fb140fc09ca105b5ba24007aff0bVesicle-based artificial cells as chemical microreactors with spatially segregated reaction pathwaysElani, Yuval; Law, Robert V.; Ces, OscarNature Communications (2014), 5 (), 5305CODEN: NCAOBW; ISSN:2041-1723. (Nature Publishing Group)In the discipline of bottom-up synthetic biol., vesicles define the boundaries of artificial cells and are increasingly being used as biochem. microreactors operating in physiol. environments. As the field matures, there is a need to compartmentalize processes in different spatial localities within vesicles, and for these processes to interact with one another. Here we address this by designing and constructing multi-compartment vesicles within which an engineered multi-step enzymic pathway is carried out. The individual steps are isolated in distinct compartments, and their products traverse into adjacent compartments with the aid of transmembrane protein pores, initiating subsequent steps. Thus, an engineered signalling cascade is recreated in an artificial cellular system. Importantly, by allowing different steps of a chem. pathway to be sepd. in space, this platform bridges the gap between table-top chem. and chem. that is performed within vesicles.
- 184Liu, S.; Zhang, Y.; He, X.; Li, M.; Huang, J.; Yang, X.; Wang, K.; Mann, S.; Liu, J. Signal processing and generation of bioactive nitric oxide in a model prototissue. Nat. Commun. 2022, 13 (1), 5254, DOI: 10.1038/s41467-022-32941-6184https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB38XitlGmu7rJ&md5=bbb2f20a4eb05bec0e3c200942cfd753Signal processing and generation of bioactive nitric oxide in a model prototissueLiu, Songyang; Zhang, Yanwen; He, Xiaoxiao; Li, Mei; Huang, Jin; Yang, Xiaohai; Wang, Kemin; Mann, Stephen; Liu, JianboNature Communications (2022), 13 (1), 5254CODEN: NCAOBW; ISSN:2041-1723. (Nature Portfolio)The design and construction of synthetic prototissues from integrated assemblies of artificial protocells is an important challenge for synthetic biol. and bioengineering. Here we spatially segregate chem. communicating populations of enzyme-decorated phospholipid-enveloped polymer/DNA coacervate protocells in hydrogel modules to construct a tubular prototissue-like vessel capable of modulating the output of bioactive nitric oxide (NO). By decorating the protocells with glucose oxidase, horseradish peroxidase or catalase and arranging different modules concentrically, a glucose/hydroxyurea dual input leads to logic-gate signal processing under reaction-diffusion conditions, which results in a distinct NO output in the internal lumen of the model prototissue. The NO output is exploited to inhibit platelet activation and blood clot formation in samples of plasma and whole blood located in the internal channel of the device, thereby demonstrating proof-of-concept use of the prototissue-like vessel for anticoagulation applications. Our results highlight opportunities for the development of spatially organized synthetic prototissue modules from assemblages of artificial protocells and provide a step towards the organization of biochem. processes in integrated micro-compartmentalized media, micro-reactor technol. and soft functional materials.
- 185Sengupta, S.; Patra, D.; Ortiz-Rivera, I.; Agrawal, A.; Shklyaev, S.; Dey, K. K.; Cordova-Figueroa, U.; Mallouk, T. E.; Sen, A. Self-powered enzyme micropumps. Nat. Chem. 2014, 6 (5), 415– 422, DOI: 10.1038/nchem.1895185https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2cXltFOmsbk%253D&md5=1284c8170f2970206bed7455783c9a08Self-powered enzyme micropumpsSengupta, Samudra; Patra, Debabrata; Ortiz-Rivera, Isamar; Agrawal, Arjun; Shklyaev, Sergey; Dey, Krishna K.; Cordova-Figueroa, Ubaldo; Mallouk, Thomas E.; Sen, AyusmanNature Chemistry (2014), 6 (5), 415-422CODEN: NCAHBB; ISSN:1755-4330. (Nature Publishing Group)Non-mech. nano- and microscale pumps that function without the aid of an external power source and provide precise control over the flow rate in response to specific signals are needed for the development of new autonomous nano- and microscale systems. Here we show that surface-immobilized enzymes that are independent of ATP function as self-powered micropumps in the presence of their resp. substrates. In the four cases studied (catalase, lipase, urease and glucose oxidase), the flow is driven by a gradient in fluid d. generated by the enzymic reaction. The pumping velocity increases with increasing substrate concn. and reaction rate. These rechargeable pumps can be triggered by the presence of specific analytes, which enables the design of enzyme-based devices that act both as sensor and pump. Finally, we show proof-of-concept enzyme-powered devices that autonomously deliver small mols. and proteins in response to specific chem. stimuli, including the release of insulin in response to glucose.
- 186Maiti, S.; Shklyaev, O. E.; Balazs, A. C.; Sen, A. Self-organization of fluids in a multienzymatic pump system. Langmuir 2019, 35 (10), 3724– 3732, DOI: 10.1021/acs.langmuir.8b03607186https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC1MXisFyit7c%253D&md5=0cea9a84182475fa2413607c984bd86dSelf-organization of fluids in a multienzymatic pump systemMaiti, Subhabrata; Shklyaev, Oleg E.; Balazs, Anna C.; Sen, AyusmanLangmuir (2019), 35 (10), 3724-3732CODEN: LANGD5; ISSN:0743-7463. (American Chemical Society)The nascent field of microscale flow chem. focuses on harnessing flowing fluids to optimize chem. reactions in microchambers and establish new routes for chem. synthesis. With enzymes and other catalysts anchored to the surface of microchambers, the catalytic reactions can act as pumps and propel the fluids through the containers. Hence, the flows not only affect the catalytic reactions, but these reactions also affect the flows. Understanding this dynamic interplay is vital to enhancing the accuracy and utility of flow technol. Through expts. and simulation, we design a system of three different enzymes, immobilized in sep. gels, on the surface of a microchamber; with the appropriate reactants in the soln., each enzyme-filled gel acts as a pump. The system also exploits a reaction cascade that controls the temporal interactions between two pumps. With three pumps in a triangular arrangement, the spatio-temporal interactions among the chem. reactions become highly coordinated and produce well-defined fluid streams, which transport chems. and form a fluidic "circuit". The circuit layout and flow direction of each constituent stream can be controlled through the no. and placement of the gels and the types of catalysts localized in the gels. These studies provide a new route for forming self-organizing and bifurcating fluids that can yield fundamental insight into nonequil., dynamical systems. Because the flows and fluidic circuits are generated by internal chem. reactions, the fluids can autonomously transport cargo to specific locations in the device. Hence, the findings also provide guidelines to facilitate further automation of microfluidic devices.
- 187Ortiz-Rivera, I.; Courtney, T. M.; Sen, A. Enzyme micropump-based inhibitor assays. Adv. Funct. Mater. 2016, 26, 2135– 2142, DOI: 10.1002/adfm.201504619187https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC28XivVCls70%253D&md5=8c6b301f04b8ef1825eab2ed3ddc04abEnzyme Micropump-Based Inhibitor AssaysOrtiz-Rivera, Isamar; Courtney, Taylor M.; Sen, AyusmanAdvanced Functional Materials (2016), 26 (13), 2135-2142CODEN: AFMDC6; ISSN:1616-301X. (Wiley-VCH Verlag GmbH & Co. KGaA)Rapid, easy-to-use, and portable devices that can provide a read-out without the need for expensive equipment represent the future of sensing technol., with applications in areas like environmental, food, chem., and biol. safety. Enzymes immobilized on a surface function as micropumps in the presence of species (e.g., substrate, cofactor, or biomarker) that trigger the enzymic reaction. The flow speed in these devices increases with increasing reaction rate. This allows the detection of substances that inhibit the enzymic reaction. Using this principle, sensors for toxic substances, like mercury, cadmium, cyanide, and azide, were designed using urease and catalase-powered pumps, resp., with limits of detection well below the concns. permitted by the Environmental Protection Agency. The study was also extended to other inhibitors for these enzymes. The sensing range of fluid flow-based inhibitor assays depends on the type of inhibition, the enzyme concn. on the sensing platform, and, for competitive inhibition, the concn. of substrate used.
- 188Manna, R. K.; Shklyaev, O. E.; Balazs, A. C. Chemically driven multimodal locomotion of active, flexible sheets. Langmuir 2023, 39 (2), 780– 789, DOI: 10.1021/acs.langmuir.2c02666188https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB3sXjtlSltg%253D%253D&md5=8634c838911bc4433cc43d24ff40b905Chemically Driven Multimodal Locomotion of Active, Flexible SheetsManna, Raj Kumar; Shklyaev, Oleg E.; Balazs, Anna C.Langmuir (2023), 39 (2), 780-789CODEN: LANGD5; ISSN:0743-7463. (American Chemical Society)The inhibitor-promoter feedback loop is a vital component in regulatory pathways that controls functionality in living systems. In this loop, the prodn. of chem. A at one site promotes the prodn. of chem. B at another site, but B inhibits the prodn. of A. In soln., differences in the vols. of the reactants and products of this reaction can generate buoyancy-driven fluid flows, which will deform neighboring soft material. To probe the intrinsic interrelationship among chem., hydrodynamics, and fluid-structure interactions, we model a bio-inspired system where a flexible sheet immersed in soln. encompasses two spatially sepd. catalytic patches, which drive the A-B inhibitor-promotor reaction. The convective rolls of fluid generated above the patches can circulate inward or outward depending on the chem. environment. Within the regime displaying chem. oscillations, the dynamic fluid-structure interactions morph the shape of the sheet to periodically "fly", "crawl", or "swim" along the bottom of the confining chamber, revealing an intimate coupling between form and function in this system. The oscillations in the sheet's motion in turn affect the chem. oscillations in the soln. In the regime with non-oscillatory chem., the induced flow still morphs the shape of the sheet, but now, the fluid simply translates the sheet along the length of the chamber. The findings reveal the potential for enzymic reactions in the body to generate hydrodynamic behavior that modifies the shape of neighboring soft tissue, which in turn modifies both the fluid dynamics and the enzymic reaction. The findings indicate that this non-linear dynamic behavior can be playing a crit. role in the functioning of regulatory pathways in living systems.
- 189Simo, C.; Serra-Casablancas, M.; Hortelao, A. C.; Di Carlo, V.; Guallar-Garrido, S.; Plaza-Garcia, S.; Rabanal, R. M.; Ramos-Cabrer, P.; Yague, B.; Aguado, L.; Bardia, L.; Tosi, S.; Gomez-Vallejo, V.; Martin, A.; Patino, T.; Julian, E.; Colombelli, J.; Llop, J.; Sanchez, S. Urease-powered nanobots for radionuclide bladder cancer therapy. Nat. Nanotechnol. 2024, DOI: 10.1038/s41565-023-01577-y .There is no corresponding record for this reference.
- 190Rollin, J. A.; Tam, T. K.; Zhang, Y. H. P. New biotechnology paradigm: cell-free biosystems for biomanufacturing. Green Chem. 2013, 15 (7), 1708– 1719, DOI: 10.1039/c3gc40625c190https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3sXpvVejtrg%253D&md5=36c724ad829eccb7f6b178b2949dad90New biotechnology paradigm: cell-free biosystems for biomanufacturingRollin, Joseph A.; Tam, Tsz Kin; Zhang, Y.-H. PercivalGreen Chemistry (2013), 15 (7), 1708-1719CODEN: GRCHFJ; ISSN:1463-9262. (Royal Society of Chemistry)A review. Cost-efficient prodn. of sustainably-derived biochems. and biofuels is a crit. goal of modern biotechnol. The current predominant biotransformation method is mainly based on microbial fermn., but this system suffers from a mismatch of engineering and cellular objectives, appropriates a significant fraction of substrate/energy sources for self-replication, and poses significant scaling challenges. Cell-free biosystems for biomanufg. (CFB2)-complex, cell-free systems that catalyze the conversion of renewable substrates to a variety of products-are emerging as an alternative to fermn. Within this burgeoning field, a new application is the prodn. of low-value and high-impact biocommodities by CFB2. In this subset, synthetic enzymic networks are capable of producing numerous desired biocommodities, with the advantages of higher product yields, faster reaction rates, and reduced interference from toxic compds., among others. In this review, CFB2, with an emphasis on biocommodities prodn., is compared with microbial fermn.; current applications are presented, illustrating some of the advantages of the system; and remaining challenges are discussed with the path forward for each.
- 191Zhang, Y.; Hess, H. Toward rational design of high-efficiency enzyme cascades. ACS Catal. 2017, 7 (9), 6018– 6027, DOI: 10.1021/acscatal.7b01766191https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2sXht1eks7zI&md5=ee1f1ce70682bace08e88f77ec132b37Toward Rational Design of High-efficiency Enzyme CascadesZhang, Yifei; Hess, HenryACS Catalysis (2017), 7 (9), 6018-6027CODEN: ACCACS; ISSN:2155-5435. (American Chemical Society)A review. The construction of an enzyme cascade with enhanced activity is desirable in biocatalysis, synthetic biol. and other fields. Although many researchers have found that immobilization of enzyme cascades on scaffolds leads to an enhanced activity, the underlying mechanisms still remain controversial. In this Viewpoint, we describe and discuss the frequently used strategies to achieve activity enhancement. We first reiterate that the proximity does not contribute to the increased overall activity of the sequential nor coenzyme regenerating cascade, and suggest that the reported obsd. enhancements in the majority of publications were caused by the influence of the scaffolds. Then we discuss the benefits and limitations of other strategies including bridging the enzymes, co-immobilization, compartmentalization. Finally, we highlight that balancing the stoichiometry, improving the individual activity, overlapping the operating temp. and pH max. are necessary for achieving a high-efficiency enzyme cascade.
- 192Shi, J.; Wu, Y.; Zhang, S.; Tian, Y.; Yang, D.; Jiang, Z. Bioinspired construction of multi-enzyme catalytic systems. Chem. Soc. Rev. 2018, 47, 4295– 4313, DOI: 10.1039/C7CS00914C192https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC1cXptValtbo%253D&md5=c3794d904054aee96bf89e4e2c11392eBioinspired construction of multi-enzyme catalytic systemsShi, Jiafu; Wu, Yizhou; Zhang, Shaohua; Tian, Yu; Yang, Dong; Jiang, ZhongyiChemical Society Reviews (2018), 47 (12), 4295-4313CODEN: CSRVBR; ISSN:0306-0012. (Royal Society of Chemistry)A review. Enzyme catalysis, as a green, efficient process, displays exceptional functionality, adaptivity and sustainability. Multi-enzyme catalysis, which can accomplish the tandem synthesis of valuable materials/chems. from renewable feedstocks, establishes a bridge between single-enzyme catalysis and whole-cell catalysis. Multi-enzyme catalysis occupies a unique and indispensable position in the realm of biol. reactions for energy and environmental applications. Two complementary strategies, i.e., compartmentalization and substrate channeling, have been evolved by living organisms for implementing the complex in vivo multi-enzyme reactions (MERs), which have been applied to construct multi-enzyme catalytic systems (MECSs) with superior catalytic activity and stabilities in practical biocatalysis. This tutorial review aims to present the recent advances and future prospects in this burgeoning research area, stressing the features and applications of the two strategies for constructing MECSs and implementing in vitro MERs. The concluding remarks are presented with a perspective on the construction of MECSs through rational combination of compartmentalization and substrate channeling.
- 193Wang, Z.; Sundara Sekar, B.; Li, Z. Recent advances in artificial enzyme cascades for the production of value-added chemicals. Bioresour. Technol. 2021, 323, 124551, DOI: 10.1016/j.biortech.2020.124551193https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB3cXis12iu7zI&md5=c362ef01381c078529cd552465831127Recent advances in artificial enzyme cascades for the production of value-added chemicalsWang, Zilong; Sundara Sekar, Balaji; Li, ZhiBioresource Technology (2021), 323 (), 124551CODEN: BIRTEB; ISSN:0960-8524. (Elsevier Ltd.)A review. Enzyme cascades are efficient tools to perform multi-step synthesis in one-pot in a green and sustainable manner, enabling non-natural synthesis of valuable chems. from easily available substrates by artificially combining two or more enzymes. Bioprodn. of many high-value chems. such as chiral and highly functionalised mols. have been achieved by developing new enzyme cascades. This review summarizes recent advances on engineering and application of enzyme cascades to produce high-value chems. (alcs., aldehydes, ketones, amines, carboxylic acids, etc) from simple starting materials. While 2-step enzyme cascades are developed for versatile enantioselective synthesis, multi-step enzyme cascades are engineered to functionalise basic chems., such as styrenes, cyclic alkanes, and arom. compds. New cascade reactions have also been developed for producing valuable chems. from bio-based substrates, such as L-phenylalanine, and renewable feedstocks such as glucose and glycerol. The challenges in current process and future outlooks in the development of enzyme cascades are also addressed.
- 194Mordhorst, S.; Andexer, J. N. Round, round we go – strategies for enzymatic cofactor regeneration. Nat. Prod. Rep. 2020, 37, 1316– 1333, DOI: 10.1039/D0NP00004C194https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB3cXht1CksrrI&md5=17a3b901eeb97ba98e33b9e27dae7899Round, round we go - strategies for enzymatic cofactor regenerationMordhorst, Silja; Andexer, Jennifer N.Natural Product Reports (2020), 37 (10), 1316-1333CODEN: NPRRDF; ISSN:0265-0568. (Royal Society of Chemistry)Covering: up to the beginning of 2020Enzymes depending on cofactors are essential in many biosynthetic pathways of natural products. They are often involved in key steps: catalytic conversions that are difficult to achieve purely with synthetic org. chem. Hence, cofactor-dependent enzymes have great potential for biocatalysis, on the condition that a corresponding cofactor regeneration system is available. For some cofactors, these regeneration systems require multiple steps; such complex enzyme cascades/multi-enzyme systems are (still) challenging for in vitro biocatalysis. Further, artificial cofactor analogs have been synthesized that are more stable, show an altered reaction range, or act as inhibitors. The development of bio-orthogonal systems that can be used for the prodn. of modified natural products in vivo is an ongoing challenge. In light of the recent progress in this field, this review aims to provide an overview of general strategies involving enzyme cofactors, cofactor analogs, and regeneration systems; highlighting the current possibilities for application of enzymes using some of the most common cofactors.
- 195Bachosz, K.; Zdarta, J.; Bilal, M.; Meyer, A. S.; Jesionowski, T. Enzymatic cofactor regeneration systems: A new perspective on efficiency assessment. Sci. Total Environ. 2023, 868, 161630, DOI: 10.1016/j.scitotenv.2023.161630195https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB3sXhs1Onur0%253D&md5=3f105ac482e65f0bb8daa60544c860dcEnzymatic cofactor regeneration systems: A new perspective on efficiency assessmentBachosz, Karolina; Zdarta, Jakub; Bilal, Muhammad; Meyer, Anne S.; Jesionowski, TeofilScience of the Total Environment (2023), 868 (), 161630CODEN: STENDL; ISSN:0048-9697. (Elsevier B.V.)A review. Nowadays, the specificity of enzymic processes makes them more and more important every year, and their usage on an industrial scale seems to be necessary. Enzymic cofactors, however, play a crucial part in the prospective applications of enzymes, because they are indispensable for conducting highly effective biocatalytic activities. Due to the relatively high cost of these compds. and their consumption during the processes carried out, it has become crucial to develop systems for cofactor regeneration. Therefore, in this review, an attempt was made to summarize current knowledge on enzymic regeneration methods, which are characterized by high specificity, non-toxicity and reported to be highly efficient. The regeneration of cofactors, such as nicotinamide dinucleotides, CoA, ATP and flavin nucleotides, which are necessary for the proper functioning of a large no. of enzymes, is discussed, as well as potential directions for further development of these systems are highlighted. This review discusses a range of highly effective cofactor regeneration systems along with the productive synthesis of many useful chems., including the simultaneous renewal of several cofactors at the same time. Addnl., the impact of the enzyme immobilization process on improving the stability and the potential for multiple uses of the developed cofactor regeneration systems was also presented. Moreover, an attempt was made to emphasize the importance of the presented research, as well as the identification of research gaps, which mainly result from the lack of available literature on this topic.
- 196Monck, C.; Elani, Y.; Ceroni, F. Cell-free protein synthesis: biomedical applications and future perspectives. Chem. Eng. Res. Des. 2022, 177, 653– 658, DOI: 10.1016/j.cherd.2021.11.025196https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB3MXisF2gurnN&md5=6ffac1e510aba2ea50c266eaa681a4f5Cell-free protein synthesis: biomedical applications and future perspectivesMonck, Carolina; Elani, Yuval; Ceroni, FrancescaChemical Engineering Research and Design (2022), 177 (), 653-658CODEN: CERDEE; ISSN:1744-3563. (Elsevier B.V.)A review. The use of cell-free protein synthesis (CFPS) has become increasingly widespread in synthetic biol. over recent years, providing an effective platform for the study and engineering of cellular processes. The versatility and portability of CFPS systems have also boosted their potential for usage outside of the lab. in a wide no. of applications, from construct prototyping to bioprodn. CFPS is particularly well suited to biomedical applications, such as the prodn. of clin. mols. and vaccines. It can also be integrated with addnl. technologies such as microfluidics and liposomal encapsulation to provide a new route for on-demand therapeutic expression. In this we outline the key features of CFPS that make it a powerful platform for biomedical applications. We also discuss existing limitations with respect to the use of CFPS in the prodn. of complex protein products and the limited prodn. capacity of current systems. Addressing these will be integral in expanding the application of CFPS in biotherapy.
- 197Perez, J. G.; Stark, J. C.; Jewett, M. C. Cell-free synthetic biology: engineering beyond the cell. Cold Spring Harb. Perspect. Biol. 2016, 8 (12), a023853, DOI: 10.1101/cshperspect.a023853197https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC1cXhtFKms7bO&md5=7f222c69d86efff730fdafaaa5941469Cell-free synthetic biology: engineering beyond the cellPerez, Jessica G.; Stark, Jessica C.; Jewett, Michael C.Cold Spring Harbor Perspectives in Biology (2016), 8 (12), a023853/1-a023853/26CODEN: CSHPEU; ISSN:1943-0264. (Cold Spring Harbor Laboratory Press)Cell-free protein synthesis (CFPS) technologies have enabled inexpensive and rapid recombinant protein expression. Numerous highly active CFPS platforms are now available and have recently been used for synthetic biol. applications. In this review, we focus on the ability of CFPS to expand our understanding of biol. systems and its applications in the synthetic biol. field. First, we outline a variety of CFPS platforms that provide alternative and complementary methods for expressing proteins from different organisms, compared with in vivo approaches. Next, we review the types of proteins, protein complexes, and protein modifications that have been achieved using CFPS systems. Finally, we introduce recent work on genetic networks in cell-free systems and the use of cell-free systems for rapid prototyping of in vivo networks. Given the flexibility of cell-free systems, CFPS holds promise to be a powerful tool for synthetic biol. as well as a protein prodn. technol. in years to come.
- 198Silverman, A. D.; Karim, A. S.; Jewett, M. C. Cell-free gene expression: an expanded repertoire of applications. Nat. Rev. Genet. 2020, 21, 151– 170, DOI: 10.1038/s41576-019-0186-3198https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC1MXit1OitbbO&md5=e227ddcf47b0b961b2b423d414fa3795Cell-free gene expression: an expanded repertoire of applicationsSilverman, Adam D.; Karim, Ashty S.; Jewett, Michael C.Nature Reviews Genetics (2020), 21 (3), 151-170CODEN: NRGAAM; ISSN:1471-0056. (Nature Research)Cell-free biol. is the activation of biol. processes without the use of intact living cells. It has been used for more than 50 years across the life sciences as a foundational research tool, but a recent tech. renaissance has facilitated high-yielding (grams of protein per L), cell-free gene expression systems from model bacteria, the development of cell-free platforms from non-model organisms and multiplexed strategies for rapidly assessing biol. design. These advances provide exciting opportunities to profoundly transform synthetic biol. by enabling new approaches to the model-driven design of synthetic gene networks, the fast and portable sensing of compds., on-demand biomanufg., building cells from the bottom up, and next-generation educational kits.
- 199Korman, T. P.; Sahachartsiri, B.; Li, D.; Vinokur, J. M.; Eisenberg, D.; Bowie, J. U. A synthetic biochemistry system for the in vitro production of isoprene from glycolysis intermediates. Protein Sci. 2014, 23 (5), 576– 585, DOI: 10.1002/pro.2436199https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2cXmsFWls7o%253D&md5=1c21b2170ee9c47872e24879a980623eA synthetic biochemistry system for the in vitro production of isoprene from glycolysis intermediatesKorman, Tyler P.; Sahachartsiri, Bobby; Li, Dan; Vinokur, Jeffrey M.; Eisenberg, David; Bowie, James U.Protein Science (2014), 23 (5), 576-585CODEN: PRCIEI; ISSN:1469-896X. (Wiley-Blackwell)The high yields required for the economical prodn. of chems. and fuels using microbes can be difficult to achieve due to the complexities of cellular metab. An alternative to performing biochem. transformations in microbes is to build biochem. pathways in vitro, an approach we call synthetic biochem. Here we test whether the full mevalonate pathway can be reconstituted in vitro and used to produce the commodity chem. isoprene. We construct an in vitro synthetic biochem. pathway that uses the carbon and ATP produced from the glycolysis intermediate phosphoenolpyruvate to run the mevalonate pathway. The system involves 12 enzymes to perform the complex transformation, while providing and balancing the ATP, NADPH, and acetyl-CoA cofactors. The optimized system produces isoprene from phosphoenolpyruvate in ∼100% molar yield. Thus, by inserting the isoprene pathway into previously developed glycolysis modules it may be possible to produce isoprene and other acetyl-CoA derived isoprenoids from glucose in vitro.
- 200Korman, T. P.; Opgenorth, P. H.; Bowie, J. U. A synthetic biochemistry platform for cell free production of monoterpenes from glucose. Nat. Commun. 2017, 8, 15526, DOI: 10.1038/ncomms15526200https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2sXot12gs78%253D&md5=d792e214239beb42398f0d503db94bfeA synthetic biochemistry platform for cell free production of monoterpenes from glucoseKorman, Tyler P.; Opgenorth, Paul H.; Bowie, James U.Nature Communications (2017), 8 (), 15526CODEN: NCAOBW; ISSN:2041-1723. (Nature Publishing Group)Cell-free systems designed to perform complex chem. conversions of biomass to biofuels or commodity chems. are emerging as promising alternatives to the metabolic engineering of living cells. Here we design a system comprises 27 enzymes for the conversion of glucose into monoterpenes that generates both NAD(P)H and ATP in a modified glucose breakdown module and utilizes both cofactors for building terpenes. Different monoterpenes are produced in our system by changing the terpene synthase enzyme. The system is stable for the prodn. of limonene, pinene and sabinene, and can operate continuously for at least 5 days from a single addn. of glucose. We obtain conversion yields >95% and titers >15 g l-1. The titers are an order of magnitude over cellular toxicity limits and thus difficult to achieve using cell-based systems. Overall, these results highlight the potential of synthetic biochem. approaches for producing bio-based chems.
- 201Opgenorth, P. H.; Korman, T. P.; Bowie, J. U. A synthetic biochemistry molecular purge valve module that maintains redox balance. Nat. Commun. 2014, 5, 4113, DOI: 10.1038/ncomms5113201https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2cXitVShsb%252FE&md5=030c8d73c345febb8aebb33914fc80b4A synthetic biochemistry molecular purge valve module that maintains redox balanceOpgenorth, Paul H.; Korman, Tyler P.; Bowie, James U.Nature Communications (2014), 5 (), 4113CODEN: NCAOBW; ISSN:2041-1723. (Nature Publishing Group)The greatest potential environmental benefit of metabolic engineering would be the prodn. of high-vol. commodity chems., such as biofuels. Yet, the high yields required for the economic viability of low-value chems. is particularly hard to achieve in microbes owing to the myriad competing biochem. pathways. An alternative approach, which we call synthetic biochem., is to eliminate the organism by constructing biochem. pathways in vitro. Viable synthetic biochem., however, will require simple methods to replace the cellular circuitry that maintains cofactor balance. Here we design a simple purge valve module for maintaining NADP+/NADPH balance. We test the purge valve in the prodn. of polyhydroxybutyryl bioplastic and isoprene-pathways where cofactor generation and utilization are unbalanced. We find that the regulatory system is highly robust to variations in cofactor levels and readily transportable. The mol. purge valve provides a step towards developing continuously operating, sustainable synthetic biochem. systems.
- 202Opgenorth, P. H.; Korman, T. P.; Bowie, J. U. A synthetic biochemistry module for production of bio-based chemicals from glucose. Nat. Chem. Biol. 2016, 12 (6), 393– 395, DOI: 10.1038/nchembio.2062202https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC28XlsFOhsL8%253D&md5=813355b68b089d81528532dc2f004b91A synthetic biochemistry module for production of bio-based chemicals from glucoseOpgenorth, Paul H.; Korman, Tyler P.; Bowie, James U.Nature Chemical Biology (2016), 12 (6), 393-395CODEN: NCBABT; ISSN:1552-4450. (Nature Publishing Group)Synthetic biochem., the cell-free prodn. of biol. based chems., is a potentially high-yield, flexible alternative to in vivo metabolic engineering. To limit costs, cell-free systems must be designed to operate continuously with minimal addn. of feedstock chems. We describe a robust, efficient synthetic glucose breakdown pathway and implement it for the prodn. of bioplastic. The system's performance suggests that synthetic biochem. has the potential to become a viable industrial alternative.
- 203Valliere, M. A.; Korman, T. P.; Woodall, N. B.; Khitrov, G. A.; Taylor, R. E.; Baker, D.; Bowie, J. U. A cell-free platform for the prenylation of natural products and application to cannabinoid production. Nat. Commun. 2019, 10 (1), 565, DOI: 10.1038/s41467-019-08448-y203https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC1MXntlKktL8%253D&md5=e43f4406f322ca8307256c80a4c961cdA cell-free platform for the prenylation of natural products and application to cannabinoid productionValliere, Meaghan A.; Korman, Tyler P.; Woodall, Nicholas B.; Khitrov, Gregory A.; Taylor, Robert E.; Baker, David; Bowie, James U.Nature Communications (2019), 10 (1), 565CODEN: NCAOBW; ISSN:2041-1723. (Nature Research)Prenylation of natural compds. adds structural diversity, alters biol. activity, and enhances therapeutic potential. Because prenylated compds. often have a low natural abundance, alternative prodn. methods are needed. Metabolic engineering enables natural product biosynthesis from inexpensive biomass, but is limited by the complexity of secondary metabolite pathways, intermediate and product toxicities, and substrate accessibility. Alternatively, enzyme catalyzed prenyl transfer provides excellent regio- and stereo-specificity, but requires expensive isoprenyl pyrophosphate substrates. Here we develop a flexible cell-free enzymic prenylating system that generates isoprenyl pyrophosphate substrates from glucose to prenylate an array of natural products. The system provides an efficient route to cannabinoid precursors cannabigerolic acid (CBGA) and cannabigerovarinic acid (CBGVA) at >1 g/L, and a single enzymic step converts the precursors into cannabidiolic acid (CBDA) and cannabidivarinic acid (CBDVA). Cell-free methods may provide a powerful alternative to metabolic engineering for chems. that are hard to produce in living organisms.
- 204Sarria, S.; Wong, B.; Martin, H. G.; Keasling, J. D.; Peralta-Yahya, P. Microbial synthesis of pinene. ACS Synth. Biol. 2014, 3 (7), 466– 475, DOI: 10.1021/sb4001382204https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2cXjt1ymsro%253D&md5=b83fc0bf6e20fdedef4cb039eb49ed86Microbial Synthesis of PineneSarria, Stephen; Wong, Betty; Martin, Hector Garcia; Keasling, Jay D.; Peralta-Yahya, PamelaACS Synthetic Biology (2014), 3 (7), 466-475CODEN: ASBCD6; ISSN:2161-5063. (American Chemical Society)The volumetric heating values of today's biofuels are too low to power energy-intensive aircraft, rockets, and missiles. Recently, pinene dimers were shown to have a volumetric heating value similar to that of the tactical fuel JP-10. To provide a sustainable source of pinene, we engineered Escherichia coli for pinene prodn. We combinatorially expressed three pinene synthases (PS) and three geranyl diphosphate synthases (GPPS), with the best combination achieving ∼28 mg/L of pinene. We speculated that pinene toxicity was limiting prodn.; however, toxicity should not be limiting at current titers. Because GPPS is inhibited by geranyl diphosphate (GPP) and to increase flux through the pathway, we combinatorially constructed GPPS-PS protein fusions. The Abies grandis GPPS-PS fusion produced 32 mg/L of pinene, a 6-fold improvement over the highest titer previously reported in engineered E. coli. Finally, we investigated the pinene isomer ratio of our pinene-producing microbe and discovered that the isomer profile is detd. not only by the identity of the PS used but also by the identity of the GPPS with which the PS is paired. We demonstrated that the GPP concn. available to PS for cyclization alters the pinene isomer ratio.
- 205Karim, A. S.; Jewett, M. C. A cell-free framework for rapid biosynthetic pathway prototyping and enzyme discovery. Metab. Eng. 2016, 36, 116– 126, DOI: 10.1016/j.ymben.2016.03.002205https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC28Xls1KrsL8%253D&md5=6d833a0a1996ede9015197d1b48d5793A cell-free framework for rapid biosynthetic pathway prototyping and enzyme discoveryKarim, Ashty S.; Jewett, Michael C.Metabolic Engineering (2016), 36 (), 116-126CODEN: MEENFM; ISSN:1096-7176. (Elsevier B. V.)Speeding up design-build-test (DBT) cycles is a fundamental challenge facing biochem. engineering. To address this challenge, we report a new cell-free protein synthesis driven metabolic engineering (CFPS-ME) framework for rapid biosynthetic pathway prototyping. In our framework, cell-free cocktails for synthesizing target small mols. are assembled in a mix-and-match fashion from crude cell lysates either contg. selectively enriched pathway enzymes from heterologous overexpression or directly producing pathway enzymes in lysates by CFPS. As a model, we apply our approach to n-butanol biosynthesis showing that Escherichia coli lysates support a highly active 17-step CoA-dependent n-butanol pathway in vitro. The elevated degree of flexibility in the cell-free environment allows us to manipulate physiochem. conditions, access enzymic nodes, discover new enzymes, and prototype enzyme sets with linear DNA templates to study pathway performance. We anticipate that CFPS-ME will facilitate efforts to define, manipulate, and understand metabolic pathways for accelerated DBT cycles without the need to reengineer organisms.
- 206Dudley, Q. M.; Anderson, K. C.; Jewett, M. C. Cell-free mixing of Escherichia coli crude extracts to prototype and rationally engineer high-titer mevalonate synthesis. ACS Synth. Biol. 2016, 5 (12), 1578– 1588, DOI: 10.1021/acssynbio.6b00154206https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC28Xht1GmtrbK&md5=c45307dfb3c6873a53e0711d454aba58Cell-Free Mixing of Escherichia coli Crude Extracts to Prototype and Rationally Engineer High-Titer Mevalonate SynthesisDudley, Quentin M.; Anderson, Kim C.; Jewett, Michael C.ACS Synthetic Biology (2016), 5 (12), 1578-1588CODEN: ASBCD6; ISSN:2161-5063. (American Chemical Society)Cell-free metabolic engineering (CFME) is advancing a powerful paradigm for accelerating the design and synthesis of biosynthetic pathways. However, as most cell-free biomol. synthesis systems to date use purified enzymes, energy and cofactor balance can be limiting. To address this challenge, we report a new CFME framework for building biosynthetic pathways by mixing multiple crude lysates, or exts. In our modular approach, cell-free lysates, each selectively enriched with an overexpressed enzyme, are generated in parallel and then combinatorically mixed to construct a full biosynthetic pathway. Endogenous enzymes in the cell-free ext. fuel high-level energy and cofactor regeneration. As a model, we apply our framework to synthesize mevalonate, an intermediate in isoprenoid synthesis. We use our approach to rapidly screen enzyme variants, optimize enzyme ratios, and explore cofactor landscapes for improving pathway performance. Further, we show that genomic deletions in the source strain redirect metabolic flux in resultant lysates. In an optimized system, mevalonate was synthesized at 17.6 g·L-1 (119 mM) over 20 h, resulting in a volumetric productivity of 0.88 g·L-1·hr-1. We also demonstrate that this system can be lyophilized and retain biosynthesis capability. Our system catalyzes ∼1250 turnover events for the cofactor NAD+ and demonstrates the ability to rapidly prototype and debug enzymic pathways in vitro for compelling metabolic engineering and synthetic biol. applications.
- 207Casini, A.; Chang, F. Y.; Eluere, R.; King, A. M.; Young, E. M.; Dudley, Q. M.; Karim, A.; Pratt, K.; Bristol, C.; Forget, A. A pressure test to make 10 molecules in 90 days: external evaluation of methods to engineer biology. J. Am. Chem. Soc. 2018, 140 (12), 4302– 4316, DOI: 10.1021/jacs.7b13292207https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC1cXjt1yls78%253D&md5=9b0d5effbfa11528fb82894e7f59b5b3A Pressure Test to Make 10 Molecules in 90 Days: External Evaluation of Methods to Engineer BiologyCasini, Arturo; Chang, Fang-Yuan; Eluere, Raissa; King, Andrew M.; Young, Eric M.; Dudley, Quentin M.; Karim, Ashty; Pratt, Katelin; Bristol, Cassandra; Forget, Anthony; Ghodasara, Amar; Warden-Rothman, Robert; Gan, Rui; Cristofaro, Alexander; Borujeni, Amin Espah; Ryu, Min-Hyung; Li, Jian; Kwon, Yong-Chan; Wang, He; Tatsis, Evangelos; Rodriguez-Lopez, Carlos; O'Connor, Sarah; Mdema, Marnix H.; Fischbach, Michael A.; Jewett, Michael C.; Voigt, Christopher; Gordon, D. BenjaminJournal of the American Chemical Society (2018), 140 (12), 4302-4316CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)Centralized facilities for genetic engineering, or biofoundries, offer the potential to design organisms to address emerging needs in medicine, agriculture, industry, and defense. The field has seen rapid advances in technol., but it is difficult to gauge current capabilities or identify gaps across projects. To this end, our foundry was assessed via a timed "pressure test," in which 3 mo were given to build organisms to produce 10 mols. unknown to us in advance. By applying a diversity of new approaches, we produced the desired mol. or a closely related one for 6/10 targets during the performance period, and made advances toward prodn. of the others as well. Specifically, we increased the titers of 1-hexadecanol, pyrrolnitrin, and pacidamycin D, found novel routes to the enediyne warhead underlying powerful antimicrobials, established a cell free system for monoterpene prodn., produced an intermediate toward vincristine biosynthesis, and encoded 7,802 individually retrievable pathways to 540 bisindoles in a DNA pool. Pathways to THF and barbamide were designed and constructed but toxicity or anal. tools inhibited further progress. In sum, we constructed 1.2Mb DNA, built 215 strains spanning 5 species (Saccharomyces cerevisiae, Escherichia coli, Streptomyces albidoflavus, Streptomyces coelicolor, and Streptomyces albovinaceus), established two cell free systems, and performed 690 assays developed inhouse for the mols.
- 208Dudley, Q. M.; Nash, C. J.; Jewett, M. C. Cell-free biosynthesis of limonene using enzyme-enriched Escherichia coli lysates. Synthetic Biology 2019, 4 (1), ysz003, DOI: 10.1093/synbio/ysz003There is no corresponding record for this reference.
- 209Nielsen, D. U.; Hu, X.-M.; Daasbjerg, K.; Skrydstrup, T. Chemically and electrochemically catalysed conversion of CO2 to CO with follow-up utilization to value-added chemicals. Nat. Catal. 2018, 1 (4), 244– 254, DOI: 10.1038/s41929-018-0051-3209https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC1MXpvVyrtbc%253D&md5=beeabaa45d07b5cfb0d698e643c88949Chemically and electrochemically catalysed conversion of CO2 to CO with follow-up utilization to value-added chemicalsNielsen, Dennis U.; Hu, Xin-Ming; Daasbjerg, Kim; Skrydstrup, TroelsNature Catalysis (2018), 1 (4), 244-254CODEN: NCAACP; ISSN:2520-1158. (Nature Research)A review. Carbon dioxide is ubiquitous and a vital mol. for maintaining life on our planet. However, the ever-increasing emission of anthropogenic CO2 into our atm. has provoked dramatic climate changes. In principle, CO2 could represent an important one-carbon building block for the chem. industry, yet its high thermodn. and kinetic stability has limited its applicability to only a handful of industrial applications. On the other hand, carbon monoxide represents a more versatile reagent applied in many industrial transformations. Here we review the different methods for converting CO2 to CO with specific focus on the reverse water gas shift reaction, main element reductants, and electrochem. protocols applying homogeneous and heterogeneous catalysts. Particular emphasis is given to synthetic methods that couple the deoxygenation step with a follow-up carbonylation step for the synthesis of carbonyl-contg. mols., thus avoiding the need to handle or store this toxic but highly synthetically useful diat. gas.
- 210Long, S. P.; Ainsworth, E. A.; Rogers, A.; Ort, D. R. Rising atmospheric carbon dioxide: plants FACE the future. Annu. Rev. Plant Biol. 2004, 55, 591– 628, DOI: 10.1146/annurev.arplant.55.031903.141610210https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD2cXlvFeisb8%253D&md5=b69cfb74635972e8d2139f7d53022c20Rising atmospheric carbon dioxide: Plants FACE the futureLong, Stephen P.; Ainsworth, Elizabeth A.; Rogers, Alistair; Ort, Donald R.Annual Review of Plant Biology (2004), 55 (), 591-628, 3 plates C1-C3CODEN: ARPBDW ISSN:. (Annual Reviews Inc.)A review. Atm. CO2 concn. ([CO2]) is now higher than it was at any time in the past 26 million years and is expected to nearly double during this century. Terrestrial plants with the C3 photosynthetic pathway respond in the short term to increased [CO2] via increased net photosynthesis and decreased transpiration. In the longer term, this increase is often offset by downregulation of photosynthetic capacity. But much of what is currently known about plant responses to elevated [CO2] comes from enclosure studies, where the responses of plants may be modified by size constraints and the limited life-cycle stages that are examd. Free-Air CO2 Enrichment (FACE) was developed as a means to grow plants in the field at controlled elevation of CO2 under fully open-air field conditions. The findings of FACE expts. are quant. summarized via meta-analytic statistics and compared to findings from chamber studies. Although trends agree with parallel summaries of enclosure studies, important quant. differences emerge that have important implications both for predicting the future terrestrial biosphere and understanding how crops may need to be adapted to the changed and changing atm.
- 211Spreitzer, R. J.; Salvucci, M. E. Rubisco: structure, regulatory interactions, and possibilities for a better enzyme. Annu. Rev. Plant Biol. 2002, 53, 449– 475, DOI: 10.1146/annurev.arplant.53.100301.135233211https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD38XlsVWhurk%253D&md5=1dd37bb9e88ffe399e5a2458aa966401Rubisco: Structure, regulatory interactions, and possibilities for a better enzymeSpreitzer, Robert J.; Salvucci, Michael E.Annual Review of Plant Biology (2002), 53 (), 449-475CODEN: ARPBDW ISSN:. (Annual Reviews Inc.)A review with 159 refs. Ribulose 1,5-bisphosphate (RuBP) carboxylase/oxygenase (Rubisco) catalyzes the 1st step in net photosynthetic CO2 assimilation and photorespiratory C oxidn. The enzyme is notoriously inefficient as a catalyst for the carboxylation of RuBP and is subject to competitive inhibition by O2, inactivation by loss of carbamylation, and dead-end inhibition by RuBP. These inadequacies make Rubisco rate-limiting for photosynthesis and an obvious target for increasing agricultural productivity. The resoln. of x-ray crystal structures and detailed anal. of divergent, mutant, and hybrid enzymes have increased insight into the structure-function relations of Rubisco. The interactions and assocns. relatively far from the Rubisco active site, including regulatory interactions with Rubisco activase, may present new approaches and strategies for understanding and ultimately improving this complex enzyme.
- 212Könneke, M.; Schubert, D. M.; Brown, P. C.; Hugler, M.; Standfest, S.; Schwander, T.; Schada von Borzyskowski, L.; Erb, T. J.; Stahl, D. A.; Berg, I. A. Ammonia-oxidizing archaea use the most energy-efficient aerobic pathway for CO2 fixation. Proc. Natl. Acad. Sci. U. S. A. 2014, 111 (22), 8239– 8244, DOI: 10.1073/pnas.1402028111212https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A280%3ADC%252BC2cjktFylsA%253D%253D&md5=3fe1a4dedd0699d28106d30cd201239aAmmonia-oxidizing archaea use the most energy-efficient aerobic pathway for CO2 fixationKonneke Martin; Schubert Daniel M; Brown Philip C; Berg Ivan A; Hugler Michael; Standfest Sonja; Schwander Thomas; Schada von Borzyskowski Lennart; Erb Tobias J; Stahl David AProceedings of the National Academy of Sciences of the United States of America (2014), 111 (22), 8239-44 ISSN:.Archaea of the phylum Thaumarchaeota are among the most abundant prokaryotes on Earth and are widely distributed in marine, terrestrial, and geothermal environments. All studied Thaumarchaeota couple the oxidation of ammonia at extremely low concentrations with carbon fixation. As the predominant nitrifiers in the ocean and in various soils, ammonia-oxidizing archaea contribute significantly to the global nitrogen and carbon cycles. Here we provide biochemical evidence that thaumarchaeal ammonia oxidizers assimilate inorganic carbon via a modified version of the autotrophic hydroxypropionate/hydroxybutyrate cycle of Crenarchaeota that is far more energy efficient than any other aerobic autotrophic pathway. The identified genes of this cycle were found in the genomes of all sequenced representatives of the phylum Thaumarchaeota, indicating the environmental significance of this efficient CO2-fixation pathway. Comparative phylogenetic analysis of proteins of this pathway suggests that the hydroxypropionate/hydroxybutyrate cycle emerged independently in Crenarchaeota and Thaumarchaeota, thus supporting the hypothesis of an early evolutionary separation of both archaeal phyla. We conclude that high efficiency of anabolism exemplified by this autotrophic cycle perfectly suits the lifestyle of ammonia-oxidizing archaea, which thrive at a constantly low energy supply, thus offering a biochemical explanation for their ecological success in nutrient-limited environments.
- 213Scheffen, M.; Marchal, D. G.; Beneyton, T.; Schuller, S. K.; Klose, M.; Diehl, C.; Lehmann, J.; Pfister, P.; Carrillo, M.; He, H. A new-to-nature carboxylation module to improve natural and synthetic CO2 fixation. Nat. Catal. 2021, 4 (2), 105– 115, DOI: 10.1038/s41929-020-00557-y213https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB3MXhtFKjsLrE&md5=7f64a1dfca369fd9ca39c4c356a5034eA new-to-nature carboxylation module to improve natural and synthetic CO2 fixationScheffen, Marieke; Marchal, Daniel G.; Beneyton, Thomas; Schuller, Sandra K.; Klose, Melanie; Diehl, Christoph; Lehmann, Jessica; Pfister, Pascal; Carrillo, Martina; He, Hai; Aslan, Selcuk; Cortina, Nina S.; Claus, Peter; Bollschweiler, Daniel; Baret, Jean-Christophe; Schuller, Jan M.; Zarzycki, Jan; Bar-Even, Arren; Erb, Tobias J.Nature Catalysis (2021), 4 (2), 105-115CODEN: NCAACP; ISSN:2520-1158. (Nature Research)The capture of CO2 by carboxylases is key to sustainable biocatalysis and a carbon-neutral bio-economy, yet currently limited to few naturally existing enzymes. Here, we developed glycolyl-CoA carboxylase (GCC), a new-to-nature enzyme, by combining rational design, high-throughput microfluidics and microplate screens. During this process, GCC's catalytic efficiency improved by three orders of magnitude to match the properties of natural CO2-fixing enzymes. We verified our active-site redesign with an at.-resoln., 1.96-Å cryo-electron microscopy structure and engineered two more enzymes that, together with GCC, form a carboxylation module for the conversion of glycolate (C2) to glycerate (C3). We demonstrate how this module can be interfaced with natural photorespiration, ethylene glycol conversion and synthetic CO2 fixation. Based on stoichiometrical calcns., GCC is predicted to increase the carbon efficiency of all of these processes by up to 150% while reducing their theor. energy demand, showcasing how expanding the soln. space of natural metab. provides new opportunities for biotechnol. and agriculture.
- 214Schwander, T.; Schada von Borzyskowski, L.; Burgener, S.; Cortina, N. S.; Erb, T. J. A synthetic pathway for the fixation of carbon dioxide in vitro. Science 2016, 354, 900– 904, DOI: 10.1126/science.aah5237214https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC28XhvFGrtb%252FM&md5=eb266bffa55c2422c2ee78f1be4f2d22A synthetic pathway for the fixation of carbon dioxide in vitroSchwander, Thomas; Schada von Borzyskowski, Lennart; Burgener, Simon; Cortina, Nina Socorro; Erb, Tobias J.Science (Washington, DC, United States) (2016), 354 (6314), 900-904CODEN: SCIEAS; ISSN:0036-8075. (American Association for the Advancement of Science)Carbon dioxide (CO2) is an important carbon feedstock for a future green economy. This requiresthe development of efficient strategies for its conversion into multicarbon compds. We describe a synthetic cycle for the continuous fixation of CO2 in vitro. The crotonyl-CoA (CoA)/ethylmalonyl-CoA/hydroxybutyryl-CoA (CETCH) cycle is a reaction network of 17 enzymes that converts CO2 into org. mols. at a rate of 5 nmol of CO2 per min per mg of protein. The CETCH cycle was drafted by metabolic retrosynthesis, established with enzymes originating from nine different organisms of all three domains of life, and optimizedin several rounds by enzyme engineering and metabolic proofreading. The CETCH cycle adds a seventh, synthetic alternative to the six naturally evolved CO2 fixation pathways, thereby openingthe way for in vitro and in vivo applications.
- 215Gale, N. L.; Beck, J. V. Evidence for the Calvin cycle and hexose monophosphate pathway in Thiobacillus ferrooxidans. J. Bacteriol. 1967, 94, 1052– 1059, DOI: 10.1128/jb.94.4.1052-1059.1967215https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaF2sXltVGmtr4%253D&md5=7433f5b8394c57030679600870078889Evidence for the Calvin cycle and hexose monophosphate pathway in Thiobacillus ferrooxidansGale, Nord L.; Beck, Jay VernJournal of Bacteriology (1967), 94 (4), 1052-9CODEN: JOBAAY; ISSN:0021-9193.The enzymes of the Calvin reductive pentose phosphate cycle and the hexose monophosphate pathway were demonstrated in cell-free exts. of T. ferroxidans. This, together with analyses of the products of CO2 fixation in cell-free systems, suggests that these pathways are operative in whole cells of this bacterium. Nevertheless, the amt. of CO2 fixed in these cell-free systems was limited by the type and amt. of compd. added as substrate. The inability of cell exts. to regenerate pentose phosphates and to perpetuate the cyclic fixation of CO2 is partially attributable to low activity of triosephosphate dehydrogenase under the exptl. conditions optimal for the enzymes involved in the utilization of ribose 5-phosphate or ribulose 1,5-diphosphate as substrate for CO2 incorporation. With the exception of ribulose 1,5-diphosphate, all substrates required the addn. of ATP or ADP for CO2 fixation. Under optimal conditions, with ribose 5-phosphate serving as substrate, each micromole of ATP added resulted in the fixation of 1.5 micromoles of CO2, whereas each micromole of ADP resulted in 0.5 micromole of CO2 fixed. These values reflect the activity of adenylate kinase in the ext. prepns. The Km for ATP in the phosphoribulokinase reaction was 0.91 × 10-3M. Kinetic studies conducted with carboxydismutase showed Km values of 1.15 × 10-4M and 5 × 10-2M for ribulose 1,5-diphosphate and bicarbonate, resp. 32 references.
- 216Miller, T. E.; Beneyton, T.; Schwander, T.; Diehl, C.; Girault, M.; McLean, R.; Chotel, T.; Claus, P.; Cortina, N. S.; Baret, J.-C.; Erb, T. J. Light-powered CO2 fixation in a chloroplast mimic with natural and synthetic parts. Science 2020, 368, 649– 654, DOI: 10.1126/science.aaz6802216https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB3cXovFCqs7Y%253D&md5=e91945625b5292262a7237f8f895858bLight-powered CO2 fixation in a chloroplast mimic with natural and synthetic partsMiller, Tarryn E.; Beneyton, Thomas; Schwander, Thomas; Diehl, Christoph; Girault, Mathias; McLean, Richard; Chotel, Tanguy; Claus, Peter; Cortina, Nina Socorro; Baret, Jean-Christophe; Erb, Tobias J.Science (Washington, DC, United States) (2020), 368 (6491), 649-654CODEN: SCIEAS; ISSN:1095-9203. (American Association for the Advancement of Science)Nature integrates complex biosynthetic and energy-converting tasks within compartments such as chloroplasts and mitochondria. Chloroplasts convert light into chem. energy, driving carbon dioxide fixation. We used microfluidics to develop a chloroplast mimic by encapsulating and operating photosynthetic membranes in cell-sized droplets. These droplets can be energized by light to power enzymes or enzyme cascades and analyzed for their catalytic properties in multiplex and real time. We demonstrate how these microdroplets can be programmed and controlled by adjusting internal compns. and by using light as an external trigger. We showcase the capability of our platform by integrating the crotonyl-CoA (CoA)/ethylmalonyl-CoA/hydroxybutyryl-CoA (CETCH) cycle, a synthetic network for carbon dioxide conversion, to create an artificial photosynthetic system that interfaces the natural and the synthetic biol. worlds.
- 217Sundaram, S.; Diehl, C.; Cortina, N. S.; Bamberger, J.; Paczia, N.; Erb, T. J. A modular in vitro platform for the production of terpenes and polyketides from CO2. Angew. Chem., Int. Ed. 2021, 60 (30), 16420– 16425, DOI: 10.1002/anie.202102333217https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB3MXhsVKmt7nE&md5=70441107ebceb5dc590597e34d390b79A Modular In Vitro Platform for the Production of Terpenes and Polyketides from CO2Sundaram, Srividhya; Diehl, Christoph; Cortina, Nina Socorro; Bamberger, Jan; Paczia, Nicole; Erb, Tobias J.Angewandte Chemie, International Edition (2021), 60 (30), 16420-16425CODEN: ACIEF5; ISSN:1433-7851. (Wiley-VCH Verlag GmbH & Co. KGaA)A long-term goal in realizing a sustainable biocatalysis and org. synthesis is the direct use of the greenhouse gas CO2 as feedstock for the prodn. of bulk and fine chems., such as pharmaceuticals, fragrances and food additives. Here we developed a modular in vitro platform for the continuous conversion of CO2 into complex multi-carbon compds., such as monoterpenes (C10), sesquiterpenes (C15) and polyketides. Combining natural and synthetic metabolic pathway modules, we established a route from CO2 into the key intermediates acetyl- and malonyl-CoA, which can be subsequently diversified through the action of different terpene and polyketide synthases. Our proof-of-principle study demonstrates the simultaneous operation of different metabolic modules comprising of up to 29 enzymes in one pot, which paves the way for developing and optimizing synthesis routes for the generation of complex CO2-based chems. in the future.
- 218Diehl, C.; Gerlinger, P. D.; Paczia, N.; Erb, T. J. Synthetic anaplerotic modules for the direct synthesis of complex molecules from CO2. Nat. Chem. Biol. 2023, 19 (2), 168– 175, DOI: 10.1038/s41589-022-01179-0218https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB38XjtVCjtL%252FE&md5=408c17b934bc0342bde4ba4eb400c1f5Synthetic anaplerotic modules for the direct synthesis of complex molecules from CO2Diehl, Christoph; Gerlinger, Patrick D.; Paczia, Nicole; Erb, Tobias J.Nature Chemical Biology (2023), 19 (2), 168-175CODEN: NCBABT; ISSN:1552-4450. (Nature Portfolio)Abstr.: Anaplerosis is an essential feature of metab. that allows the continuous operation of natural metabolic networks, such as the citric acid cycle, by constantly replenishing drained intermediates. However, this concept has not been applied to synthetic in vitro metabolic networks, thus far. Here we used anaplerotic strategies to directly access the core sequence of the CETCH cycle, a new-to-nature in vitro CO2-fixation pathway that features several C3-C5 biosynthetic precursors. We drafted four different anaplerotic modules that use CO2 to replenish the CETCH cycle's intermediates and validated our designs by producing 6-deoxyerythronolide B (6-DEB), the C21-macrolide backbone of erythromycin. Our best design allowed the carbon-pos. synthesis of 6-DEB via 54 enzymic reactions in vitro at yields comparable to those with isolated 6-DEB polyketide synthase (DEBS). Our work showcases how new-to-nature anaplerotic modules can be designed and tailored to enhance and expand the synthetic capabilities of complex catalytic in vitro reaction networks. [graphic not available: see fulltext].
- 219Xiao, L.; Liu, G.; Gong, F.; Zhu, H.; Zhang, Y.; Cai, Z.; Li, Y. A minimized synthetic carbon fixation cycle. ACS Catal. 2022, 12 (1), 799– 808, DOI: 10.1021/acscatal.1c04151219https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB3MXivVeks7jP&md5=72345a4b3a3b1f07f55d553527d4386dA minimized synthetic carbon fixation cycleXiao, Lu; Liu, Guoxia; Gong, Fuyu; Zhu, Huawei; Zhang, Yanping; Cai, Zhen; Li, YinACS Catalysis (2022), 12 (1), 799-808CODEN: ACCACS; ISSN:2155-5435. (American Chemical Society)Natural CO2 fixation cycles usually comprise multiple reactions, which may reduce the efficiency of the cycle. Here, we report the design and exptl. demonstration of a minimized synthetic CO2 fixation cycle which contains only four reactions. The cycle comprises pyruvate carboxylase, oxaloacetate acetylhydrolase, acetate-CoA ligase, and pyruvate synthase and is named the POAP cycle. The POAP cycle can condense two mols. of CO2 into one mol. of oxalate in each step at the expense of two mols. of ATP and one reducing equiv. in the form of NAD(P)H. By identifying a ferredoxin from Hydrogenobacter thermophilus that can efficiently drive the rate-limiting reductive carboxylation step, the POAP cycle can be operated at 50°C under anaerobic conditions, reaching a CO2 fixation rate of 8.0 nmol CO2 min-1 mg-1 CO2-fixing enzymes. The design and demonstration of the POAP cycle may provide a model to study CO2 fixation in the earliest organisms.
- 220Bierbaumer, S.; Nattermann, M.; Schulz, L.; Zschoche, R.; Erb, T. J.; Winkler, C. K.; Tinzl, M.; Glueck, S. M. Enzymatic conversion of CO2: from natural to artificial utilization. Chem. Rev. 2023, 123 (9), 5702– 5754, DOI: 10.1021/acs.chemrev.2c00581220https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB3sXhs1GntL4%253D&md5=be4bc4e4b9fe5d5d7b2ba233dafdab61Enzymatic Conversion of CO2: From Natural to Artificial UtilizationBierbaumer, Sarah; Nattermann, Maren; Schulz, Luca; Zschoche, Reinhard; Erb, Tobias J.; Winkler, Christoph K.; Tinzl, Matthias; Glueck, Silvia M.Chemical Reviews (Washington, DC, United States) (2023), 123 (9), 5702-5754CODEN: CHREAY; ISSN:0009-2665. (American Chemical Society)A review. Enzymic carbon dioxide fixation is one of the most important metabolic reactions as it allows the capture of inorg. carbon from the atm. and converting it into org. biomass. However, due to the often unfavorable thermodn. and the difficulties assocd. with the utilization of CO2, a gaseous substrate that is found in comparatively low concns. in the atm., such reactions remain challenging for biotechnol. applications. Nature has tackled these problems by evolution of dedicated CO2-fixing enzymes, i.e., carboxylases, and embedding them in complex metabolic pathways. Biotechnol. employs such carboxylating and decarboxylating enzymes for the carboxylation of arom. and aliph. substrates either by embedding them into more complex reaction cascades or by shifting the reaction equil. via reaction engineering. This review aims to provide an overview of natural CO2-fixing enzymes and their mechanistic similarities. We also discuss biocatalytic applications of carboxylases and decarboxylases for the synthesis of valuable products and provide a sep. summary of strategies to improve the efficiency of such processes. We briefly summarize natural CO2 fixation pathways, provide a roadmap for the design and implementation of artificial carbon fixation pathways, and highlight examples of biocatalytic cascades involving carboxylases. Addnl., we suggest that biochem. utilization of reduced CO2 homologues, such as formate or methanol represents a suitable alternative to direct use of CO2 and provide several examples. Our discussion closes with a techno-economic perspective on enzymic CO2 fixation and its potential to reduce CO2-emissions.
- 221Cai, T.; Sun, H.; Qiao, J.; Zhu, L.; Zhang, F.; Zhang, J.; Tang, Z.; Wei, X.; Yang, J.; Yuan, Q. Cell-free chemo-enzymatic starch synthesis from carbon dioxide. Science 2021, 373, 1523– 152, DOI: 10.1126/science.abh4049221https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB3MXitFOisbnK&md5=81fb33a622016cbc14aacff7170dc3a1Cell-free chemoenzymatic starch synthesis from carbon dioxideCai, Tao; Sun, Hongbing; Qiao, Jing; Zhu, Leilei; Zhang, Fan; Zhang, Jie; Tang, Zijing; Wei, Xinlei; Yang, Jiangang; Yuan, Qianqian; Wang, Wangyin; Yang, Xue; Chu, Huanyu; Wang, Qian; You, Chun; Ma, Hongwu; Sun, Yuanxia; Li, Yin; Li, Can; Jiang, Huifeng; Wang, Qinhong; Ma, YanheScience (Washington, DC, United States) (2021), 373 (6562), 1523-1527CODEN: SCIEAS; ISSN:1095-9203. (American Association for the Advancement of Science)Starches, a storage form of carbohydrates, are a major source of calories in the human diet and a primary feedstock for bioindustry. We report a chem.-biochem. hybrid pathway for starch synthesis from carbon dioxide (CO2) and hydrogen in a cell-free system. The artificial starch anabolic pathway (ASAP), consisting of 11 core reactions, was drafted by computational pathway design, established through modular assembly and substitution, and optimized by protein engineering of three bottleneck-assocd. enzymes. In a chemoenzymic system with spatial and temporal segregation, ASAP, driven by hydrogen, converts CO2 to starch at a rate of 22 nmol of CO2 per min per mg of total catalyst, an ~ 8.5-fold higher rate than starch synthesis in maize. This approach opens the way toward future chemo-biohybrid starch synthesis from CO2.
- 222Zhou, J.; Tian, X.; Yang, Q.; Zhang, Z.; Chen, C.; Cui, Z.; Ji, Y.; Schwaneberg, U.; Chen, B.; Tan, T. Three multi-enzyme cascade pathways for conversion of C1 to C2/C4 compounds. Chem. Catal. 2022, 2 (10), 2675– 2690, DOI: 10.1016/j.checat.2022.07.011222https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB3sXjslOqt78%253D&md5=645bccc9f0ec5b17e95c3f31815b92e1Three multi-enzyme cascade pathways for conversion of C1 to C2/C4 compoundsZhou, Junhui; Tian, Xinyu; Yang, Qian; Zhang, Zixuan; Chen, Changjing; Cui, Ziheng; Ji, Yu; Schwaneberg, Ulrich; Chen, Biqiang; Tan, TianweiChem Catalysis (2022), 2 (10), 2675-2690CODEN: CCHAE9; ISSN:2667-1093. (Elsevier Inc.)A feasible and promising carbon redn. strategy is to convert CO2 into C1 compds. through renewable energy-driven first and then use a multi-enzyme cascade to obtain compds. with higher carbon nos. and market value. In this study, three multi-enzyme cascade pathways for prepg. important C2/C4 compds. (ethylene glycol [EG], glycolic acid [GA], and D-erythrose) from methanol were proposed. All pathways use glycolaldehyde as the key intermediate, which can be obtained from methanol through glycolaldehyde synthase and alc. oxidase cascade. Then, the target compds. can be acquired by adding different enzymes. As a result, 0.90 g/L EG, 0.33 g/L GA, and 53.65 mg/L D-erythrose were produced from methanol. Compared with the titers of EG (6.6 mM) and GA (1.2 g/L) synthesis routes from formaldehyde reported in the literature, the titers of this study (27.58 mM EG and 1.59 g/L GA from formaldehyde) were significantly improved.
- 223Zhang, J.; Liu, D.; Liu, Y.; Chu, H.; Bai, J.; Cheng, J.; Zhao, H.; Fu, S.; Liu, H.; Fu, Y. Hybrid synthesis of polyhydroxybutyrate bioplastics from carbon dioxide. Green Chem. 2023, 25 (8), 3247– 3255, DOI: 10.1039/D3GC00387F223https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB3sXmvVWltLo%253D&md5=5becb0e5eeb8c4d64bdcc5b2a6bc3510Hybrid synthesis of polyhydroxybutyrate bioplastics from carbon dioxideZhang, Jie; Liu, Dingyu; Liu, Yuwan; Chu, Huanyu; Bai, Jie; Cheng, Jian; Zhao, Haodong; Fu, Shaoping; Liu, Huihong; Fu, YuE.; Ma, Yanhe; Jiang, HuifengGreen Chemistry (2023), 25 (8), 3247-3255CODEN: GRCHFJ; ISSN:1463-9262. (Royal Society of Chemistry)Global sustainable development has intensified the demand for switching to a renewable economy with a reduced carbon footprint. Here, we report a hybrid system, coupling a chem. process for CO2 redn. with hydrogen, and a biol. process for polyhydroxybutyrate (PHB) synthesis, capable of converting CO2 into bioplastics with a theor. carbon yield of 100%. The synthetic pathway from CO2 to PHB was modularly optimized by improving the catalytic efficiency of key enzymes, avoiding the kinetic trap of metabolic flux and optimizing the whole catalytic process, resulting in 5.96 g L-1 PHB with a productivity of 1.19 g L-1 h-1 and a molar CO2 utilization efficiency of 71.8%. These results represent a promising closed-loop prodn. process from CO2 to biodegradable plastics.
- 224Li, F.; Wei, X.; Zhang, L.; Liu, C.; You, C.; Zhu, Z. Installing a green engine to drive an enzyme cascade: a light-powered in vitro biosystem for poly(3-hydroxybutyrate) synthesis. Angew. Chem., Int. Ed. 2022, 61 (1), e202111054, DOI: 10.1002/anie.202111054224https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB3MXisFOitL3M&md5=33486d9a6a565d88773c9986adecdc8fInstalling a Green Engine To Drive an Enzyme Cascade: A Light-Powered In Vitro Biosystem for Poly(3-hydroxybutyrate) SynthesisLi, Fei; Wei, Xinlei; Zhang, Lin; Liu, Cheng; You, Chun; Zhu, ZhiguangAngewandte Chemie, International Edition (2022), 61 (1), e202111054CODEN: ACIEF5; ISSN:1433-7851. (Wiley-VCH Verlag GmbH & Co. KGaA)Many existing in vitro biosystems harness power from the chem. energy contained in substrates and co-substrates, and light or elec. energy provided from abiotic parts, leading to a compromise in atom economy, incompatibility between biol. and abiotic parts, and most importantly, incapability to spatiotemporally co-regenerate ATP and NADPH. In this study, we developed a light-powered in vitro biosystem for poly(3-hydroxybutyrate) (PHB) synthesis using natural thylakoid membranes (TMs) to regenerate ATP and NADPH for a five-enzyme cascade. Through effective coupling of cofactor regeneration and mass conversion, 20 mM PHB was yielded from 50 mM sodium acetate with a molar conversion efficiency of carbon of 80.0% and a light-energy conversion efficiency of 3.04%, which are much higher than the efficiencies of similar in vitro PHB synthesis biosystems. This suggests the promise of installing TMs as a green engine to drive more enzyme cascades.
- 225Liu, H.; Arbing, M. A.; Bowie, J. U. Expanding the use of ethanol as a feedstock for cell-free synthetic biochemistry by implementing acetyl-CoA and ATP generating pathways. Sci. Rep. 2022, 12 (1), 7700, DOI: 10.1038/s41598-022-11653-3225https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB38Xht1OiurjJ&md5=0575f6683e35f5090a75b81d9d52a81cExpanding the use of ethanol as a feedstock for cell-free synthetic biochemistry by implementing acetyl-CoA and ATP generating pathwaysLiu, Hongjiang; Arbing, Mark A.; Bowie, James U.Scientific Reports (2022), 12 (1), 7700CODEN: SRCEC3; ISSN:2045-2322. (Nature Portfolio)Ethanol is a widely available carbon compd. that can be increasingly produced with a net neg. carbon balance. Carbon-neg. ethanol might therefore provide a feedstock for building a wider range of sustainable chems. Here we show how ethanol can be converted with a cell free system into acetyl-CoA, a central precursor for myriad biochems., and how we can use the energy stored in ethanol to generate ATP, another key mol. important for powering biochem. pathways. The ATP generator produces acetone as a value-added side product. Our ATP generator reached titers of 27 ± 6 mM ATP and 59 ± 15 mM acetone with max. ATP synthesis rate of 2.8 ± 0.6 mM/h and acetone of 7.8 ± 0.8 mM/h. We illustrated how the ATP generating module can power cell-free biochem. pathways by converting mevalonate into isoprenol at a titer of 12.5 ± 0.8 mM and a max. productivity of 1.0 ± 0.05 mM/h. These proof-of-principle demonstrations may ultimately find their way to the manuf. of diverse chems. from ethanol and other simple carbon compds.
- 226Bailoni, E.; Partipilo, M.; Coenradij, J.; Grundel, D. A. J.; Slotboom, D. J.; Poolman, B. Minimal out-of-equilibrium metabolism for synthetic cells: a membrane perspective. ACS Synth. Biol. 2023, 12 (4), 922– 946, DOI: 10.1021/acssynbio.3c00062226https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB3sXntVSrsLc%253D&md5=cb72d2883fc86ce12e323b5f2d8dc842Minimal Out-of-Equilibrium Metabolism for Synthetic Cells: A Membrane PerspectiveBailoni, Eleonora; Partipilo, Michele; Coenradij, Jelmer; Grundel, Douwe A. J.; Slotboom, Dirk J.; Poolman, BertACS Synthetic Biology (2023), 12 (4), 922-946CODEN: ASBCD6; ISSN:2161-5063. (American Chemical Society)A review. Life-like systems need to maintain a basal metab., which includes importing a variety of building blocks required for macromol. synthesis, exporting dead-end products, and recycling cofactors and metabolic intermediates, while maintaining steady internal phys. and chem. conditions (physicochem. homeostasis). A compartment, such as a unilamellar vesicle, functionalized with membrane-embedded transport proteins and metabolic enzymes encapsulated in the lumen meets these requirements. Here, we identify four modules designed for a minimal metab. in a synthetic cell with a lipid bilayer boundary: energy provision and conversion, physicochem. homeostasis, metabolite transport, and membrane expansion. We review design strategies that can be used to fulfill these functions with a focus on the lipid and membrane protein compn. of a cell. We compare our bottom-up design with the equiv. essential modules of JCVI-syn3a, a top-down genome-minimized living cell with a size comparable to that of large unilamellar vesicles. Finally, we discuss the bottlenecks related to the insertion of a complex mixt. of membrane proteins into lipid bilayers and provide a semiquant. est. of the relative surface area and lipid-to-protein mass ratios (i.e., the minimal no. of membrane proteins) that are required for the construction of a synthetic cell.
- 227Lee, K. Y.; Park, S. J.; Lee, K. A.; Kim, S. H.; Kim, H.; Meroz, Y.; Mahadevan, L.; Jung, K. H.; Ahn, T. K.; Parker, K. K. Photosynthetic artificial organelles sustain and control ATP-dependent reactions in a protocellular system. Nat. Biotechnol. 2018, 36 (6), 530– 535, DOI: 10.1038/nbt.4140227https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC1cXhtVWktrzM&md5=38cc9b7aa604f285b5d84c793c9e26cfPhotosynthetic artificial organelles sustain and control ATP-dependent reactions in a protocellular systemLee, Keel Yong; Park, Sung-Jin; Lee, Keon Ah; Kim, Se-Hwan; Kim, Heeyeon; Meroz, Yasmine; Mahadevan, L.; Jung, Kwang-Hwan; Ahn, Tae Kyu; Parker, Kevin Kit; Shin, KwanwooNature Biotechnology (2018), 36 (6), 530-535CODEN: NABIF9; ISSN:1087-0156. (Nature Research)Inside cells, complex metabolic reactions are distributed across the modular compartments of organelles. Reactions in organelles have been recapitulated in vitro by reconstituting functional protein machineries into membrane systems. However, maintaining and controlling these reactions is challenging. Here we designed, built, and tested a switchable, light-harvesting organelle that provides both a sustainable energy source and a means of directing intravesicular reactions. An ATP (ATP) synthase and two photoconverters (plant-derived photosystem II and bacteria-derived proteorhodopsin) enable ATP synthesis. Independent optical activation of the two photoconverters allows dynamic control of ATP synthesis: red light facilitates and green light impedes ATP synthesis. We encapsulated the photosynthetic organelles in a giant vesicle to form a protocellular system and demonstrated optical control of two ATP-dependent reactions, carbon fixation and actin polymn., with the latter altering outer vesicle morphol. Switchable photosynthetic organelles may enable the development of biomimetic vesicle systems with regulatory networks that exhibit homeostasis and complex cellular behaviors.
- 228Berhanu, S.; Ueda, T.; Kuruma, Y. Artificial photosynthetic cell producing energy for protein synthesis. Nat. Commun. 2019, 10 (1), 1325, DOI: 10.1038/s41467-019-09147-4228https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A280%3ADC%252BB3cbnt1yjsw%253D%253D&md5=706ef019ff2af417bb362cf8bb29d3b0Artificial photosynthetic cell producing energy for protein synthesisBerhanu Samuel; Ueda Takuya; Kuruma Yutetsu; Kuruma YutetsuNature communications (2019), 10 (1), 1325 ISSN:.Attempts to construct an artificial cell have widened our understanding of living organisms. Many intracellular systems have been reconstructed by assembling molecules, however the mechanism to synthesize its own constituents by self-sufficient energy has to the best of our knowledge not been developed. Here, we combine a cell-free protein synthesis system and small proteoliposomes, which consist of purified ATP synthase and bacteriorhodopsin, inside a giant unilamellar vesicle to synthesize protein by the production of ATP by light. The photo-synthesized ATP is consumed as a substrate for transcription and as an energy for translation, eventually driving the synthesis of bacteriorhodopsin or constituent proteins of ATP synthase, the original essential components of the proteoliposome. The de novo photosynthesized bacteriorhodopsin and the parts of ATP synthase integrate into the artificial photosynthetic organelle and enhance its ATP photosynthetic activity through the positive feedback of the products. Our artificial photosynthetic cell system paves the way to construct an energetically independent artificial cell.
- 229Bailoni, E.; Poolman, B. ATP recycling fuels sustainable glycerol 3-phosphate formation in synthetic cells fed by dynamic dialysis. ACS Synth. Biol. 2022, 11 (7), 2348– 2360, DOI: 10.1021/acssynbio.2c00075229https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB38Xos1KitLo%253D&md5=24e104c5d768995c265e6adac1cc131eATP Recycling Fuels Sustainable Glycerol 3-Phosphate Formation in Synthetic Cells Fed by Dynamic DialysisBailoni, Eleonora; Poolman, BertACS Synthetic Biology (2022), 11 (7), 2348-2360CODEN: ASBCD6; ISSN:2161-5063. (American Chemical Society)The bottom-up construction of an autonomously growing, self-reproducing cell represents a great challenge for synthetic biol. Synthetic cellular systems are envisioned as out-of-equil. enzymic networks encompassed by a selectively open phospholipid bilayer allowing for protein-mediated communication; internal metabolite recycling is another key aspect of a sustainable metab. Importantly, gaining tight control over the external medium is essential to avoid thermodn. equil. due to nutrient depletion or waste buildup in a closed compartment (e.g., a test tube). Implementing a sustainable strategy for phospholipid biosynthesis is key to expanding the cellular boundaries. However, phospholipid biosynthesis is currently limited by substrate availability, e.g., of glycerol 3-phosphate, the essential core of phospholipid headgroups. Here, we reconstitute an enzymic network for sustainable glycerol 3-phosphate synthesis inside large unilamellar vesicles. We exploit the Escherichia coli glycerol kinase GlpK to synthesize glycerol 3-phosphate from externally supplied glycerol. We fuel phospholipid headgroup formation by sustainable L-arginine breakdown. In addn., we design and characterize a dynamic dialysis setup optimized for synthetic cells, which is used to control the external medium compn. and to achieve sustainable glycerol 3-phosphate synthesis.
- 230Blanken, D.; Foschepoth, D.; Serrao, A. C.; Danelon, C. Genetically controlled membrane synthesis in liposomes. Nat. Commun. 2020, 11 (1), 4317, DOI: 10.1038/s41467-020-17863-5230https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB3cXhslejtL3L&md5=cf776e04404b4d227a23041a0acef3c8Genetically controlled membrane synthesis in liposomesBlanken, Duco; Foschepoth, David; Serrao, Adriana Calaca; Danelon, ChristopheNature Communications (2020), 11 (1), 4317CODEN: NCAOBW; ISSN:2041-1723. (Nature Research)Lipid membranes, nucleic acids, proteins, and metab. are essential for modern cellular life. Synthetic systems emulating the fundamental properties of living cells must therefore be built upon these functional elements. In this work, phospholipid-producing enzymes encoded in a synthetic minigenome are cell-free expressed within liposome compartments. The de novo synthesized metabolic pathway converts precursors into a variety of lipids, including the constituents of the parental liposome. Balanced prodn. of phosphatidylethanolamine and phosphatidylglycerol is realized, owing to transcriptional regulation of the activity of specific genes combined with a metabolic feedback mechanism. Fluorescence-based methods are developed to image the synthesis and membrane incorporation of phosphatidylserine at the single liposome level. Our results provide exptl. evidence for DNA-programed membrane synthesis in a minimal cell model. Strategies are discussed to alleviate current limitations toward effective liposome growth and self-reprodn.
- 231Partipilo, M.; Ewins, E. J.; Frallicciardi, J.; Robinson, T.; Poolman, B.; Slotboom, D. J. Minimal pathway for the regeneration of redox cofactors. JACS Au 2021, 1 (12), 2280– 2293, DOI: 10.1021/jacsau.1c00406231https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB3MXisVKnsrjJ&md5=cd8bcc68d0494b701fad84d2f816f644Minimal Pathway for the Regeneration of Redox CofactorsPartipilo, Michele; Ewins, Eleanor J.; Frallicciardi, Jacopo; Robinson, Tom; Poolman, Bert; Slotboom, Dirk JanJACS Au (2021), 1 (12), 2280-2293CODEN: JAAUCR; ISSN:2691-3704. (American Chemical Society)Effective metabolic pathways are essential for the construction of in vitro systems mimicking the biochem. complexity of living cells. Such pathways require the inclusion of a metabolic branch that ensures the availability of reducing equiv. Here, we built a minimal enzymic pathway confinable in the lumen of liposomes, in which the redox status of the nicotinamide cofactors NADH and NADPH is controlled by an externally provided formate. Formic acid permeates the membrane where a luminal formate dehydrogenase uses NAD+ to form NADH and carbon dioxide. Carbon dioxide diffuses out of the liposomes, leaving only the reducing equiv. in the lumen. A sol. transhydrogenase subsequently utilizes NADH for redn. of NADP+ thereby making NAD+ available again for the first reaction. The pathway is functional in liposomes ranging from a few hundred nanometers in diam. (large unilamellar vesicles) up to several tens of micrometers (giant unilamellar vesicles) and remains active over a period of 7 days. We demonstrate that the downstream biochem. process of redn. of glutathione disulfide can be driven by the transfer of reducing equiv. from formate via NAD(P)H, thereby providing a versatile set of electron donors for reductive metab.
- 232Pols, T.; Sikkema, H. R.; Gaastra, B. F.; Frallicciardi, J.; Smigiel, W. M.; Singh, S.; Poolman, B. A synthetic metabolic network for physicochemical homeostasis. Nat. Commun. 2019, 10 (1), 4239, DOI: 10.1038/s41467-019-12287-2232https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A280%3ADC%252BB3MngtVOrtA%253D%253D&md5=9738665e6507488772d12472354a902eA synthetic metabolic network for physicochemical homeostasisPols Tjeerd; Sikkema Hendrik R; Gaastra Bauke F; Frallicciardi Jacopo; Smigiel Wojciech M; Singh Shubham; Poolman BertNature communications (2019), 10 (1), 4239 ISSN:.One of the grand challenges in chemistry is the construction of functional out-of-equilibrium networks, which are typical of living cells. Building such a system from molecular components requires control over the formation and degradation of the interacting chemicals and homeostasis of the internal physical-chemical conditions. The provision and consumption of ATP lies at the heart of this challenge. Here we report the in vitro construction of a pathway in vesicles for sustained ATP production that is maintained away from equilibrium by control of energy dissipation. We maintain a constant level of ATP with varying load on the system. The pathway enables us to control the transmembrane fluxes of osmolytes and to demonstrate basic physicochemical homeostasis. Our work demonstrates metabolic energy conservation and cell volume regulatory mechanisms in a cell-like system at a level of complexity minimally needed for life.
- 233Katz, E.; Privman, V. Enzyme-based logic systems for information processing. Chem. Soc. Rev. 2010, 39 (5), 1835– 1857, DOI: 10.1039/b806038j233https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3cXltFKgsb8%253D&md5=8761554ed19bd8f3938689902d6b78c0Enzyme-based logic systems for information processingKatz, Evgeny; Privman, VladimirChemical Society Reviews (2010), 39 (5), 1835-1857CODEN: CSRVBR; ISSN:0306-0012. (Royal Society of Chemistry)A review. In this crit. review the authors review enzymic systems which involve biocatalytic reactions utilized for information processing (biocomputing). Extensive ongoing research in biocomputing, mimicking Boolean logic gates has been motivated by potential applications in biotechnol. and medicine. Furthermore, novel sensor concepts have been contemplated with multiple inputs processed biochem. before the final output is coupled to transducing "smart-material" electrodes and other systems. These applications have warranted recent emphasis on networking of biocomputing gates. First few-gate networks have been exptl. realized, including coupling, for instance, to signal-responsive electrodes for signal readout. To achieve scalable, stable network design and functioning, considerations of noise propagation and control have been initiated as a new research direction. Optimization of single enzyme-based gates for avoiding analog noise amplification has been explored, as were certain network-optimization concepts. The authors review and exemplify these developments, as well as offer an outlook for possible future research foci. The latter include design and uses of non-Boolean network elements, e.g., filters, as well as other developments motivated by potential novel sensor and biotechnol. applications (136 refs.).
- 234Benenson, Y. Biomolecular computing systems: principles, progress and potential. Nat. Rev. Genet. 2012, 13 (7), 455– 468, DOI: 10.1038/nrg3197234https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC38XotlCku7Y%253D&md5=df2cf7bb6f50782328b62cdcccdd2160Biomolecular computing systems: principles, progress and potentialBenenson, YaakovNature Reviews Genetics (2012), 13 (7), 455-468CODEN: NRGAAM; ISSN:1471-0056. (Nature Publishing Group)A review. The task of information processing, or computation, can be performed by natural and man-made 'devices'. Man-made computers are made from silicon chips, whereas natural 'computers', such as the brain, use cells and mols. Computation also occurs on a much smaller scale in regulatory and signalling pathways in individual cells and even within single biomols. Indeed, much of what we recognize as life results from the remarkable capacity of biol. building blocks to compute in highly sophisticated ways. Rational design and engineering of biol. computing systems can greatly enhance our ability to study and to control biol. systems. Potential applications include tissue engineering and regeneration and medical treatments. This Review introduces key concepts and discusses recent progress that has been made in biomol. computing.
- 235Grozinger, L.; Amos, M.; Gorochowski, T. E.; Carbonell, P.; Oyarzun, D. A.; Stoof, R.; Fellermann, H.; Zuliani, P.; Tas, H.; Goni-Moreno, A. Pathways to cellular supremacy in biocomputing. Nat. Commun. 2019, 10 (1), 5250, DOI: 10.1038/s41467-019-13232-z235https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A280%3ADC%252BB3Mfht12rtA%253D%253D&md5=3890719db11fbfe37974c513492740baPathways to cellular supremacy in biocomputingGrozinger Lewis; Stoof Ruud; Fellermann Harold; Zuliani Paolo; Goni-Moreno Angel; Amos Martyn; Gorochowski Thomas E; Gorochowski Thomas E; Carbonell Pablo; Oyarzun Diego A; Oyarzun Diego A; Tas HuseyinNature communications (2019), 10 (1), 5250 ISSN:.Synthetic biology uses living cells as the substrate for performing human-defined computations. Many current implementations of cellular computing are based on the "genetic circuit" metaphor, an approximation of the operation of silicon-based computers. Although this conceptual mapping has been relatively successful, we argue that it fundamentally limits the types of computation that may be engineered inside the cell, and fails to exploit the rich and diverse functionality available in natural living systems. We propose the notion of "cellular supremacy" to focus attention on domains in which biocomputing might offer superior performance over traditional computers. We consider potential pathways toward cellular supremacy, and suggest application areas in which it may be found.
- 236Ivanov, N. M.; Baltussen, M. G.; Regueiro, C. L. F.; Derks, M.; Huck, W. T. S. Computing arithmetic functions using immobilised enzymatic reaction networks. Angew. Chem., Int. Ed. 2023, 62 (7), e202215759, DOI: 10.1002/anie.202215759236https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB3sXhtFGitrg%253D&md5=d8a6cb44f9286028dba214a8be43f00bComputing Arithmetic Functions Using Immobilised Enzymatic Reaction NetworksIvanov, Nikita M.; Baltussen, Mathieu G.; Regueiro, Cristina Lia Fernandez; Derks, Max T. G. M.; Huck, Wilhelm T. S.Angewandte Chemie, International Edition (2023), 62 (7), e202215759CODEN: ACIEF5; ISSN:1433-7851. (Wiley-VCH Verlag GmbH & Co. KGaA)Living systems use enzymic reaction networks to process biochem. information and make decisions in response to external or internal stimuli. Herein, we present a modular and reusable platform for mol. information processing using enzymes immobilized in hydrogel beads and compartmentalised in a continuous stirred tank reactor. We demonstrate how this setup allows us to perform simple arithmetic operations, such as addn., subtraction and multiplication, using various concns. of substrates or inhibitors as inputs and the prodn. of a fluorescent mol. as the readout.
- 237Genot, A. J.; Fujii, T.; Rondelez, Y. Computing with competition in biochemical networks. Phys. Rev. Lett. 2012, 109 (20), 208102, DOI: 10.1103/PhysRevLett.109.208102237https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC38XhvVCmurrL&md5=df4657c49b9599ee143da5f25b160f72Computing with competition in biochemical networksGenot, Anthony J.; Fujii, Teruo; Rondelez, YannickPhysical Review Letters (2012), 109 (20), 208102/1-208102/5CODEN: PRLTAO; ISSN:0031-9007. (American Physical Society)Cells rely on limited resources such as enzymes or transcription factors to process signals and make decisions. However, independent cellular pathways often compete for a common mol. resource. Competition is difficult to analyze because of its nonlinear global nature, and its role remains unclear. Here we show how decision pathways such as transcription networks may exploit competition to process information. Competition for one resource leads to the recognition of convex sets of patterns, whereas competition for several resources (overlapping or cascaded regulons) allows even more general pattern recognition. Competition also generates surprising couplings, such as correlating species that share no resource but a common competitor. The mechanism we propose relies on three primitives that are ubiquitous in cells: multiinput motifs, competition for a resource, and pos. feedback loops.
- 238Pandi, A.; Koch, M.; Voyvodic, P. L.; Soudier, P.; Bonnet, J.; Kushwaha, M.; Faulon, J. L. Metabolic perceptrons for neural computing in biological systems. Nat. Commun. 2019, 10 (1), 3880, DOI: 10.1038/s41467-019-11889-0238https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A280%3ADC%252BB3MrivFCkuw%253D%253D&md5=709c8f35b4f3ed72a901ad0bfefd03ecMetabolic perceptrons for neural computing in biological systemsPandi Amir; Koch Mathilde; Soudier Paul; Kushwaha Manish; Faulon Jean-Loup; Voyvodic Peter L; Bonnet Jerome; Soudier Paul; Faulon Jean-Loup; Faulon Jean-LoupNature communications (2019), 10 (1), 3880 ISSN:.Synthetic biological circuits are promising tools for developing sophisticated systems for medical, industrial, and environmental applications. So far, circuit implementations commonly rely on gene expression regulation for information processing using digital logic. Here, we present a different approach for biological computation through metabolic circuits designed by computer-aided tools, implemented in both whole-cell and cell-free systems. We first combine metabolic transducers to build an analog adder, a device that sums up the concentrations of multiple input metabolites. Next, we build a weighted adder where the contributions of the different metabolites to the sum can be adjusted. Using a computational model fitted on experimental data, we finally implement two four-input perceptrons for desired binary classification of metabolite combinations by applying model-predicted weights to the metabolic perceptron. The perceptron-mediated neural computing introduced here lays the groundwork for more advanced metabolic circuits for rapid and scalable multiplex sensing.
- 239Okumura, S.; Gines, G.; Lobato-Dauzier, N.; Baccouche, A.; Deteix, R.; Fujii, T.; Rondelez, Y.; Genot, A. J. Nonlinear decision-making with enzymatic neural networks. Nature 2022, 610 (7932), 496– 501, DOI: 10.1038/s41586-022-05218-7239https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB38Xis1Onu7rJ&md5=e6485c7b357ad006c2f01ab78a791e80Nonlinear decision-making with enzymatic neural networksOkumura, S.; Gines, G.; Lobato-Dauzier, N.; Baccouche, A.; Deteix, R.; Fujii, T.; Rondelez, Y.; Genot, A. J.Nature (London, United Kingdom) (2022), 610 (7932), 496-501CODEN: NATUAS; ISSN:1476-4687. (Nature Portfolio)Atificial neural networks have revolutionized electronic computing. Similarly, mol. networks with neuromorphic architectures may enable mol. decision-making on a level comparable to gene regulatory networks1,2. Non-enzymic networks could in principle support neuromorphic architectures, and seminal proofs-of-principle have been reported3,4. However, leakages (i.e., the unwanted release of species), as well as issues with sensitivity, speed, prepn. and the lack of strong nonlinear responses, make the compn. of layers delicate, and mol. classifications equiv. to a multilayer neural network remain elusive (for example, the partitioning of a concn. space into regions that cannot be linearly sepd.). Here we introduce DNA-encoded enzymic neurons with tuneable wts. and biases, and which are assembled in multilayer architectures to classify nonlinearly separable regions. We first leverage the sharp decision margin of a neuron to compute various majority functions on 10 bits. We then compose neurons into a two-layer network and synthesize a parametric family of rectangular functions on a microRNA input. Finally, we connect neural and logical computations into a hybrid circuit that recursively partitions a concn. plane according to a decision tree in cell-sized droplets. This computational power and extreme miniaturization open avenues to query and manage mol. systems with complex contents, such as liq. biopsies or DNA databases.
- 240Hathcock, D.; Sheehy, J.; Weisenberger, C.; Ilker, E.; Hinczewski, M. Noise filtering and prediction in biological signaling networks. IEEE Transactions on Molecular, Biological and Multi-Scale Communications 2016, 2 (1), 16– 30, DOI: 10.1109/TMBMC.2016.2633269There is no corresponding record for this reference.
- 241O’Brien, J.; Murugan, A. Temporal pattern recognition through analog molecular computation. ACS Synth. Biol. 2019, 8 (4), 826– 832, DOI: 10.1021/acssynbio.8b00503241https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC1MXktFahsr8%253D&md5=ac507d10d6d2c3ab067d76b0859dde63Temporal Pattern Recognition through Analog Molecular ComputationO'Brien, Jackson; Murugan, ArvindACS Synthetic Biology (2019), 8 (4), 826-832CODEN: ASBCD6; ISSN:2161-5063. (American Chemical Society)Living cells communicate information about physiol. conditions by producing signaling mols. in a specific timed manner. Different conditions can result in the same total amt. of a signaling mol., differing only in the pattern of the mol. concn. over time. Such temporally coded information can be completely invisible to even state-of-the-art mol. sensors with high chem. specificity that respond only to the total amt. of the signaling mol. Here, we demonstrate design principles for circuits with temporal specificity, i.e., mol. circuits that respond to specific temporal patterns in a mol. concn. We consider pulsatile patterns in a mol. concn. characterized by three fundamental temporal features: time period, duty fraction, and no. of pulses. We develop circuits that respond to each one of these features while being insensitive to the others. We demonstrate our design principles using general chem. reaction networks and with explicit simulations of DNA strand displacement reactions. In this way, our work develops building blocks for temporal pattern recognition through mol. computation.
- 242McGrath, T.; Jones, N. S.; Ten Wolde, P. R.; Ouldridge, T. E. Biochemical machines for the interconversion of mutual information and work. Phys. Rev. Lett. 2017, 118 (2), 028101 DOI: 10.1103/PhysRevLett.118.028101242https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2sXpt1Cktbg%253D&md5=f366eb71b2d50e88c3253359c37127b7Biochemical machines for the interconversion of mutual information and workMcGrath, Thomas; Jones, Nick S.; ten Wolde, Pieter Rein; Ouldridge, Thomas E.Physical Review Letters (2017), 118 (2), 028101/1-028101/5CODEN: PRLTAO; ISSN:1079-7114. (American Physical Society)We propose a phys. realizable information-driven device consisting of an enzyme in a chem. bath, interacting with pairs of mols. prepd. in correlated states. These correlations persist without direct interaction and thus store free energy equal to the mutual information. The enzyme can harness this free energy, and that stored in the individual mol. states, to do chem. work. Alternatively, the enzyme can use the chem. driving to create mutual information. A modified system can function without external intervention, approaching biol. systems more closely.
- 243Stern, M.; Murugan, A. Learning without neurons in physical systems. Annu. Rev. Condens. Matter Phys. 2023, 14, 417– 41, DOI: 10.1146/annurev-conmatphys-040821-113439There is no corresponding record for this reference.
- 244Shklyaev, O. E.; Balazs, A. C. Interlinking spatial dimensions and kinetic processes in dissipative materials to create synthetic systems with lifelike functionality. Nat. Nanotechnol. 2024, 19, 146, DOI: 10.1038/s41565-023-01530-z244https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB3sXisFGgsrvF&md5=267ff1ebfaaa2263ab3cae55a61cd399Interlinking spatial dimensions and kinetic processes in dissipative materials to create synthetic systems with lifelike functionalityShklyaev, Oleg E.; Balazs, Anna C.Nature Nanotechnology (2024), 19 (2), 146-159CODEN: NNAABX; ISSN:1748-3387. (Nature Portfolio)Abstr.: Biol. systems spontaneously convert energy input into the actions necessary to survive. Motivated by the efficacy of these processes, researchers aim to forge materials systems that exhibit the self-sustained and autonomous functionality found in nature. Success in this effort will require synthetic analogs of the following: a metab. to generate energy, a vasculature to transport energy and materials, a nervous system to transmit 'commands', a musculoskeletal system to translate commands into phys. action, regulatory networks to monitor the entire enterprise, and a mechanism to convert 'nutrients' into growing materials. Design rules must interconnect the material's structural and kinetic properties over ranges of length (that can vary from the nano- to mesoscale) and timescales to enable local energy dissipations to power global functionality. Moreover, by harnessing dynamic interactions intrinsic to the material, the system itself can perform the work needed for its own functionality. Here, we assess the advances and challenges in dissipative materials design and at the same time aim to spur developments in next-generation functional, 'living' materials.
- 245Bray, D. Protein molecules as computational elements in living cells. Nature 1995, 376, 307– 312, DOI: 10.1038/376307a0245https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaK2MXnt1Sjsrg%253D&md5=f124baf6c54d1697e41fb0f9f57a81b2Protein molecules as computational elements in living cellsBray, DennisNature (London) (1995), 376 (6538), 307-12CODEN: NATUAS; ISSN:0028-0836. (Macmillan Magazines)A review with 40 refs. Many proteins in living cells appear to have as their primary function the transfer and processing of information, rather than the chem. transformation of metabolic intermediates or the building of cellular structures. Such protein are functionally linked through allosteric or other mechanisms into biochem. 'circuits' that perform a variety of simple computational tasks including amplification, integration and information storage.
- 246van Sluijs, B.; Zhou, T.; Helwig, B.; Baltussen, M. G.; Nelissen, F. H. T.; Heus, H. A.; Huck, W. T. S. Inverse design of enzymatic reaction network states. Nat. Commun. 2024, 15, 1602, DOI: 10.1038/s41467-024-45886-9246https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB2cXksVeitL0%253D&md5=b30fd58feb0af469782485178069090aIterative design of training data to control intricate enzymatic reaction networksvan Sluijs, Bob; Zhou, Tao; Helwig, Britta; Baltussen, Mathieu G.; Nelissen, Frank H. T.; Heus, Hans A.; Huck, Wilhelm T. S.Nature Communications (2024), 15 (1), 1602CODEN: NCAOBW; ISSN:2041-1723. (Nature Portfolio)Abstr.: Kinetic modeling of in vitro enzymic reaction networks is vital to understand and control the complex behaviors emerging from the nonlinear interactions inside. However, modeling is severely hampered by the lack of training data. Here, we introduce a methodol. that combines an active learning-like approach and flow chem. to efficiently create optimized datasets for a highly interconnected enzymic reactions network with multiple sub-pathways. The optimal exptl. design (OED) algorithm designs a sequence of out-of-equil. perturbations to maximize the information about the reaction kinetics, yielding a descriptive model that allows control of the output of the network towards any cost function. We exptl. validate the model by forcing the network to produce different product ratios while maintaining a min. level of overall conversion efficiency. Our workflow scales with the complexity of the system and enables the optimization of previously unobtainable network outputs.
- 247Zaikin, A. N.; Zhabotinsky, A. M. Concentration wave propagation in two-dimensional liquid-phase self-oscillating system. Nature 1970, 225, 535– 537, DOI: 10.1038/225535b0247https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaE3cXhtVCgsLc%253D&md5=7ef8bca1d462b0284614ebd74939b503Concentration wave propagation in two-dimensional liquid-phase self-oscillating systemZaikin, A. N.; Zhabotinskii, A. M.Nature (London, United Kingdom) (1970), 225 (5232), 535-7CODEN: NATUAS; ISSN:0028-0836.A study on oscillating chem. reactions in the system bromate-bromomalonic acid-ferroin (indicator and catalyst) was made. The reaction was carried out in a thin layer of soln. at 20°. Photographs were taken at 1 min intervals. In the 1st photograph, the catalyst is completely reduced, and subsequent photographs show it starting to be oxidized at particular points (leading points) from which circular waves of oxidn. are propagated. The 4th photograph shows oxidn. taking place in areas not reached by these waves. The next photographs show waves coming from leading centers oxidizing all the space step by step. Radial sym. patterns are also obsd. The obsd. phenomenon is characterized by the occurrence of progressive concn. waves and by a space structure supported at the expense of redox reaction energy. A model for the wave propagation is proposed.