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Perspective

Enabling Broader Adoption of Biocatalysis in Organic Chemistry
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  • Evan O. Romero
    Evan O. Romero
    Life Sciences Institute & Department of Chemistry, University of Michigan, Ann Arbor, Michigan 48109, United States
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    Orcidhttps://orcid.org/0000-0001-8553-1238
  • Anthony T. Saucedo
    Anthony T. Saucedo
    Life Sciences Institute & Department of Chemistry, University of Michigan, Ann Arbor, Michigan 48109, United States
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  • José R. Hernández-Meléndez
    José R. Hernández-Meléndez
    Life Sciences Institute & Department of Chemistry, University of Michigan, Ann Arbor, Michigan 48109, United States
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  • Di Yang
    Di Yang
    Life Sciences Institute & Department of Chemistry, University of Michigan, Ann Arbor, Michigan 48109, United States
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  • Suman Chakrabarty
    Suman Chakrabarty
    Life Sciences Institute & Department of Chemistry, University of Michigan, Ann Arbor, Michigan 48109, United States
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    Orcidhttps://orcid.org/0000-0002-6611-3839
  • Alison R. H. Narayan*
    Alison R. H. Narayan
    Life Sciences Institute & Department of Chemistry, University of Michigan, Ann Arbor, Michigan 48109, United States
    *Email: [email protected]. Phone: +1 (734) 615-5505.
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    Orcidhttps://orcid.org/0000-0001-8290-0077
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JACS Au

Cite this: JACS Au 2023, 3, 8, 2073–2085
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https://doi.org/10.1021/jacsau.3c00263
Published July 19, 2023

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Abstract

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Biocatalysis is becoming an increasingly impactful method in contemporary synthetic chemistry for target molecule synthesis. The selectivity imparted by enzymes has been leveraged to complete previously intractable chemical transformations and improve synthetic routes toward complex molecules. However, the implementation of biocatalysis in mainstream organic chemistry has been gradual to this point. This is partly due to a set of historical and technological barriers that have prevented chemists from using biocatalysis as a synthetic tool with utility that parallels alternative modes of catalysis. In this Perspective, we discuss these barriers and how they have hindered the adoption of enzyme catalysts into synthetic strategies. We also summarize tools and resources that already enable organic chemists to use biocatalysts. Furthermore, we discuss ways to further lower the barriers for the adoption of biocatalysis by the broader synthetic organic chemistry community through the dissemination of resources, demystifying biocatalytic reactions, and increasing collaboration across the field.

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1. Introduction

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Throughout history, humans have employed complex molecules for therapeutic purposes. In modern times, the ability to synthesize these molecules through chemical methods has expanded access to both naturally occurring and synthetic medicinal compounds. (1,2) As a result, synthetic strategies toward complex molecules have evolved tremendously over the past two centuries to arrive at a level of sophistication that directly impacts medicine, agriculture, and other industries. (3,4) People have used enzymes through fermentation since ancient history, but in the 21st century, especially, the use of enzymes for selective synthesis of complex molecules has expanded. (5−7) In particular, the use of enzymes in chemical synthesis has seen a resurgence in the past decade with increasing demand for cleaner and greener reactions. (8) The potential of biocatalysis in complex molecule synthesis is aptly described by Professor Brian Stoltz, a leader and practitioner of organic synthesis: “As a synthetic organic chemist primarily interested in accessing complex structures, I believe that we need to be open to using any and all tools available to build the bonds we desire to make. Enzymes and biocatalysts offer a unique and often orthogonal set of instruments to accomplish this molecular surgery. Recent advances in enzyme engineering bolster our ability to employ these catalysts in our day-to-day laboratory experience. I can only imagine that our toolset of enzymes and biocatalytic transformations will greatly increase in the future.”
The precision and efficiency possible with enzyme catalysts has led to the incorporation of biocatalytic strategies into synthetic campaigns in both academic and industrial spheres. (9,10) Enzymes can operate under ambient conditions and possess the potential for high chemo- and site-selectivity which can override the need to protect nonparticipating functional groups in complex substrates. (11,12) Additionally, the ambient and low-temperature reaction conditions typical for enzymatic reactions enhance their suitability for academic and industrial settings by improving the safety profile, reducing the energy required, and maximizing the overall procedural simplicity. (11−14) Consequently, biocatalytic methods are increasingly being embraced by mainstream organic chemists. (6,7,15−17) The advantages of leveraging enzymes in synthesis can be seen in academic settings, with chemoenzymatic synthesis becoming another tool for elegant total syntheses of complex molecules. (7,14,18−25) For example, biocatalysis has been used to form challenging C–C bonds (Figure 1A), (7,17,19) to introduce C–O bonds in a selective fashion (Figure 1B–D), and to carry out other difficult transformations. (14,21,22,24−26) Furthermore, biocatalytic methods are also becoming commonplace on process scale in the industrial production of pharmaceutical compounds, which is exemplified by Merck’s multienzyme cascade synthesis of islatravir (18) among a growing set of contemporary examples (Figure 1E). (13,14,27,28)

Figure 1

Figure 1. Select examples of chemical structures accessed by using biocatalysis. (A) Compounds formed through C–C bond forming reactions. (B) Compounds accessed using C–H hydroxylation reactions. (C) Hydroxylative dearomatization in the total synthesis of azaphilone natural products. (D) Amino acid C–H hydroxylation in the synthesis of manzacidin C. (E) Multienzyme cascade toward the process-scale total synthesis of islatravir.

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Despite these elegant examples, the organic chemistry community’s overall adoption of biocatalytic methods has been slow; however, with emerging technological advances, biocatalysis is becoming a tool that anyone can integrate into their syntheses. In this Perspective, we provide an analysis of how the use of enzymes in organic chemistry has evolved and become accessible to chemists in the lab. We begin our discussion by highlighting the historical use of enzymes and the technological barriers that have slowed the implementation of enzymatic catalysis in organic chemistry. We then provide insight into potential solutions to these barriers by highlighting powerful resources that currently exist and new steps that can be implemented to broaden the incorporation of biocatalytic and hybrid chemoenzymatic tools in mainstream chemical synthesis.

2. Historical Barriers for Biocatalysis

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2.1. Biocatalysis throughout History

Enzymatic methods have played a pivotal role in the food and beverage industries throughout history, primarily through fermentation processes relying on microorganisms. (29) However, it was not until the 1800s that scientists began to uncover the chemical processes occurring within microorganisms. Crucially, in 1897, Eduard Buchner demonstrated that yeast cell-free extracts could still produce alcohol from glucose, indicating that living cells were not necessary for the process. (30) This discovery was essential in identifying the biomolecules responsible for these reactions and paved the way for further studies of enzyme-mediated processes. Further discoveries in molecular biology and enzymology propelled the field forward as individual enzymes were identified and their mechanisms were uncovered. (31)
Even with these advances, a distinct limitation of the application of biocatalysis in organic chemistry was the accessibility of enzymes capable of performing the desired reactions (Figure 2A). Traditionally, enzymes were accessed using the cells or cell-free extract of the organism where the enzyme is found natively. (32) This meant that researchers would have to obtain the specific strain of an organism. For example, in 1981 Oliveri and co-workers found that a strain of Agrobacterium radiobacter could perform two sequential stereospecific hydrolyses of racemic hydantoins to produce d-amino acids (Figure 2B), molecules which were difficult to access with chemical methods. (33) These products were highly useful for synthesizing antibiotics; however, this enzymatic reaction required a specific microorganism, limiting the widespread applicability of this work. (34)

Figure 2

Figure 2. (A) Historical access to enzymes and enzyme products was a time-consuming process. The understanding of biological systems and the lack of enabling technologies make it difficult to efficiently develop new biocatalysts. (B) Example of an early application of biocatalysts in the synthesis of d-amino acids. This process required the use of a specific strain of bacteria to complete the transformation.

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Although enzyme access could be a barrier for research, the mid- to late-1900s saw an increase in research in this field. Seminal examples of enzyme catalysis include dynamic resolutions, hydrolyses, oxidations, reductions, and ligations. (31) Around this time, more companies also began to sell enzymes extracted from organisms, making biocatalytic transformations slightly more accessible. (31) Scientists also found that individual enzymes could be immobilized to produce desired products more efficiently, such as in the industrial manufacture of penicillanic acid. (35)
Despite this early adoption of enzymes in the production of valuable molecules, the broader implementation of biocatalysis in synthesis was slow due in part to the time it took to discover and understand new enzymes. This is exemplified in the discovery and subsequent understanding of the biosynthesis of morphine, a vital pain medication that has been used for centuries. (36) The structure of morphine was first determined in 1925, (37) but the enzymes involved in its biosynthesis were not elucidated until the 1960s. (36) Clearly, Nature had assembled a group of powerful enzymes that could stitch together a highly complex structure, but researchers did not have the resources or understanding to determine precisely how this was accomplished.
Overall, enzymes have been employed extensively throughout history. Foundational advances in enzymology allowed for specific enzymes to be used for synthetic transformations and laid the groundwork for biocatalysis in organic synthesis.

2.2. Effects of Technological Barriers

In the past, limited knowledge of enzyme synthesis, structure, and function has been a significant barrier to the advancement of biocatalysis. Because enzymes could only be accessed from specific organisms, the breadth of enzymes that could be characterized was meager compared to the millions of proteins that are present in Nature. (38) Furthermore, many of the reports of enzymes throughout history have focused on determining the function of various enzymes, with little emphasis on the breadth of individual enzyme’s capabilities. (31) Consequently, there has been a distinct lack of data covering the overall reactivity of enzymes. This information gap has traditionally hindered the applications of enzymes in organic synthesis as it limits the known starting points for integrating enzymes into synthetic campaigns.
Paralleling this lack of enzyme reactivity and the limited examples of enzymes in organic synthesis, biocatalysis has not traditionally been incorporated into organic chemistry education. Even though enzymes can offer distinct advantages in organic synthesis, they rarely find their way into an organic chemistry classroom or introductory laboratory course. (39) Consequently, students have limited exposure to this valuable strategy of building molecules. This may cause developing scientists to view biocatalysis as a niche tool rather than one they could use in the laboratory.

3. Biocatalysis Today

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Although the aforementioned barriers have historically limited the access to enzymes and their application in organic synthesis laboratories, within the past 50 years there has been significant progress in how we conduct research with enzymes. Many of these advances are centered around determining DNA sequence, access to DNA for genes of interest, and methods for producing enzymes efficiently. (31) The technological innovations developed for these purposes have drastically expanded our knowledge of enzymes and lowered the barriers for obtaining an enzyme of interest.
Knowing the exact sequence of a molecule of DNA was once a massive hurdle; however, the development of robust sequencing technologies means that an exponentially growing number of genomes are publicly available, and that DNA sequencing can be done within individual laboratories, core facilities, and a growing number of companies. (40) As a result, researchers can send a DNA sample to one of the many companies that conduct the sequencing and have sequence data in 1–2 days.
These advances in DNA sequencing technology have led to an explosion of publicly available genetic information. Partial or entire organism genome sequences are consistently being added to online databases, resulting in billions of individual gene sequence entries. (41,42) These DNA sequences of individual annotated genes can be automatically translated to afford the sequence of individual proteins. In most cases, these protein sequences can then be automatically assigned to a protein family based on their similarity to previously characterized proteins. In addition, protein sequences are housed in online databases, such as UniProt, where users can access the sequence and any other information about the proteins. (43) As a result, there is now a wealth of data that researchers can access when choosing enzymes for synthesis.
We recognize that the experimental techniques and equipment used to access biocatalysts and run reactions may be an unknown territory for many synthetic chemists. Below, we outline the basic steps to acquire a biocatalyst and to run biocatalytic reactions (Figure 3). This is not a comprehensive tutorial, but it is intended to provide helpful context to synthetic chemists that may not have previously interacted with this field. Additionally, as there are an increasing number of technologies and companies that are lowering barriers within this process, different entry points will be highlighted to emphasize the growing accessibility of obtaining enzymes.

Figure 3

Figure 3. Accessing biocatalysts with today’s methods. (A) General workflow for producing enzymes from the gene encoding for an enzyme of interest. The various entry points where a scientist could step into the process are highlighted. (B) Enzymes can be used in biocatalytic reactions at various levels of purity.

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3.1. Access to DNA for Genes of Interest

The connection between genetic information and enzymes is crucial for understanding and manipulating enzymes. It was not until the mid-20th century that the link between DNA and proteins was established. This discovery was a major breakthrough in the field of molecular biology as it enabled scientists to understand how genetic information is encoded in DNA and translated into proteins. Even after this discovery, the efficient synthesis of DNA remained a significant challenge, as the technologies to enable this had not yet been developed.
That all changed with the advancement in DNA synthesis technologies such as microarray-based gene synthesis, (44) Gibson assembly, (45) as well as engineering of Terminal deoxynucleotidyl Transferase (TdT) (46) DNA that encode biocatalysts of interest which can be synthesized with high efficiency and high throughput. The DNA fragments can then be cloned into an expression vector of choice for downstream recombinant protein production. Several companies provide DNA synthesis services at low cost and high efficiency. DNA fragments from 300 base pairs up to 10 kilobase pairs can be synthesized, cloned into expression vectors, and delivered to users within a short time frame. At the time of publication of this Perspective, a 1000 base pair gene cloned into a standard expression vector would cost approximately $200.

3.2. Transformation and Heterologous Expression

Heterologous protein expression (also known as recombinant protein expression) can be performed to produce an enzyme of interest. First, the gene that encodes the biocatalyst of interest must be cloned into an expression vector to construct a plasmid, a self-replicating circular DNA with specific selection markers that can be used for gene expression. The gene can then be delivered into the host of choice through transformation, transfection, or transduction. (47) Common heterologous protein expression hosts include Escherichia coli (E. coli), (48) yeast (Pichia pastoris), (49) and mammalian cells. (50) Cells carrying the gene of interest can be selected using the selection marker and protein production can be induced by the addition of a specific chemical or by using self-inducing media. (51) All of the cell strains that are required for heterologous protein expression and the chemicals needed for selection and induction are commercially available, making the process streamlined and straightforward to execute.
This method for producing enzymes represents a significant improvement compared with the traditional way of accessing enzymes, which required isolation from the native organism. Heterologous protein expression allows for a standardized way of producing large quantities of protein. An emerging method for protein production is cell-free expression, where cell lysate containing the molecular machinery for transcription and translation is used in vitro with DNA as the template. This strategy may offer advantages such as more efficient protein production and enhanced control overexpression conditions. (52) The development of protein production methods has been highly enabling in biocatalysis research as it gives researchers a way to rapidly access a protein of interest.

3.3. Preparing Biocatalysts for Reactions

Once cultures have been grown and used to produce the desired protein, it is time to decide how to use the catalyst (Figure 3B). A biocatalyst can be added to reactions in the form of whole cells, cell lysate, or purified protein with each of these methods successively increasing the time and equipment required. The simplest method is to leave the enzyme in the cells and use the whole cell to deliver the catalyst. This method does not require any additional equipment and uses minimal resources. (53) Some situations require the enzyme to be present in solution, which can be achieved by lysing cells mechanically or chemically. This produces what is known as a crude cell lysate, which is a common form of enzyme used in biocatalytic reactions. The lysate can also be lyophilized to produce a powder that can be easier to handle and store. (54) Finally, the most rigorous way to prepare the catalyst is to purify the enzyme from the lysate solution. This is commonly achieved by using affinity chromatography. An example of this is using a Nickel resin that binds a polyhistidine tag which is appended to the enzyme of interest, followed by washing and eluting steps that result in purified protein. (55) Having your target enzyme in the purified form allows for direct quantification of the amount of enzyme and eliminates potentially problematic impurities. That said, the required time and equipment needed to obtain purified protein create a barrier to using protein in this form. Fortunately, running reactions in a whole cell or crude cell lysate format can often be equally as effective as a pure enzyme. (53,54)

3.4. Entry Points for Accessing Biocatalysts

There are numerous entry points available for researchers to participate in the process of obtaining an enzyme. If an enzyme is not commercially available, it can be produced from the DNA that encodes the enzyme of interest. This process starts by obtaining the gene (“Entry Point 1”, Figure 3A), which can be custom ordered from several different vendors. Next, the gene can be inserted into a plasmid using the cloning process described above to give the recombinant plasmid.
Recombinant plasmids can also be readily obtained from commercial sources (“Entry Point 2”, Figure 3A), through either a plasmid repository or custom-made services. Plasmid repositories, such as Addgene, have an inventory of plasmids containing many enzymes of interest. These plasmids are often stocked by research laboratories that want to make their plasmids available to the broader research community. For example, the Addgene repository has over 100,000 plasmids available for purchase. Although these repositories have many plasmids, an enzyme of interest may not be available in a presynthesized plasmid. Fortunately, with the recent advances in gene synthesis, (56) there are now several companies, such as Twist Bioscience and Gene Script, that can routinely make and provide synthetic plasmid within a few weeks. This growing set of vendors removes several technical biochemical steps and is facilitating a wider use of biocatalysis.
With the recombinant plasmid in hand, the process can continue with transformation of the host organism. These transformed recombinant cells can be used directly or stored as glycerol stocks that can be later used to produce enzymes. If an enzyme is discussed in the literature, the research group may have a glycerol stock containing a plasmid for the enzyme of interest that they would be willing to send to other groups (“Entry Point 3”, Figure 3A). In this case, a simple request may allow a researcher to quickly access a strain capable of producing the enzyme of interest.
Some enzymes can be obtained directly from a chemical vendor, similar to ordering a commercially available transition metal catalyst (“Entry Point 4”, Figure 3A). Several companies offer a range of enzymes such as reductases, dehydrogenases, transaminases, and lipases. These do not require researchers to produce the biocatalysts themselves, and instead receive the enzyme as a lyophilized powder that can be directly used in synthesis. These collections of enzymes cover the most commonly used enzyme classes and can be sold in kits that allow nonexperts to screen for biocatalysts that could be applied in valuable functional group interconversions. Furthermore, some companies, such as Prozomix, allow academic researchers to license their enzymes to be sold. This allows commercial access to enzymes that are not typically available through chemical vendors.

3.5. Carrying out and Optimizing Enzymatic Reactions

With protein in hand, setting up a biocatalytic reaction is relatively straightforward and is analogous to a standard “dump and stir” small-molecule chemical reaction. In general, the process is to add buffered solvent, substrate, cosolvent (if necessary), cofactors, and the biocatalyst to a flask. (57) As biocatalytic reactions are often done under ambient conditions in aqueous solvent, rigorous glassware drying or solvent preparation is often unnecessary. Reactions can easily be tracked by common analysis techniques such as thin-layer chromatography, high-performance liquid chromatography, and NMR. (58−60)
Optimizing an enzymatic reaction can be as simple as screening reaction component concentrations, time, and temperature─just as one would for a small-molecule organic reaction. Enzymes can be sensitive and easily denatured outside of their optimal reaction environments (i.e., temperature, pH, solvent, etc.), (61,62) and these factors can often be addressed with simple variations of the reaction conditions. Additionally, new tools and methods are being developed to improve the way biocatalytic reactions are set up. For example, enzyme immobilization is a tool that has enabled the use of biocatalysis in a variety of commonplace organic solvents. (63) Furthermore, this technique has allowed for increased thermal stability, use in one-pot multistep processes, easier catalyst reuse, and improved purification of reaction products. (64) The use of these immobilization techniques has even extended to continuous flow methods, which have extensively demonstrated the robustness of these catalysts in both academic and industrial applications. (65)
In some cases, a given substrate may not be compatible with biocatalytic reaction conditions or with the enzyme itself. For example, organic substrates may have limited solubility in water, but this can often be solved by using a cosolvent or by modulating the substrate to be more soluble in water. In some instances, even poorly water-soluble substrates can still deliver high yields on process scales. (66) In addition, if the enzyme is inefficient at catalyzing a desired reaction, the substrate can sometimes be engineered to more closely match the native substrate of the enzyme. (67) This is straightforward for a synthetic laboratory to achieve and requires only limited biology knowledge and resources.
When developing small-molecule methods, substrate generality is often a point of emphasis. Conversely, in Nature, chemical transformations are often highly selective and enzymes may struggle to transform substrates outside of those that structurally resemble their native substrate. (62) For many synthetic organic chemists, this poses a great barrier to developing methods and syntheses using biocatalysis. However, many tools have recently been developed that may help researchers choose and optimize an enzyme’s reactivity. Some of these tools are summarized below in section 4, and many can be used by a researcher with limited biochemical knowledge.
Another established tool for improving a biocatalytic reaction is enzyme engineering and directed evolution. (68) This powerful strategy has been used to improve enzyme properties, increase selectivity, increase catalytic efficiency, and optimize non-native functions. (69) Unfortunately, enzyme engineering requires specialized technical skills in molecular biology, (70) which makes this approach less accessible to synthetic chemists seeking an improved biocatalyst. There are several companies that can assist with engineering campaigns, and these organizations will surely become more commonplace in the near future. Additionally, many academic groups are conducting research in biocatalyst engineering and are open to collaborations.

3.6. Reaction Miniaturization

Because biocatalysis spans chemistry and biochemistry, a host of tools are available from both fields that can be used to accelerate research. In particular, biochemistry laboratories commonly use workflows that enable researchers to study systems on a very small scale. This allows for high-throughput experiments and rapid analysis of a large set of conditions. Analogously, biocatalytic reactions can easily be miniaturized and subsequently screened to identify ideal conditions, enzymes, or substrates. (71) A common way to accomplish this is by using 96-well plates, (72) but systems utilizing 384-well plates, (73) and even microfluidics (74) have also been demonstrated. These high-throughput platforms allow for other enabling technologies, such as liquid-handling robotics, (75) that have greatly accelerated the pace at which competent biocatalysts can be developed.

3.7. Equipment Requirements

An essential element of performing science in a laboratory is having the necessary equipment. Very little synthetic chemistry can be done without a functioning rotary evaporator, fume hood, or even something as simple as a stir plate. Biocatalysis is not an exception when it comes to the need for specific equipment; however, we acknowledge that this extra expense may be a hindrance for groups interested in using biocatalysts. In some cases, standard laboratory equipment can serve as a suitable substitution for biocatalytic reactions (such as a flask/hot plate instead of an incubator shaker). In situations requiring more technical biochemical equipment, it is likely that other laboratories in most chemistry and biology departments have the basic equipment described in this section. In this case, only a knock on the door or a quick email to a colleague could simplify the way synthetic chemists can access the wide range of reactivity enzymes offer.

4. Current Resources Aiding Biocatalysis

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4.1. Resources for Choosing Biocatalysts

Bioinformatics and computational resources have greatly assisted in the rapid development and data storage of enzyme-mediated reactions. Using tools such as online databases, synthetic organic chemists have rapid and thorough access to increasingly prevalent information about biocatalytic reactions, as they would with traditional synthetic method reports.
To aid synthetic planning and adoption, information about biocatalytic methods should be accessible through existing databases currently utilized by organic chemists. In recent years, two prominent databases, Reaxys and SciFinder, have formatted their search engine features to allow specific searches for biocatalytic transformations. For example, when filtering through reaction results in Reaxys, users can specify “catalyst classes” and limit search results to “organism/enzymes.” Within this specification, users can select the reaction type (i.e., oxidation/reduction, hydrolysis, isomerization, etc.) they would like to investigate. Although SciFinder’s enzymatic reaction search is not as detailed as Reaxys’, this platform does allow its users to utilize a “Biosequence Search” tool, which enables one to search through vast amounts of sequence data (over 500 million biosequences to date). Although the integration of information relating to biocatalytic reactions into these databases is helpful to organic chemists, the depth of information relating to the enzymes necessary for these reactions is lackluster compared to nonbiological reagents. In order to make these biological tools more accessible to chemists, details related to areas like preparation, purification, physical data, source, etc. should be readily available through Reaxys’ and SciFinder’s platforms.
Outside of these platforms familiar to synthetic chemists, other impactful database tools seek to better incorporate biocatalysis into synthetic organic chemistry (Table 1). One example is RetroBioCat, a tool largely curated by William Finnigan and co-workers from Nicholas Turner’s group at the University of Manchester. (75) This online tool provides users with two efficient methods for designing biocatalytic reactions: retrosynthetic planning and known enzyme reactivity tools. Through the retrosynthetic planning tool, users submit a target compound’s SMILES string and generate a network of possible precursors that can be modified via known enzymatic transformations. Additionally, the pathway explorer tool allows users to submit a SMILES string, from which the program will generate pathways to the compound of interest over several steps set by the user. From this, the program ranks each generated pathway based on parameters that the user can also set.
Table 1. Useful Online Resources for Biocatalysis
databasedescription
RetroBioCat (retrobiocat.com)aids in design of biocatalytic reactions/cascades through retrosynthetic approach
EFI-EST/Cytoscape (efi.igb.illinois.edu/efi-est/; cytoscape.org)enables users to generate SSNs and view these generated networks for identification of enzyme homologues and orthologues
PrenDB (prendb.pharmazie.uni-marburg.de/prendb/home)enzyme database with information related to prenyltransferase enzymes
BioCatNet Databases (www.biocatnet.de)database with sequence, structure, and biocatalytic data pertaining to a variety of protein families
BioCyc Collection of Pathway/Genome Databases (www.biocyc.org)database containing extensive sequence data and a variety of bioinformatic tools
Expasy (www.expasy.org)a wide collection of bioinformatic prediction and analysis tools
MACiE (www.ebi.ac.uk/thornton-srv/m-csa)database with extensive details pertaining to enzymatic reaction mechanisms
GTD (randr.nist.gov/enzyme/Default.aspx)database providing details related to thermodynamic parameters of enzymatic reactions
UniProt (www.uniprot.org)database with information pertaining to structure and function of proteins
Protein Data Bank (www.rcsb.org)database that provides reported 3D structures of proteins
EAWAG-BBD (eawag-bbd.ethz.ch/index.html)database with information regarding microbial biocatalytic reactions and biodegradation pathways for chemical compounds
ExplorEnz (www.enzyme-database.org)enzyme database that emphasizes enzyme nomenclature and classification
ESTHER (bioweb.supagro.inra.fr/ESTHER/general?what=index)enzyme database with information related to superfamily of alpha/beta-hydrolases
MEROPS (www.ebi.ac.uk/merops/index.shtml)enzyme database with information related to peptidase enzymes
Lipase database (www.led.uni-stuttgart.de)enzyme database with information related to lipase enzymes
CAZy (www.cazy.org)enzyme database with information related to carbohydrate-active enzymes
RedoxiBase (peroxibase.toulouse.inra.fr)enzyme database with information related to oxidoreductase enzymes
In addition to its retrosynthetic planning tools, RetroBioCat offers an enzyme database that enables users to explore known enzymatic reactions and to explore the application of these enzymes on non-native substrates. The site also offers users the opportunity to contribute additional information to its enzyme database, which allows for increased diversity and greater accessibility to the enzymatic reactions available to synthetic organic chemists.
In cases where a researcher finds an enzyme that is known to perform their reaction of interest but is not capable of converting their substrate, there are bioinformatic tools that can be used to identify similar enzymes that might act on their substrate. One such tool is the National Center for Biotechnology Information’s (NCBI) Basic Local Alignment Search Tool (BLAST). (76) Through this resource, users are able to identify proteins with high sequence similarity to their enzymes of interest via an advanced algorithm that pulls information from enzyme databases hosted by NCBI. The output of this tool is a list of proteins and their corresponding sequence alignment scores relative to the enzyme of interest. These related enzymes can then be produced and assayed to look for the modulated activity.
Phylogenetic trees are another useful bioinformatic tool that scientists can use to look for similar enzymes and define the relationship between enzymes in a family. (77) The generated diagrams offer information related to the evolutionary history of protein families and enable chemists to explore highly related enzymes that may offer improved or alternate reactivity from a the initial query enzyme. (78−80) Phylogenetic trees may be constructed and visualized readily using free programs such as Molecular Evolutionary Genetics Analysis (MEGA) and Ensembl. (81−83)
Sequence similarity networks (SSNs) are also highly valuable in the development of biocatalytic reactions, as they allow users to compare an extensive collection of protein sequences available in online databases. (84,85) Fundamentally, SSNs are a visualization tool that assists scientists in organizing large volumes of protein sequence data, which enables them to better determine sequence-function relationships and identify enzymes for a target transformation. (84) This process is valuable to synthetic organic chemists who wish to explore libraries of enzymes for developing reactions with wide substrate scopes in which enzyme promiscuity or differential reactivity/selectivity is crucial. (26,86−91) SSNs have become widely accessible with the development of the Enzyme Function Initiative - Enzyme Similarity Tool (EFI-EST) provided by the University of Illinois at Urbana–Champaign. (92) This tool allows chemists to easily input sequence data from proteins of interest and generate SSNs within minutes. Furthermore, these generated SSNs can be viewed and using Cytoscape, an open-source platform for processing complex network data. (93) To further assist chemists, Cytoscape also offers many tutorials and demonstrations for new users in processing SSNs. In addition to these tools, key examples of other publicly available enzyme database tools are summarized in Table 1.

4.2. Literature Resources

A growing number of literary resources are available to organic chemists interested in exploring biocatalysis. (72) In his recent perspective, Roger Sheldon, a pioneer in green chemistry and biocatalysis, offers a guide on how traditional organic chemists can access biocatalysts, what enzymes are and how they operate, and discusses how chemists can employ these biological tools for a variety of chemical transformations. (10) In a recent review from the laboratories of the renowned synthetic chemist, Erick Carreira, and innovator in biocatalysis, Nicholas Turner, the authors provide a framework for the implementation of biocatalysis in synthetic organic chemistry through the scope of retrosynthetic analysis. The concept of retrosynthetic analysis has shaped the logic of synthetic planning for organic chemists and has enabled access to countless synthetic targets, and including biocatalysis further expands its usefulness. (94) Furthermore, the Supporting Information documents that are included with biocatalysis or chemoenzymatic reports in the literature may give detailed descriptions of how enzymes were accessed and used in reactions. These are great starting points for becoming familiar with some of the technical aspects of biocatalysis.
In addition to the variety of published articles detailing the opportunity to incorporate biocatalytic methods in synthetic organic chemistry, an assortment of books provide more in-depth information and ideas to assist organic chemists in implementing biocatalysis in areas such as complex molecule synthesis. (95,96) In his recently published book, “Biocatalysis in Organic Synthesis: The Retrosynthesis Approach,” Turner further provides organic chemists a framework for implementing biocatalytically driven transformations based on the familiar technique of retrosynthesis. (97)

4.3. Industry Investment in Biocatalysis

Industrial science often requires optimization for large scale processes which puts them in a position to drive innovation and fine-tune methods, making the tools even more useful for the synthetic community. Biocatalysis is increasingly being adopted by companies, with new examples of large scale enzymatic/chemoenzymatic methods being published every year. (27) These syntheses are often on process scale and can significantly improve previous syntheses. For example, the COVID-19 antiviral drug Molnupiravir was synthesized by Merck with two out of the three total steps of the synthesis using enzymes with a 69% overall yield (Figure 4). (28) Similarly, Pfizer used commercially available enzymes to perform a multikilogram chemoenzymatic synthesis of a selective γ-secretase inhibitor with potential antitumor activity. (98) The continued investment of companies in biocatalysis will surely push forward the development of new technologies and processes that will broaden the applicability of biocatalysis in synthesis.

Figure 4

Figure 4. Chemoenzymatic synthesis of Molnupiravir demonstrated by Merck (right) compared to the previous small-molecule route (left).

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5. How Can We Make Biocatalysis More Accessible?

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Although these resources and tools have started to bring biocatalysis into the realm of synthetic organic chemistry, significant work remains to make enzyme catalysis more accessible to the average chemist. Broadly, there needs to be an expansion of the educational curriculum, technical biocatalysis resources, data repository development, access to a wider selection of enzymes, and increased cross-disciplinary collaboration. As these changes occur, biocatalysis will become a more accessible tool for synthetic organic chemistry laboratories.

5.1. Undergraduate Education and Training

Perhaps one of the most important ways to impact the chemistry community is the early exposure of the next generation of chemists to topics through undergraduate education and laboratory training. In many cases, classroom and laboratory courses are students’ first exposure to the various subsets of chemistry and can be foundational for their engagement with the topics as they progress through their education and careers. There have been significant strides in synthetic organic chemistry in the past 40 years, yet many modern techniques are slow to be incorporated into classroom or laboratory courses. (99,100) This remains true for enzymatic catalysis as there is a distinct lack of biocatalysis in chemistry curricula.
There are many ways that biocatalysis could be introduced to students. One simple way to do this is by presenting biocatalytic methods in parallel with small molecule methods when discussing a given chemical reaction. For example, most introductory organic chemistry courses include a section on carbonyl reduction where students learn traditional racemic and enantioselective methods for accomplishing this transformation. (101) Dehydrogenase enzymes could be presented in parallel with the textbook small molecule reducing agents as a complementary method for carbonyl reduction. (102) This particular example also has the opportunity to bring in meaningful historical context for the use of natural enzymes. (5,103)
Organic chemistry laboratory courses are also a highly impactful way for students to engage in synthetic methods; however, few courses include biocatalysis experiments. Biocatalysis is relatively easy to include in laboratory courses. This hands-on exposure to enzymes would begin dismantling the “black box” in which biocatalysis is often placed. It is possible to design an experiment that requires only standard laboratory equipment, a commercially available enzyme, and the requisite substrate(s).
Laboratory units like this have been designed and implemented previously and successfully teach many different aspects of organic synthesis. (104,105) For example, Johnston and co-workers published a laboratory unit on a multistep chemoenzymatic synthesis off endogenous cannabinergic ligand 2-arachidonoylglycerol (Figure 5). (104) This synthesis introduced students to enzymatic catalysis and also reinforced other essential synthetic techniques.

Figure 5

Figure 5. Example of a chemoenzymatic synthesis used in an undergraduate chemistry laboratory course.

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Every chemistry department should consider evolving their introductory courses to include biocatalysis as the curriculum is updated. The examples referenced herein would be an excellent starting point for instructors who wish to incorporate biocatalysis into their laboratory curriculum.

5.2. Tutorials

Clear, comprehensive, and accessible explanations of what it takes to perform biocatalysis have, to our knowledge, not yet been consolidated as a resource for the community. The books, articles, and Supporting Information documents listed earlier in this Perspective contain excellent basic descriptions of how to do biocatalytic transformations; however, they lack a complete picture of the whole process. This points to the need for a readily available and centralized compilation of technical biocatalysis resources analogous to Not Voodoo, a web site developed by Alison Frontier at the University of Rochester that is an excellent resource for conducting traditional organic chemistry reactions. The tagline for the web site is “Demystifying synthetic organic chemistry since 2004”─a goal it has undoubtedly accomplished with its many visitors over the years. (106) The web site is a compendium of information geared toward enhancing the experimental skills of synthetic organic chemists and includes sections like Tips and Tricks, Chromatography, Workup, Troubleshooting, and several more. This web site offers valuable information for the expert and novice synthetic alike.
A similar resource for biocatalysis would begin to demystify the technical aspects of enzymatic reactions that can be a barrier to its use in the laboratory. A biocatalysis-focused web site could compile practical tips, describe how to find enzymes, address common issues, and any other information useful for enabling the incorporation of enzymes in synthetic chemistry. Furthermore, this web site could include video demonstrations of the various steps of the biocatalysis process to make it even more accessible. An additional component that could be useful is a forum where community members can ask questions and have them answered by biocatalysis experts. This would further the accessibility of enzyme catalysis, while also encouraging discourse that would progress the field.

5.3. Enzyme Activity Data Repositories

As previously noted in this perspective, there are select examples of databases that catalog enzyme activity data. However, many of these are still in their infancy. For example, the data currently represented in RetroBioCat (https://retrobiocat.com/leaderboard) comes from 420 papers with a total of 1773 enzyme sequences. Considering the many institutions and groups publishing on enzymatic reactions, these are representatively small totals.
For these databases to become more broadly useful, they need a more robust foundation of data. This will be accomplished, in part, over time as data continue to be collected and submitted to the database; however, a more proactive approach should be adopted to rapidly make these tools useful to chemists. If all groups participating in biocatalysis or fundamental enzyme research upload experimental enzyme activity data to these repositories, it would improve the utility of these resources for the chemistry community. The cooperative efforts of the chemistry and biochemistry communities will enable these data collections to grow and improve.
In addition to bolstering the effectiveness of enzyme-specific databases, popular chemistry repositories (i.e., Scifinder and Reaxys) must be modified to best facilitate the indexing and presentation of biocatalytic reactions. Although the functionality to search by organisms/enzymes exists, many publications that are focused on biocatalysts are not indexed in the same manner as more conventional organic chemistry. For example, a search in Reaxys for a publication from our lab that describes biocatalytic α-deuteration of amino acids and methyl esters reveals that none of the many “Substances” or “Reactions” have been cataloged. (107) This is in stark contrast to a publication from the same journal in the same year on photocatalytic construction of α-functionalized amine products, (108) which has 66 “Substances” and 35 “Reactions” indexed. These papers contain a similar number of chemical structures and schemes in both the article and Supporting Information, and there are no clear differences that would lead to this difference in indexing.
These types of large databases are often the first step in a chemist’s journey to implement new chemistries in their syntheses. If there is a disparity in the classes of reactions represented, the underrepresented methods will naturally be incorporated much slower. Furthermore, robust data availability is critically important for developing predictive models that can aid in the enzyme catalyst choice. Therefore, we enthusiastically encourage chemical search and retrieval systems like Reaxys and SciFinder to evaluate their methods of indexing new publications and ensure that all types of chemistry are represented in their databases. Scientists conducting research in enzymatic catalysis should also make certain their publications are included in these databases by contacting them if the data are not accurately reflected.

5.4. Continued Interdisciplinary Collaboration and Acceptance

Biocatalysis is a highly interdisciplinary field that combines aspects of biology, engineering, and organic chemistry. Because of this, it often leads to collaboration among experts in separate fields. In particular, to progress the use of biocatalysis in chemical synthesis, continued collaboration between biocatalysis and synthetic organic chemistry groups will be critical. This will progress the field of chemoenzymatic synthesis while also destigmatizing the use of enzymes in synthesis.
Indeed, these types of collaborations have become more regular over the past few decades, and prominent members of the organic chemistry and biocatalysis communities have worked together to demonstrate high-impacting science. For example, in 2020 Todd Hyster, David MacMillan, and co-workers presented a chemoenzymatic method for dynamic kinetic resolution of β-substituted ketones, combining the utility of modern photoredox catalysis and biocatalysis into a useful synthetic platform (Figure 6A). (109) Francis Arnold, Brian Stoltz, and co-workers also joined forces in their enantioselective chemoenzymatic total synthesis of norditerpenoid alkaloid nigelladine A. (15) The synthesis relied on late-stage C–H oxidation, which was not possible with current chemical methods. This was solved by implementing an engineered P450 enzyme which completed the desired oxidation in a regio- and stereoselective manner (Figure 6B).

Figure 6

Figure 6. Examples of chemoenzymatic and enzymatic methods that result from collaborations between organic and biocatalysis research groups.

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In addition to these strong collaborations, some well-established members of the organic chemistry community have taken large steps into biocatalysis and have subsequently made major advances in the field. One such chemist is John Hartwig, who started with biocatalysis collaborations and whose group conducts research on biocatalysis in their own laboratory, making major contributions to the active research area of artificial metalloenzymes. (110,111) An example of this work is displayed in a recent publication where they use a non-natural iridium porphyrin with cytochromes P450 to site-selectively functionalize (sp3) C–H bonds (Figure 6C). (112)
When we asked him about the place of enzymes in organic chemistry, Hartwig commented, “The catalysts produced by Nature possess many advantages, but they also possess many disadvantages and limitations, relative to the catalysts produced by chemists in the laboratory. High selectivity for the creation of complex chemical architectures and genetic encoding that allows evolution are two important attributes of Nature’s catalysts, but the limited range of chemical transformations is one liability. For years, I, and presumably others, have dreamed of interleaving chemical and enzymatic catalysts to create complex structures biosynthetically in an organism and of an ability to evolve organometallic catalysts in the lab or in a living organism. Researchers in the field of artificial metalloenzymes are progressing toward this mutual vision, and one can imagine a future in which synthetic chemists are equally comfortable with small-molecule catalysts and large-molecule enzymes. Remember, it wasn’t so long ago that synthetic organic chemists considered a palladium catalyst useful as a last resort, an iridium catalyst too expensive, and gold best for a children’s bed-time story.”
These collaborations and new ventures from seasoned organic chemists represent only a small fraction of the potential for biocatalytic and chemoenzymatic synthetic strategies. Many early career scientists have already developed robust research programs focused on biocatalysis and are paving the way for others to join the community.

6. Conclusion

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Biocatalysis is a highly beneficial tool for organic synthesis. Despite this, historical barriers have slowed the adoption of enzyme catalysts in the broader organic chemistry toolbox. In this Perspective, we shared how such obstacles can be addressed and how new developments are empowering every organic chemist to use enzymes.
The best way to encourage growth for both organic chemistry and biocatalysis is by collaborating with one another. We encourage every organic chemist to consider contacting colleagues who research biocatalysis to help solve challenging chemical problems. Likewise, we urge those studying enzymes to join synthetic chemists in envisioning new ways to use biocatalysts in complex molecule synthesis and chemoenzymatic methods. Through strategic collaboration, existing barriers will be dismantled and biocatalysts will quickly become a tool not relegated to niche communities, but rather, the door will be open to all organic chemists.

Author Information

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  • Corresponding Author
  • Authors
    • Evan O. Romero - Life Sciences Institute & Department of Chemistry, University of Michigan, Ann Arbor, Michigan 48109, United States;  Orcidhttps://orcid.org/0000-0001-8553-1238
    • Anthony T. Saucedo - Life Sciences Institute & Department of Chemistry, University of Michigan, Ann Arbor, Michigan 48109, United States
    • José R. Hernández-Meléndez - Life Sciences Institute & Department of Chemistry, University of Michigan, Ann Arbor, Michigan 48109, United States
    • Di Yang - Life Sciences Institute & Department of Chemistry, University of Michigan, Ann Arbor, Michigan 48109, United States
    • Suman Chakrabarty - Life Sciences Institute & Department of Chemistry, University of Michigan, Ann Arbor, Michigan 48109, United States;  Orcidhttps://orcid.org/0000-0002-6611-3839
  • Author Contributions

    CRediT: Evan O. Romero writing-original draft, writing-review & editing; Anthony T. Saucedo writing-original draft, writing-review & editing; José R. Hernández-Meléndez writing-original draft, writing-review & editing; Di Yang writing-original draft, writing-review & editing; Suman Chakrabarty writing-original draft, writing-review & editing; Alison R. H. Narayan writing-original draft, writing-review & editing.

  • Notes
    The authors declare no competing financial interest.

Acknowledgments

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The authors acknowledge support from the University of Michigan Life Sciences Institute, University of Michigan Department of Chemistry, the National Institutes of Health Grant R35 GM124880, and NSF 2221346. E.O.R. and A.T.S acknowledge support from the NSF graduate research fellowship (DGE 1841052). J.R.H.M. acknowledges support from the NIH Chemistry-Biology Interface training program (5T32GM132046-03). The authors graciously thank Professor Brian Stoltz and Professor John Hartwig for providing statements included in this piece.

References

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    A review. This crit. review presented an introduction to biocatalysis for synthetic chemists. Advances in biocatalysis of the past 5 years illustrate the breadth of applications for these powerful and selective catalysts in conducting key reaction steps. Asym. synthesis of value-added targets and other reaction types were covered, with an emphasis on pharmaceutical intermediates and bulk chems. Resources of interest for the non-initiated are provided, including specialized web-sites and service providers to facilitate identification of suitable biocatalysts, as well as refs. to recent vols. and reviews for more detailed biocatalytic procedures. Challenges related to the application of biocatalysts were discussed, including how green a biocatalytic reaction may be, and trends in biocatalyst improvement through enzyme engineering were presented (152 refs.).
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  10. 10
    Sheldon, R. A.; Brady, D.; Bode, M. L. The Hitchhiker’s guide to biocatalysis: recent advances in the use of enzymes in organic synthesis. Chem. Sci. 2020, 11, 2587– 2605,  DOI: 10.1039/C9SC05746C
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    The Hitchhiker's guide to biocatalysis: recent advances in the use of enzymes in organic synthesis
    Sheldon Roger A; Brady Dean; Bode Moira L; Sheldon Roger A
    Chemical science (2020), 11 (10), 2587-2605 ISSN:2041-6520.
    Enzymes are excellent catalysts that are increasingly being used in industry and academia. This perspective is primarily aimed at synthetic organic chemists with limited experience using enzymes and provides a general and practical guide to enzymes and their synthetic potential, with particular focus on recent applications.
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    Abdelraheem, E. M. M.; Busch, H.; Hanefeld, U.; Tonin, F. Biocatalysis explained: from pharmaceutical to bulk chemical production. React. Chem. Eng. 2019, 4, 1878– 1894,  DOI: 10.1039/C9RE00301K
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    Biocatalysis explained: from pharmaceutical to bulk chemical production
    Abdelraheem, Eman M. M.; Busch, Hanna; Hanefeld, Ulf; Tonin, Fabio
    Reaction Chemistry & Engineering (2019), 4 (11), 1878-1894CODEN: RCEEBW; ISSN:2058-9883. (Royal Society of Chemistry)
    A review. Biocatalysis is one of the most promising technologies for the sustainable synthesis of mols. for pharmaceutical, biotechnol. and industrial purposes. From the gram to the ton scale, biocatalysis is employed with success. This is underpinned by the fact that the global enzyme market is predicted to increase from $7 billion to $10 billion by 2024. This review concs. on showing the strong benefits that biocatalysis and the use of enzymes can provide to synthetic chem. Several examples of successful implementations of enzymes are discussed highlighting not only high-value pharmaceutical processes but also low-cost bulk products. Thus, biocatalytic methods make the chem. more environmentally friendly and product specific.
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    Sheldon, R. A.; Brady, D. Broadening the Scope of Biocatalysis in Sustainable Organic Synthesis. ChemSusChem 2019, 12, 2859– 2881,  DOI: 10.1002/cssc.201900351
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    Broadening the scope of biocatalysis in sustainable organic synthesis
    Sheldon, Roger A.; Brady, Dean
    ChemSusChem (2019), 12 (13), 2859-2881CODEN: CHEMIZ; ISSN:1864-5631. (Wiley-VCH Verlag GmbH & Co. KGaA)
    This Review is aimed at synthetic org. chemists who may be familiar with organometallic catalysis but have no experience with biocatalysis, and seeks to provide an answer to the perennial question: if it is so attractive, why wasn't it extensively used in the past. The development of biocatalysis in industrial org. synthesis is traced from the middle of the last century. Advances in mol. biol. in the last two decades, in particular genome sequencing, gene synthesis and directed evolution of proteins, have enabled remarkable improvements in scope and substantially reduced biocatalyst development times and cost contributions. Addnl., improvements in biocatalyst recovery and reuse have been facilitated by developments in enzyme immobilization technologies. Biocatalysis has become eminently competitive with chemocatalysis and the biocatalytic prodn. of important pharmaceutical intermediates, such as enantiopure alcs. and amines, has become mainstream org. synthesis. The synthetic space of biocatalysis has significantly expanded and is currently being extended even further to include new-to-nature biocatalytic reactions.
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    Hughes, G.; Lewis, J. C. Introduction: Biocatalysis in Industry. Chem. Rev. 2018, 118, 1– 3,  DOI: 10.1021/acs.chemrev.7b00741
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    Introduction: Biocatalysis in Industry
    Hughes, Greg; Lewis, Jared C.
    Chemical Reviews (Washington, DC, United States) (2018), 118 (1), 1-3CODEN: CHREAY; ISSN:0009-2665. (American Chemical Society)
    There is no expanded citation for this reference.
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    Huffman, M. A.; Fryszkowska, A.; Alvizo, O.; Borra-Garske, M.; Campos, K. R.; Canada, K. A.; Devine, P. N.; Duan, D.; Forstater, J. H.; Grosser, S. T.; Halsey, H. M.; Hughes, G. J.; Jo, J.; Joyce, L. A.; Kolev, J. N.; Liang, J.; Maloney, K. M.; Mann, B. F.; Marshall, N. M.; McLaughlin, M.; Moore, J. C.; Murphy, G. S.; Nawrat, C. C.; Nazor, J.; Novick, S.; Patel, N. R.; Rodriguez-Granillo, A.; Robaire, S. A.; Sherer, E. C.; Truppo, M. D.; Whittaker, A. M.; Verma, D.; Xiao, L.; Xu, Y.; Yang, H. Design of an in vitro biocatalytic cascade for the manufacture of islatravir. Science 2019, 366, 1255– 1259,  DOI: 10.1126/science.aay8484
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    Design of an in vitro biocatalytic cascade for the manufacture of islatravir
    Huffman, Mark A.; Fryszkowska, Anna; Alvizo, Oscar; Borra-Garske, Margie; Campos, Kevin R.; Canada, Keith A.; Devine, Paul N.; Duan, Da; Forstater, Jacob H.; Grosser, Shane T.; Halsey, Holst M.; Hughes, Gregory J.; Jo, Junyong; Joyce, Leo A.; Kolev, Joshua N.; Liang, Jack; Maloney, Kevin M.; Mann, Benjamin F.; Marshall, Nicholas M.; McLaughlin, Mark; Moore, Jeffrey C.; Murphy, Grant S.; Nawrat, Christopher C.; Nazor, Jovana; Novick, Scott; Patel, Niki R.; Rodriguez-Granillo, Agustina; Robaire, Sandra A.; Sherer, Edward C.; Truppo, Matthew D.; Whittaker, Aaron M.; Verma, Deeptak; Xiao, Li; Xu, Yingju; Yang, Hao
    Science (Washington, DC, United States) (2019), 366 (6470), 1255-1259CODEN: SCIEAS; ISSN:1095-9203. (American Association for the Advancement of Science)
    Enzyme-catalyzed reactions have begun to transform pharmaceutical manufg., offering levels of selectivity and tunability that can dramatically improve chem. synthesis. Combining enzymic reactions into multistep biocatalytic cascades brings addnl. benefits. Cascades avoid the waste generated by purifn. of intermediates. They also allow reactions to be linked together to overcome an unfavorable equil. or avoid the accumulation of unstable or inhibitory intermediates. We report an in vitro biocatalytic cascade synthesis of the investigational HIV treatment islatravir. Five enzymes were engineered through directed evolution to act on non-natural substrates. These were combined with four auxiliary enzymes to construct islatravir from simple building blocks in a three-step biocatalytic cascade. The overall synthesis requires fewer than half the no. of steps of the previously reported routes.
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    Loskot, S. A.; Romney, D. K.; Arnold, F. H.; Stoltz, B. M. Enantioselective Total Synthesis of Nigelladine A via Late-Stage C–H Oxidation Enabled by an Engineered P450 Enzyme. J. Am. Chem. Soc. 2017, 139, 10196– 10199,  DOI: 10.1021/jacs.7b05196
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    15
    Enantioselective Total Synthesis of Nigelladine A via Late-Stage C-H Oxidation Enabled by an Engineered P450 Enzyme
    Loskot, Steven A.; Romney, David K.; Arnold, Frances H.; Stoltz, Brian M.
    Journal of the American Chemical Society (2017), 139 (30), 10196-10199CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)
    An enantioselective total synthesis of the norditerpenoid alkaloid nigelladine A is described. Strategically, the synthesis relies on a late-stage C-H oxidn. of an advanced intermediate. While traditional chem. methods failed to deliver the desired outcome, an engineered cytochrome P 450 enzyme was employed to effect a chemo- and regioselective allylic C-H oxidn. in the presence of four oxidizable positions. The enzyme variant was readily identified from a focused library of three enzymes, allowing for completion of the synthesis without the need for extensive screening.
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    Chen, K.; Huang, X.; Kan, S. B. J.; Zhang, R. K.; Arnold, F. H. Enzymatic construction of highly strained carbocycles. Science 2018, 360, 71– 75,  DOI: 10.1126/science.aar4239
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    Enzymatic construction of highly strained carbocycles
    Chen, Kai; Huang, Xiongyi; Kan, S. B. Jennifer; Zhang, Ruijie K.; Arnold, Frances H.
    Science (Washington, DC, United States) (2018), 360 (6384), 71-75CODEN: SCIEAS; ISSN:0036-8075. (American Association for the Advancement of Science)
    Small carbocycles are structurally rigid and possess high intrinsic energy due to their ring strain. These features lead to broad applications but also create challenges for their construction. We report the engineering of a cytochrome P 450 variant (designated P411) that catalyze the formation of chiral bicyclobutanes, one of the most strained four-membered systems, via successive carbene addn. to unsatd. carbon-carbon bonds. Enzymes that produce cyclopropenes, putative intermediates to the bicyclobutanes, were also identified. These genetically encoded proteins are readily optimized by directed evolution, function in Escherichia coli, and act on structurally diverse substrates with high efficiency and selectivity, providing an effective route to many chiral strained structures. This biotransformation is easily performed at preparative scale, and the resulting strained carbocycles can be derivatized, opening myriad potential applications.
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    Zhang, X.; King-Smith, E.; Dong, L.-B.; Yang, L.-C.; Rudolf, J. D.; Shen, B.; Renata, H. Divergent synthesis of complex diterpenes through a hybrid oxidative approach. Science 2020, 369, 799– 806,  DOI: 10.1126/science.abb8271
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    Divergent synthesis of complex diterpenes through a hybrid oxidative approach
    Zhang, Xiao; King-Smith, Emma; Dong, Liao-Bin; Yang, Li-Cheng; Rudolf, Jeffrey D.; Shen, Ben; Renata, Hans
    Science (Washington, DC, United States) (2020), 369 (6505), 799-806CODEN: SCIEAS; ISSN:1095-9203. (American Association for the Advancement of Science)
    Polycyclic diterpenes exhibit many important biol. activities, but de novo synthetic access to these mols. is highly challenging because of their structural complexity. Semisynthetic access has also been limited by the lack of chem. tools for scaffold modifications. We report a chemoenzymic platform to access highly oxidized diterpenes by a hybrid oxidative approach that strategically combines chem. and enzymic oxidn. methods. This approach allows for selective oxidns. of previously inaccessible sites on the parent carbocycles and enables abiotic skeletal rearrangements to addnl. underlying architectures. We synthesized a total of nine complex natural products with rich oxygenation patterns and skeletal diversity in 10 steps or less from ent-steviol.
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    Nakamura, H.; Schultz, E. E.; Balskus, E. P. A new strategy for aromatic ring alkylation in cylindrocyclophane biosynthesis. Nat. Chem. Biol. 2017, 13, 916– 921,  DOI: 10.1038/nchembio.2421
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    A new strategy for aromatic ring alkylation in cylindrocyclophane biosynthesis
    Nakamura, Hitomi; Schultz, Erica E.; Balskus, Emily P.
    Nature Chemical Biology (2017), 13 (8), 916-921CODEN: NCBABT; ISSN:1552-4450. (Nature Publishing Group)
    Alkylation of arom. rings with alkyl halides is an important transformation in org. synthesis, yet an enzymic equiv. is unknown. Here, we report that cylindrocyclophane biosynthesis in Cylindrospermum licheniforme ATCC 29412 involves chlorination of an unactivated carbon center by a novel halogenase, followed by a previously uncharacterized enzymic dimerization reaction featuring sequential, stereospecific alkylations of resorcinol arom. rings. Discovery of the enzymic machinery underlying this unique biosynthetic carbon-carbon bond formation has implications for biocatalysis and metabolic engineering.
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    Schultz, E. E.; Braffman, N. R.; Luescher, M. U.; Hager, H. H.; Balskus, E. P. Biocatalytic Friedel–Crafts Alkylation Using a Promiscuous Biosynthetic Enzyme. Angew. Chem., Int. Ed. 2019, 58, 3151– 3155,  DOI: 10.1002/anie.201814016
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    Biocatalytic Friedel-Crafts Alkylation Using a Promiscuous Biosynthetic Enzyme
    Schultz, Erica E.; Braffman, Nathaniel R.; Luescher, Michael U.; Hager, Harry H.; Balskus, Emily P.
    Angewandte Chemie, International Edition (2019), 58 (10), 3151-3155CODEN: ACIEF5; ISSN:1433-7851. (Wiley-VCH Verlag GmbH & Co. KGaA)
    The Friedel-Crafts alkylation is commonly used in org. synthesis to form aryl-alkyl C-C linkages. However, this reaction lacks the stereospecificity and regiocontrol of enzymic catalysis. Here, we describe a stereospecific, biocatalytic Friedel-Crafts alkylation of the 2-position of resorcinol rings using the cylindrocyclophane biosynthetic enzyme CylK. This regioselectivity is distinct from that of the classical Friedel-Crafts reaction. Numerous secondary alkyl halides are accepted by this enzyme, as are resorcinol rings with a variety of substitution patterns. Finally, we have been able to use this transformation to access novel analogs of the clin. drug candidate benvitimod that are challenging to construct with existing synthetic methods. These findings highlight the promise of enzymic catalysis for enabling mild and selective C-C bond-forming synthetic methodol.
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    Lau, W.; Sattely, E. S. Six enzymes from mayapple that complete the biosynthetic pathway to the etoposide aglycone. Science 2015, 349, 1224– 1228,  DOI: 10.1126/science.aac7202
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    20
    Six enzymes from mayapple that complete the biosynthetic pathway to the etoposide aglycone
    Lau, Warren; Sattely, Elizabeth S.
    Science (Washington, DC, United States) (2015), 349 (6253), 1224-1228CODEN: SCIEAS; ISSN:0036-8075. (American Association for the Advancement of Science)
    Podophyllotoxin is the natural product precursor of the chemotherapeutic etoposide, yet only part of its biosynthetic pathway is known. We used transcriptome mining in Podophyllum hexandrum (mayapple) to identify biosynthetic genes in the podophyllotoxin pathway. We selected 29 candidate genes to combinatorially express in Nicotiana benthamiana (tobacco) and identified six pathway enzymes, including an oxoglutarate-dependent dioxygenase that closes the core cyclohexane ring of the aryltetralin scaffold. By coexpressing 10 genes in tobacco-these 6 plus 4 previously discovered-we reconstitute the pathway to (-)-4'-desmethylepipodophyllotoxin (the etoposide aglycon), a naturally occurring lignan that is the immediate precursor of etoposide and, unlike podophyllotoxin, a potent topoisomerase inhibitor. Our results enable prodn. of the etoposide aglycon in tobacco and circumvent the need for cultivation of mayapple and semisynthetic epimerization and demethylation of podophyllotoxin.
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    Lowell, A. N.; DeMars, M. D.; Slocum, S. T.; Yu, F.; Anand, K.; Chemler, J. A.; Korakavi, N.; Priessnitz, J. K.; Park, S. R.; Koch, A. A.; Schultz, P. J.; Sherman, D. H. Chemoenzymatic Total Synthesis and Structural Diversification of Tylactone-Based Macrolide Antibiotics through Late-Stage Polyketide Assembly, Tailoring, and C─H Functionalization. J. Am. Chem. Soc. 2017, 139, 7913– 7920,  DOI: 10.1021/jacs.7b02875
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    21
    Chemoenzymatic Total Synthesis and Structural Diversification of Tylactone-Based Macrolide Antibiotics through Late-Stage Polyketide Assembly, Tailoring, and C-H Functionalization
    Lowell, Andrew N.; DeMars, Matthew D.; Slocum, Samuel T.; Yu, Fengan; Anand, Krithika; Chemler, Joseph A.; Korakavi, Nisha; Priessnitz, Jennifer K.; Park, Sung Ryeol; Koch, Aaron A.; Schultz, Pamela J.; Sherman, David H.
    Journal of the American Chemical Society (2017), 139 (23), 7913-7920CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)
    Polyketide synthases (PKSs) represent a powerful catalytic platform capable of effecting multiple carbon-carbon bond forming reactions and oxidn. state adjustments. We explored the functionality of two terminal PKS modules that produce the 16-membered tylosin macrocycle, using them as biocatalysts in the chemoenzymic synthesis of tylactone and its subsequent elaboration to complete the first total synthesis of the juvenimicin, M-4365, and rosamicin classes of macrolide antibiotics via late-stage diversification. Synthetic chem. was employed to generate the tylactone hexaketide chain elongation intermediate that was accepted by the juvenimicin (Juv) ketosynthase of the penultimate JuvEIV PKS module. The hexaketide is processed through two complete modules (JuvEIV and JuvEV) in vitro, which catalyze elongation and functionalization of two ketide units followed by cyclization of the resulting octaketide into tylactone. After macrolactonization, a combination of in vivo glycosylation, selective in vitro cytochrome P 450-mediated oxidn., and chem. oxidn. was used to complete the scalable construction of a series of macrolide natural products in as few as 15 linear steps (21 total) with an overall yield of 4.6%.
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  22. 22
    Lukowski, A. L.; Denomme, N.; Hinze, M. E.; Hall, S.; Isom, L. L.; Narayan, A. R. H. Biocatalytic Detoxification of Paralytic Shellfish Toxins. ACS Chem. Biol. 2019, 14, 941– 948,  DOI: 10.1021/acschembio.9b00123
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    Biocatalytic Detoxification of Paralytic Shellfish Toxins
    Lukowski, April L.; Denomme, Nicholas; Hinze, Meagan E.; Hall, Sherwood; Isom, Lori L.; Narayan, Alison R. H.
    ACS Chemical Biology (2019), 14 (5), 941-948CODEN: ACBCCT; ISSN:1554-8929. (American Chemical Society)
    Small mols. that bind to voltage-gated sodium channels (VGSCs) are promising leads in the treatment of numerous neurodegenerative diseases and pain. Nature is a highly skilled medicinal chemist in this regard, designing potent VGSC ligands capable of binding to and blocking the channel, thereby offering compds. of potential therapeutic interest. Paralytic shellfish toxins (PSTs), produced by cyanobacteria and marine dinoflagellates, are examples of these naturally occurring small mol. VGSC blockers that can potentially be leveraged to solve human health concerns. Unfortunately, the remarkable potency of these natural products results in equally exceptional toxicity, presenting a significant challenge for the therapeutic application of these compds. Identifying less potent analogs and convenient methods for accessing them therefore provides an attractive approach to developing mols. with enhanced therapeutic potential. Fortunately, Nature has evolved tools to modulate the toxicity of PSTs through selective hydroxylation, sulfation, and desulfation of the core scaffold. Function of enzymes encoded in cyanobacterial PST biosynthetic gene clusters that have evolved specifically for the sulfation of highly functionalized PSTs, the substrate scope of these enzymes, and elucidate the biosynthetic route from saxitoxin to monosulfated gonyautoxins and disulfated C-toxins. Finally, the binding affinities of the nonsulfated, monosulfated, and disulfated products of these enzymic reactions have been evaluated for VGSC binding affinity using mouse whole brain membrane prepns. to provide an assessment of relative toxicity. These data demonstrate the unique detoxification effect of sulfotransferases in PST biosynthesis, providing a potential mechanism for the development of more attractive PST-derived therapeutic analogs.
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    Wang, J.; Zhang, Y.; Liu, H.; Shang, Y.; Zhou, L.; Wei, P.; Yin, W.-B.; Deng, Z.; Qu, X.; Zhou, Q. A biocatalytic hydroxylation-enabled unified approach to C19-hydroxylated steroids. Nat. Commun. 2019, 10, 3378,  DOI: 10.1038/s41467-019-11344-0
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    A biocatalytic hydroxylation-enabled unified approach to C19-hydroxylated steroids
    Wang Junlin; Shang Yong; Zhou Qianghui; Zhang Yanan; Liu Huanhuan; Zhou Linjun; Deng Zixin; Qu Xudong; Wei Penglin; Yin Wen-Bing
    Nature communications (2019), 10 (1), 3378 ISSN:.
    Steroidal C19-hydroxylation is pivotal to the synthesis of naturally occurring bioactive C19-OH steroids and 19-norsteroidal pharmaceuticals. However, realizing this transformation is proved to be challenging through either chemical or biological synthesis. Herein, we report a highly efficient method to synthesize 19-OH-cortexolone in 80% efficiency at the multi-gram scale. The obtained C19-OH-cortexolone can be readily transformed to various synthetically useful intermediates including the industrially valuable 19-OH-androstenedione, which can serve as a basis for synthesis of C19-functionalized steroids as well as 19-nor steroidal drugs. Using this biocatalytic C19-hydroxylation method, the unified synthesis of six C19-hydroxylated pregnanes is achieved in just 4 to 9 steps. In addition, the structure of sclerosteroid B is revised on the basis of our synthesis.
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    Pyser, J. B.; Baker Dockrey, S. A.; Benítez, A. R.; Joyce, L. A.; Wiscons, R. A.; Smith, J. L.; Narayan, A. R. H. Stereodivergent, Chemoenzymatic Synthesis of Azaphilone Natural Products. J. Am. Chem. Soc. 2019, 141, 18551– 18559,  DOI: 10.1021/jacs.9b09385
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    Stereodivergent, Chemoenzymatic Synthesis of Azaphilone Natural Products
    Pyser, Joshua B.; Baker Dockrey, Summer A.; Benitez, Attabey Rodriguez; Joyce, Leo A.; Wiscons, Ren A.; Smith, Janet L.; Narayan, Alison R. H.
    Journal of the American Chemical Society (2019), 141 (46), 18551-18559CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)
    Selective access to a targeted isomer is often crit. in the synthesis of biol. active mols. Whereas small-mol. reagents and catalysts often act with anticipated site- and stereoselectivity, this predictability does not extend to enzymes. Further, the lack of access to catalysts that provide complementary selectivity creates a challenge in the application of biocatalysis in synthesis. Here, we report an approach for accessing biocatalysts with complementary selectivity that is orthogonal to protein engineering. Through the use of a sequence similarity network (SSN), a no. of sequences were selected, and the corresponding biocatalysts were evaluated for reactivity and selectivity. With a no. of biocatalysts identified that operate with complementary site- and stereoselectivity, these catalysts were employed in the stereodivergent, chemoenzymic synthesis of azaphilone natural products. Specifically, the first syntheses of trichoflectin, deflectin-1a, and lunatoic acid A were achieved. In addn., chemoenzymic syntheses of these azaphilones supplied enantioenriched material for reassignment of the abs. configuration of trichoflectin and deflectin-1a based on optical rotation, CD spectra, and X-ray crystallog.
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    Zwick, C. R.; Renata, H. Remote C–H Hydroxylation by an α-Ketoglutarate-Dependent Dioxygenase Enables Efficient Chemoenzymatic Synthesis of Manzacidin C and Proline Analogs. J. Am. Chem. Soc. 2018, 140, 1165– 1169,  DOI: 10.1021/jacs.7b12918
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    25
    Remote C-H Hydroxylation by an α-Ketoglutarate-Dependent Dioxygenase Enables Efficient Chemoenzymatic Synthesis of Manzacidin C and Proline Analogs
    Zwick, Christian R.; Renata, Hans
    Journal of the American Chemical Society (2018), 140 (3), 1165-1169CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)
    Selective C-H functionalization at distal positions remains a highly challenging problem in org. synthesis. Though Nature has evolved a myriad of enzymes capable of such feat, their synthetic utility has largely been overlooked. Here, we functionally characterize an α-ketoglutarate-dependent dioxygenase (Fe/αKG) that selectively hydroxylates the δ position of various aliph. amino acids. Kinetic anal. and substrate profiling of the enzyme show superior catalytic efficiency and substrate promiscuity relative to other Fe/αKGs that catalyze similar reactions. We demonstrate the practical utility of this transformation in the concise syntheses of a rare alkaloid, manzacidin C, and densely substituted amino acid derivs. with remarkable step efficiency. This work provides a blueprint for future applications of Fe/αKG hydroxylation in complex mol. synthesis and the development of powerful synthetic paradigms centered on enzymic C-H functionalization logic.
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    Lukowski, A. L.; Liu, J.; Bridwell-Rabb, J.; Narayan, A. R. H. Structural basis for divergent C–H hydroxylation selectivity in two Rieske oxygenases. Nat. Commun. 2020, 11, 2991,  DOI: 10.1038/s41467-020-16729-0
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    26
    Structural basis for divergent C-H hydroxylation selectivity in two Rieske oxygenases
    Lukowski, April L.; Liu, Jianxin; Bridwell-Rabb, Jennifer; Narayan, Alison R. H.
    Nature Communications (2020), 11 (1), 2991CODEN: NCAOBW; ISSN:2041-1723. (Nature Research)
    Abstr.: Biocatalysts that perform C-H hydroxylation exhibit exceptional substrate specificity and site-selectivity, often through the use of high valent oxidants to activate these inert bonds. Rieske oxygenases are examples of enzymes with the ability to perform precise mono- or dioxygenation reactions on a variety of substrates. Understanding the structural features of Rieske oxygenases responsible for control over selectivity is essential to enable the development of this class of enzymes for biocatalytic applications. Decades of research has illuminated the crit. features common to Rieske oxygenases, however, structural information for enzymes that functionalize diverse scaffolds is limited. Here, we report the structures of two Rieske monooxygenases involved in the biosynthesis of paralytic shellfish toxins (PSTs), SxtT and GxtA, adding to the short list of structurally characterized Rieske oxygenases. Based on these structures, substrate-bound structures, and mutagenesis expts., we implicate specific residues in substrate positioning and the divergent reaction selectivity obsd. in these two enzymes.
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    Wu, S.; Snajdrova, R.; Moore, J. C.; Baldenius, K.; Bornscheuer, U. T. Biocatalysis: Enzymatic Synthesis for Industrial Applications. Angew. Chem., Int. Ed. 2021, 60, 88– 119,  DOI: 10.1002/anie.202006648
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    Biocatalysis: Enzymatic Synthesis for Industrial Applications
    Wu, Shuke; Snajdrova, Radka; Moore, Jeffrey C.; Baldenius, Kai; Bornscheuer, Uwe T.
    Angewandte Chemie, International Edition (2021), 60 (1), 88-119CODEN: ACIEF5; ISSN:1433-7851. (Wiley-VCH Verlag GmbH & Co. KGaA)
    A review. Biocatalysis has found numerous applications in various fields as an alternative to chem. catalysis. The use of enzymes in org. synthesis, esp. to make chiral compds. for pharmaceuticals as well for the flavors and fragrance industry, are the most prominent examples. In addn., biocatalysts are used on a large scale to make specialty and even bulk chems. This review intends to give illustrative examples in this field with a special focus on scalable chem. prodn. using enzymes. It also discusses the opportunities and limitations of enzymic syntheses using distinct examples and provides an outlook on emerging enzyme classes.
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  28. 28
    McIntosh, J. A.; Benkovics, T.; Silverman, S. M.; Huffman, M. A.; Kong, J.; Maligres, P. E.; Itoh, T.; Yang, H.; Verma, D.; Pan, W.; Ho, H.-I.; Vroom, J.; Knight, A. M.; Hurtak, J. A.; Klapars, A.; Fryszkowska, A.; Morris, W. J.; Strotman, N. A.; Murphy, G. S.; Maloney, K. M.; Fier, P. S. Engineered Ribosyl-1-Kinase Enables Concise Synthesis of Molnupiravir, an Antiviral for COVID-19. ACS Cent. Sci. 2021, 7, 1980– 1985,  DOI: 10.1021/acscentsci.1c00608
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    Engineered ribosyl-1-kinase enables concise synthesis of molnupiravir, an antiviral for COVID-19
    McIntosh, John A.; Benkovics, Tamas; Silverman, Steven M.; Huffman, Mark A.; Kong, Jongrock; Maligres, Peter E.; Itoh, Tetsuji; Yang, Hao; Verma, Deeptak; Pan, Weilan; Ho, Hsing-I.; Vroom, Jonathan; Knight, Anders M.; Hurtak, Jessica A.; Klapars, Artis; Fryszkowska, Anna; Morris, William J.; Strotman, Neil A.; Murphy, Grant S.; Maloney, Kevin M.; Fier, Patrick S.
    ACS Central Science (2021), 7 (12), 1980-1985CODEN: ACSCII; ISSN:2374-7951. (American Chemical Society)
    Molnupiravir (MK-4482) is an investigational antiviral agent that is under development for the treatment of COVID-19. Given the potential high demand and urgency for this compd., it was crit. to develop a short and sustainable synthesis from simple raw materials that would minimize the time needed to manuf. and supply molnupiravir. The route reported here is enabled through the invention of a novel biocatalytic cascade featuring an engineered ribosyl-1-kinase and uridine phosphorylase. These engineered enzymes were deployed with a pyruvate-oxidase-enabled phosphate recycling strategy. Compared to the initial route, this synthesis of molnupiravir is 70% shorter and approx. 7-fold higher yielding. Looking forward, the biocatalytic approach to molnupiravir outlined here is anticipated to have broad applications for streamlining the synthesis of nucleosides in general.
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  29. 29
    Bornscheuer, U. T.; Buchholz, K. Highlights in Biocatalysis – Historical Landmarks and Current Trends. Eng. Life Sci. 2005, 5, 309– 323,  DOI: 10.1002/elsc.200520089
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    Highlights in biocatalysis - historical landmarks and current trends
    Bornscheuer, U. T.; Buchholz, K.
    Engineering in Life Sciences (2005), 5 (4), 309-323CODEN: ELSNAE; ISSN:1618-0240. (Wiley-VCH Verlag GmbH & Co. KGaA)
    A review. Biocatalysis has ancient roots, yet it is developing into a key tool for synthesis in a wide range of applications. Important events in the history of enzyme technol. from the 19th century onwards are highlighted. Considering the most relevant progress steps, the prodn. of penicillanic acid and the impact of genetic engineering are traced in more detail. Applied biocatalysis has been defined as the application of a biocatalyst to achieve a desired conversion selectively, under controlled, mild conditions in a bioreactor. Biocatalysts are currently used to produce a wide range of products in the fields of food manuf. (such as bread, cheese, beer), fine chems. (e.g., amino acids, vitamins), and pharmaceuticals (e.g., derivs. of antibiotics). They not only provide access to innovative products and processes, but also meet criteria of sustainability. In org. synthesis, recombinant technologies and biocatalysts have greatly widened the scope of application. Examples of current applications and processes are given. Recent developments and trends are presented as a survey, covering new methods for accessing biodiversity with new enzymes, directed evolution for improving enzymes, designed cells, and integrated downstream processing.
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    Buchner, E. Alkoholische Gährung ohne Hefezellen. Berichte der deutschen chemischen Gesellschaft 1897, 30, 117– 124,  DOI: 10.1002/cber.18970300121
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    Alcoholic fermentation without yeast cells
    Buchner, Eduard
    Berichte der Deutschen Chemischen Gesellschaft (1897), 30 (), 117-24CODEN: BDCGAS ISSN:.
    When brewery yeast, to which no starch has been added, is ground with quartz sand and kieselguhr, moistened with water and pressed, the liquid which is obtained has the power of producing the fermentation of sugar, although it appears to be quite free from yeast cells. It has a sp. gr. of 1.0416, contains about 10 per cent. of residue, and gelatinises when boiled. This liquid produces alcoholic fermentation in solutions of cane-sugar, maltose, glucose, and fructose, but does not ferment either lactose or mannitol. Fermentation continued in many cases for two weeks, even at the temperature of 0°, and was not stopped by nitration of the liquid through a Berkefeldt filter. Plate cultures showed that in some cases small numbers of micro-organisms were present, but yeast cells were in no case detected. The author gives the name zymase to the substance which produces the fermentation. This appears to be a proteid, since the fermentative power of the solution is practically destroyed when it is heated for an hour at 40-50° and the coagulated albumin filtered off. The dried precipitate produced by alcohol does not yield any ferment to water.
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    Heckmann, C. M.; Paradisi, F. Looking Back: A Short History of the Discovery of Enzymes and How They Became Powerful Chemical Tools. ChemCatChem. 2020, 12, 6082– 6102,  DOI: 10.1002/cctc.202001107
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    Looking Back: A Short History of the Discovery of Enzymes and How They Became Powerful Chemical Tools
    Heckmann, Christian M.; Paradisi, Francesca
    ChemCatChem (2020), 12 (24), 6082-6102CODEN: CHEMK3; ISSN:1867-3880. (Wiley-VCH Verlag GmbH & Co. KGaA)
    A review. Enzymic approaches to challenges in chem. synthesis are increasingly popular and very attractive to industry given their green nature and high efficiency compared to traditional methods. In this historical review the authors highlight the developments across several fields that were necessary to create the modern field of biocatalysis, with enzyme engineering and directed evolution at its core. The authors exemplify the modular, incremental, and highly unpredictable nature of scientific discovery, driven by curiosity, and showcase the resulting examples of cutting-edge enzymic applications in industry.
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    Whitesides, G. M. Applications of Cell-Free Enzymes in Organic Synthesis. In Ciba Foundation Symposium 111 - Enzymes in Organic Synthesis; Pitman: London, 1985; pp 76– 96.
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    Olivieri, R.; Fascetti, E.; Angelini, L.; Degen, L. Microbial transformation of racemic hydantoins to d-amino acids. Biotechnol. Bioeng. 1981, 23, 2173– 2183,  DOI: 10.1002/bit.260231002
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    Microbial transformation of racemic hydantoins to D-amino acids
    Olivieri, R.; Fascetti, E.; Angelini, L.; Degen, L.
    Biotechnology and Bioengineering (1981), 23 (10), 2173-83CODEN: BIBIAU; ISSN:0006-3592.
    Resting cells of Agrobacterium radiobacter catalyze a sequence of 2 stereospecific hydrolytic reactions leading to the complete transformation of racemic hydantoins to D-amino acids. These hydantoinase [9030-74-4] and N-carbamoyl-D-amino acid amidohydrolase [71768-08-6] activities and their potential application for the prodn. of some D-amino acids, which are used as intermediates in the prepn. of semisynthetic penicillins and cephalosporins, are described.
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    Liu, Y.; Zhu, L.; Qi, W.; Yu, B. Biocatalytic production of D-p-hydroxyphenylglycine by optimizing protein expression and cell wall engineering in Escherichia coli. Appl. Microbiol. Biotechnol. 2019, 103, 8839– 8851,  DOI: 10.1007/s00253-019-10155-z
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    Biocatalytic production of D-p-hydroxyphenylglycine by optimizing protein expression and cell wall engineering in Escherichia coli
    Liu, Yang; Zhu, Lingfeng; Qi, Wenpeng; Yu, Bo
    Applied Microbiology and Biotechnology (2019), 103 (21-22), 8839-8851CODEN: AMBIDG; ISSN:0175-7598. (Springer)
    D-p-hydroxyphenylglycine (D-HPG) functions as an intermediate and has important value in antibiotic industries. The high pollution and costs from chem. processes make biotechnol. route for D-HPG highly desirable. Here, a whole-cell transformation process by D-hydantoinase(Hase) and D-carbamoylase(Case) was developed to produce D-HPG from DL-hydroxyphenylhydantoin(DL-HPH) in Escherichia coli. The artificially designed ribosome binding site with strong intensity significantly facilitated the protein expression of limiting step enzyme Case. Next, the cell wall permeability was improved by disturbing the peptidoglycan structure by overprodn. of D,D-carboxypeptidases without obviously affecting cell growth, to increase the bioavailability of low sol. hydantoin substrate. By fine-tuning regulation of expression level of D,D-carboxypeptidase DacB, the final prodn. yield of D-HPG increased to 100% with 140 mM DL-HPH substrate under the optimized transformation conditions. This is the first example to enhance bio-productivity of chems. by cell wall engineering and creates a new vision on biotransformation of sparingly sol. substrates. Addnl., the newly demonstrated 'hydroxyl occupancy' phenomenon when Case reacts with hydroxyl substrates provides a referential information for the enzyme engineering in future.
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    Buchholz, K. A breakthrough in enzyme technology to fight penicillin resistance─industrial application of penicillin amidase. Appl. Microbiol. Biotechnol. 2016, 100, 3825– 3839,  DOI: 10.1007/s00253-016-7399-6
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    A breakthrough in enzyme technology to fight penicillin resistance-industrial application of penicillin amidase
    Buchholz, Klaus
    Applied Microbiology and Biotechnology (2016), 100 (9), 3825-3839CODEN: AMBIDG; ISSN:0175-7598. (Springer)
    A review. Enzymic penicillin hydrolysis by penicillin amidase (also penicillin acylase, PA) represents a Landmark: the first industrially and economically highly important process using an immobilized biocatalyst. Resistance of infective bacteria to antibiotics had become a major topic of research and industrial activities. Solns. to this problem, the antibiotics resistance of infective microorganisms, required the search for new antibiotics, but also the development of derivs., notably penicillin derivs., that overcame resistance. An obvious route was to hydrolyze penicillin to 6-aminopenicillanic acid (6-APA), as a first step, for the introduction via chem. synthesis of various different side chains. Hydrolysis via chem. reaction sequences was tedious requiring large amts. of toxic chems., and they were cost intensive. Enzymic hydrolysis using penicillin amidase represented a much more elegant route. The basis for such a soln. was the development of techniques for enzyme immobilization, a highly difficult task with respect to industrial application. Two pioneer groups started to develop solns. to this problem in the late 1960s and 1970s: that of Gunter Schmidt-Kastner at Bayer AG (Germany) and that of Malcolm Lilly of Imperial College London. Here, one example of this development, that at Bayer, will be presented in more detail since it illustrates well the achievement of a soln. to the problems of industrial application of enzymic processes, notably development of an immobilization method for penicillin amidase suitable for scale up to application in industrial reactors under economic conditions. A range of bottlenecks and tech. problems of large-scale application had to be overcome. Data giving an inside view of this pioneer achievement in the early phase of the new field of biocatalysis are presented. The development finally resulted in a highly innovative and com. important enzymic process to produce 6-APA that created a new antibiotics industry and that opened the way for the establishment of over 100 industrial processes with immobilized biocatalysts worldwide today.
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    Wicks, C.; Hudlicky, T.; Rinner, U. Morphine alkaloids: History, biology, and synthesis. In The Alkaloids: Chemistry and Biology; Knölker, H.-J., Ed.; Academic Press: 2021; Vol. 86, Ch. 2, pp 145– 342.
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    Gulland, J. M.; Robinson, R. Constitution of codeine and thebaine. Mem. Proc. Manchester Lit. Philos. Soc. 1925, 69, 79– 86
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    Constitution of codeine and thebaine
    Gulland, J. M.; Robinson, R.
    (1925), 69 (), 79-86 ISSN:.
    Hydroxycodeinone (I) and its dihydro deriv. (II) do not reduce Fehling soln. or NH4OH-Ag2O, even on warming; both bases are recovered largely unchanged from solns. in 30% aq. H2SO4 after boiling 2.5 h. These facts argue against the formulation of I as an α-hydroxy ketone. Bromocodeinone and I yield the same I oxime, m. 279°. I is considered 14-hydroxycodeinone. Structural formulas are suggested for these compds. II condenses with piperonal to form an amorphous yellow powder, C26H25O6N, giving a red soln. in concd. HCl and a purplish red color in H2SO4. The corresponding solns. of the benzylidene deriv. are colorless and red, resp. I gives similar derivs., showing the same color reactions, but analyses indicate the occurrence of redn. as well as condensation. Both I and II condense with o-HOC6H4CHO, giving the orange-red solns. characteristic of the salts of most salicylidene-ketones. I and 6-aminopiperonal condense with EtONa to give the compd. C26H26O6N2, crystg. with 1C6H6 m. 243-4°; it gives Gadamer's test and thus contains the CH2O2 group. II gives a dianhydro-6-aminopiperonaldihydrohydroxycodeinone, C26H24O6N2, m. 282-3° (decompn.); the colorless H2SO4 soln. does not exhibit fluorescence.
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    Armstrong, E. F. Enzymes: A Discovery and its Consequences. Nature 1933, 131, 535– 537,  DOI: 10.1038/131535a0
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    Mohan, R. S.; Mejia, M. P. Environmentally Friendly Organic Chemistry Laboratory Experiments for the Undergraduate Curriculum: A Literature Survey and Assessment. J. Chem. Educ. 2020, 97, 943– 959,  DOI: 10.1021/acs.jchemed.9b00753
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    Environmentally Friendly Organic Chemistry Laboratory Experiments for the Undergraduate Curriculum: A Literature Survey and Assessment
    Mohan, Ram S.; Mejia, Maria P.
    Journal of Chemical Education (2020), 97 (4), 943-959CODEN: JCEDA8; ISSN:0021-9584. (American Chemical Society and Division of Chemical Education, Inc.)
    A review. Due to a growing awareness of environmental issues, green chem. concepts are increasingly being incorporated into the undergraduate org. chem. lecture and lab. component. This minireview summarizes environmentally friendly org. chem. expts. suitable for undergraduate labs. Whenever feasible, LD50 values for various chems. are provided to allow readers to det. the suitability of an expt. for their curriculum based on the toxicity of reagents used.
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    Heather, J. M.; Chain, B. The sequence of sequencers: The history of sequencing DNA. Genomics 2016, 107, 1– 8,  DOI: 10.1016/j.ygeno.2015.11.003
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    The sequence of sequencers: The history of sequencing DNA
    Heather, James M.; Chain, Benjamin
    Genomics (2016), 107 (1), 1-8CODEN: GNMCEP; ISSN:0888-7543. (Elsevier Inc.)
    A review. Detg. the order of nucleic acid residues in biol. samples is an integral component of a wide variety of research applications. Over the last fifty years large nos. of researchers have applied themselves to the prodn. of techniques and technologies to facilitate this feat, sequencing DNA and RNA mols. This time-scale has witnessed tremendous changes, moving from sequencing short oligonucleotides to millions of bases, from struggling towards the deduction of the coding sequence of a single gene to rapid and widely available whole genome sequencing. This article traverses those years, iterating through the different generations of sequencing technol., highlighting some of the key discoveries, researchers, and sequences along the way.
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    Baxevanis, A. D. Using Genomic Databases for Sequence-Based Biological Discovery. Mol. Med. 2003, 9, 185– 192,  DOI: 10.1007/BF03402130
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    Using genomic databases for sequence-based biological discovery
    Baxevanis, Andreas D.
    Molecular Medicine (Manhasset, NY, United States) (2003), 9 (9-12), 185-192CODEN: MOMEF3; ISSN:1076-1551. (North Shore-Long Island Jewish Research Institute)
    A review. The inherent potential underlying the sequence data produced by the International Human Genome Sequencing Consortium and other systematic sequencing projects is, obviously, tremendous. As such, it becomes increasingly important that all biologists have the ability to navigate through and cull important information from key publicly available databases. The continued rapid rise in available sequence information, particularly as model organism data is generated at breakneck speed, also underscores the necessity for all biologists to learn how to effectively make their way through the expanding "sequence information space.". This review discusses some of the more commonly used tools for sequence discovery; tools have been developed for the effective and efficient mining of sequence information. These include LocusLink, which provides a gene-centric view of sequence-based information, as well as the 3 major genome browsers: the National Center for Biotechnol. Information Map Viewer, the University of California Santa Cruz Genome Browser, and the European Bioinformatics Institute's Ensembl system. An overview of the types of information available through each of these front-ends is given, as well as information on tutorials and other documentation intended to increase the reader's familiarity with these tools.
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    The UniProt Consortium UniProt: the universal protein knowledgebase. Nucleic Acids Res. 2017, 45, D158– D169,  DOI: 10.1093/nar/gkw1099
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    LeProust, E. M.; Peck, B. J.; Spirin, K.; McCuen, H. B.; Moore, B.; Namsaraev, E.; Caruthers, M. H. Synthesis of high-quality libraries of long (150mer) oligonucleotides by a novel depurination controlled process. Nucleic Acids Res. 2010, 38, 2522– 2540,  DOI: 10.1093/nar/gkq163
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    Synthesis of high-quality libraries of long (150mer) oligonucleotides by a novel depurination controlled process
    LeProust Emily M; Peck Bill J; Spirin Konstantin; McCuen Heather Brummel; Moore Bridget; Namsaraev Eugeni; Caruthers Marvin H
    Nucleic acids research (2010), 38 (8), 2522-40 ISSN:.
    We have achieved the ability to synthesize thousands of unique, long oligonucleotides (150mers) in fmol amounts using parallel synthesis of DNA on microarrays. The sequence accuracy of the oligonucleotides in such large-scale syntheses has been limited by the yields and side reactions of the DNA synthesis process used. While there has been significant demand for libraries of long oligos (150mer and more), the yields in conventional DNA synthesis and the associated side reactions have previously limited the availability of oligonucleotide pools to lengths <100 nt. Using novel array based depurination assays, we show that the depurination side reaction is the limiting factor for the synthesis of libraries of long oligonucleotides on Agilent Technologies' SurePrint DNA microarray platform. We also demonstrate how depurination can be controlled and reduced by a novel detritylation process to enable the synthesis of high quality, long (150mer) oligonucleotide libraries and we report the characterization of synthesis efficiency for such libraries. Oligonucleotide libraries prepared with this method have changed the economics and availability of several existing applications (e.g. targeted resequencing, preparation of shRNA libraries, site-directed mutagenesis), and have the potential to enable even more novel applications (e.g. high-complexity synthetic biology).
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    Gibson, D. G.; Young, L.; Chuang, R.-Y.; Venter, J. C.; Hutchison, C. A.; Smith, H. O. Enzymatic assembly of DNA molecules up to several hundred kilobases. Nat. Methods 2009, 6, 343– 345,  DOI: 10.1038/nmeth.1318
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    Enzymatic assembly of DNA molecules up to several hundred kilobases
    Gibson, Daniel G.; Young, Lei; Chuang, Ray-Yuan; Venter, J. Craig; Hutchison, Clyde A.; Smith, Hamilton O.
    Nature Methods (2009), 6 (5), 343-345CODEN: NMAEA3; ISSN:1548-7091. (Nature Publishing Group)
    The authors describe an isothermal, single-reaction method for assembling multiple overlapping DNA mols. by the concerted action of a 5' exonuclease, a DNA polymerase and a DNA ligase. First they recessed DNA fragments, yielding single-stranded DNA overhangs that specifically annealed, and then covalently joined them. This assembly method can be used to seamlessly construct synthetic and natural genes, genetic pathways and entire genomes, and could be a useful mol. engineering tool.
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    Loftie-Eaton, W.; Heinisch, T.; Soskine, M.; Champion, E.; Godron, X.; Ybert, T. Novel Variants of Endonuclease V and Uses Thereof. WO2022/090057, 2022.
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    Moustafa, K.; Makhzoum, A.; Trémouillaux-Guiller, J. Molecular farming on rescue of pharma industry for next generations. Crit. Rev. Biotechnol. 2016, 36, 840– 850,  DOI: 10.3109/07388551.2015.1049934
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    Molecular farming on rescue of pharma industry for next generations
    Moustafa, Khaled; Makhzoum, Abdullah; Tremouillaux-Guiller, Jocelyne
    Critical Reviews in Biotechnology (2016), 36 (5), 840-850CODEN: CRBTE5; ISSN:0738-8551. (Taylor & Francis Ltd.)
    A review. Recombinant proteins expressed in plants have been emerged as a novel branch of the biopharmaceutical industry, offering practical and safety advantages over traditional approaches. Cultivable in various platforms (i.e. open field, greenhouses or bioreactors), plants hold great potential to produce different types of therapeutic proteins with reduced risks of contamination with human and animal pathogens. To maximize the yield and quality of plant-made pharmaceuticals, crucial factors should be taken into account, including host plants, expression cassettes, subcellular localization, post-translational modifications, and protein extn. and purifn. methods. DNA technol. and genetic transformation methods have also contributed to great parts with substantial improvements. To play their proper function and stability, proteins require multiple post-translational modifications such as glycosylation. Intensive glycoengineering research has been performed to reduce the immunogenicity of recombinant proteins produced in plants. Important strategies have also been developed to minimize the proteolysis effects and enhance protein accumulation. With growing human population and new epidemic threats, the need for new medications will be paramount so that the traditional pharmaceutical industry will not be alone to answer medication demands for upcoming generations. Here, we review several aspects of plant mol. pharming and outline some important challenges that hamper these ambitious biotechnol. developments.
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    Advances in Escherichia coli production of therapeutic proteins
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    Current Opinion in Biotechnology (2001), 12 (2), 195-201CODEN: CUOBE3; ISSN:0958-1669. (Elsevier Science Ltd.)
    A review with 59 refs. Escherichia coli offers a means for the rapid and economical prodn. of recombinant proteins. These advantages, coupled with a wealth of biochem. and genetic knowledge, have enabled the prodn. of such economically sensitive products as insulin and bovine growth hormone. Although significant progress has been made in transcription, translation and secretion, one of the major challenges is obtaining the product in a sol. and bioactive form. Recent progress in oxidative cytoplasmic folding and cell-free protein synthesis offers attractive alternatives to std. expression methods.
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    Karbalaei, M.; Rezaee, S. A.; Farsiani, H. Pichia pastoris: A highly successful expression system for optimal synthesis of heterologous proteins. J. Cell. Physiol. 2020, 235, 5867– 5881,  DOI: 10.1002/jcp.29583
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    Pichia pastoris: A highly successful expression system for optimal synthesis of heterologous proteins
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    Journal of Cellular Physiology (2020), 235 (9), 5867-5881CODEN: JCLLAX; ISSN:0021-9541. (Wiley-Blackwell)
    One of the most important branches of genetic engineering is the expression of recombinant proteins using biol. expression systems. Nowadays, different expression systems are used for the prodn. of recombinant proteins including bacteria, yeasts, molds, mammals, plants, and insects. Yeast expression systems such as Saccharomyces cerevisiae (S. cerevisiae) and Pichia pastoris (P. pastoris) are more popular. P. pastoris expression system is one of the most popular and std. tools for the prodn. of recombinant protein in mol. biol. Overall, the benefits of protein prodn. by P. pastoris system include appropriate folding (in the endoplasmic reticulum) and secretion (by Kex2 as signal peptidase) of recombinant proteins to the external environment of the cell. Moreover, in the P. pastoris expression system due to its limited prodn. of endogenous secretory proteins, the purifn. of recombinant protein is easy. It is also considered a unique host for the expression of subunit vaccines which could significantly affect the growing market of medical biotechnol. Although P. pastoris expression systems are impressive and easy to use with well-defined process protocols, some degree of process optimization is required to achieve max. prodn. of the target proteins. Methanol and sorbitol concn., Mut forms, temp. and incubation time have to be adjusted to obtain optimal conditions, which might vary among different strains and externally expressed protein. Eventually, optimal conditions for the prodn. of a recombinant protein in P. pastoris expression system differ according to the target protein.
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    Silverman, 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.
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    de Carvalho, C. C. C. R. Whole cell biocatalysts: essential workers from Nature to the industry. Microb. Biotechnol. 2017, 10, 250– 263,  DOI: 10.1111/1751-7915.12363
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    Whole cell biocatalysts: essential workers from Nature to the industry
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    Microbial biotechnology (2017), 10 (2), 250-263 ISSN:.
    Microorganisms have been exposed to a myriad of substrates and environmental conditions throughout evolution resulting in countless metabolites and enzymatic activities. Although mankind have been using these properties for centuries, we have only recently learned to control their production, to develop new biocatalysts with high stability and productivity and to improve their yields under new operational conditions. However, microbial cells still provide the best known environment for enzymes, preventing conformational changes in the protein structure in non-conventional medium and under harsh reaction conditions, while being able to efficiently regenerate necessary cofactors and to carry out cascades of reactions. Besides, a still unknown microbe is probably already producing a compound that will cure cancer, Alzeihmer's disease or kill the most resistant pathogen. In this review, the latest developments in screening desirable activities and improving production yields are discussed.
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    Alissandratos, A. In vitro multi-enzymatic cascades using recombinant lysates of E. coli: an emerging biocatalysis platform. Biophys. Rev. 2020, 12, 175– 182,  DOI: 10.1007/s12551-020-00618-3
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    In vitro multi-enzymatic cascades using recombinant lysates of E. coli: an emerging biocatalysis platform
    Alissandratos, Apostolos
    Biophysical Reviews (2020), 12 (1), 175-182CODEN: BRIECG; ISSN:1867-2450. (Springer)
    A review. Abstr.: In recent years, cell-free exts. (or lysates) have (re-)emerged as a third route to the traditional options of isolated or whole-cell biocatalysts. Advances in mol. biol. and genetic engineering enable facile prodn. of recombinant cell-free exts., where endogenous enzymes are enriched with heterologous activities. These inexpensive prepns. may be used to catalyze multistep enzymic reactions without the constraints of cell toxicity and the cell membrane or the cost and complexity assocd. with prodn. of isolated biocatalysts. Herein, we present an overview of the key advancements in cell-free synthetic biol. that have led to the emergence of cell-free exts. as a promising biocatalysis platform.
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    Gräslund, S.; Nordlund, P.; Weigelt, J.; Hallberg, B. M.; Bray, J.; Gileadi, O.; Knapp, S.; Oppermann, U.; Arrowsmith, C.; Hui, R.; Ming, J.; dhe-Paganon, S.; Park, H.-w.; Savchenko, A.; Yee, A.; Edwards, A.; Vincentelli, R.; Cambillau, C.; Kim, R.; Kim, S.-H.; Rao, Z.; Shi, Y.; Terwilliger, T. C.; Kim, C.-Y.; Hung, L.-W.; Waldo, G. S.; Peleg, Y.; Albeck, S.; Unger, T.; Dym, O.; Prilusky, J.; Sussman, J. L.; Stevens, R. C.; Lesley, S. A.; Wilson, I. A.; Joachimiak, A.; Collart, F.; Dementieva, I.; Donnelly, M. I.; Eschenfeldt, W. H.; Kim, Y.; Stols, L.; Wu, R.; Zhou, M.; Burley, S. K.; Emtage, J. S.; Sauder, J. M.; Thompson, D.; Bain, K.; Luz, J.; Gheyi, T.; Zhang, F.; Atwell, S.; Almo, S. C.; Bonanno, J. B.; Fiser, A.; Swaminathan, S.; Studier, F. W.; Chance, M. R.; Sali, A.; Acton, T. B.; Xiao, R.; Zhao, L.; Ma, L. C.; Hunt, J. F.; Tong, L.; Cunningham, K.; Inouye, M.; Anderson, S.; Janjua, H.; Shastry, R.; Ho, C. K.; Wang, D.; Wang, H.; Jiang, M.; Montelione, G. T.; Stuart, D. I.; Owens, R. J.; Daenke, S.; Schütz, A.; Heinemann, U.; Yokoyama, S.; Büssow, K.; Gunsalus, K. C.; Structural Genomics, C.; Architecture et Fonction des Macromolécules, B.; Berkeley Structural Genomics, C.; China Structural Genomics, C.; Integrated Center for, S.; Function, I.; Israel Structural Proteomics, C.; Joint Center for Structural, G.; Midwest Center for Structural, G.; New York Structural Genomi, X. R. C. f. S. G.; Northeast Structural Genomics, C.; Oxford Protein Production, F.; Protein Sample Production Facility, M. D. C. f. M. M.; Initiative, R. S. G. P.; Complexes, S. Protein production and purification. Nat. Methods 2008, 5, 135– 146,  DOI: 10.1038/nmeth.f.202
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    Protein production and purification
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    Nature methods (2008), 5 (2), 135-46 ISSN:.
    In selecting a method to produce a recombinant protein, a researcher is faced with a bewildering array of choices as to where to start. To facilitate decision-making, we describe a consensus 'what to try first' strategy based on our collective analysis of the expression and purification of over 10,000 different proteins. This review presents methods that could be applied at the outset of any project, a prioritized list of alternate strategies and a list of pitfalls that trip many new investigators.
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    Hughes, R. A.; Ellington, A. D. Synthetic DNA Synthesis and Assembly: Putting the Synthetic in Synthetic Biology. Cold Spring Harbor Perspect. Biol. 2017, 9, a023812,  DOI: 10.1101/cshperspect.a023812
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    Synthetic DNA synthesis and assembly: putting the synthetic in synthetic biology
    Hughes, Randall A.; Ellington, Andrew D.
    Cold Spring Harbor Perspectives in Biology (2017), 9 (1), a023812/1-a023812/18CODEN: CSHPEU; ISSN:1943-0264. (Cold Spring Harbor Laboratory Press)
    The chem. synthesis of DNA oligonucleotides and their assembly into synthons, genes, circuits, and even entire genomes by gene synthesis methods has become an enabling technol. for modern mol. biol. and enables the design, build, test, learn, and repeat cycle underpinning innovations in synthetic biol. In this perspective, we briefly review the techniques and technologies that enable the synthesis of DNA oligonucleotides and their assembly into larger DNA constructs with a focus on recent advancements that have sought to reduce synthesis cost and increase sequence fidelity. The development of lower-cost methods to produce high-quality synthetic DNA will allow for the exploration of larger biol. hypotheses by lowering the cost of use and help to close the DNA read -write cost gap.
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    Baker Dockrey, S. A.; Doyon, T. J.; Perkins, J. C.; Narayan, A. R. H. Whole-cell biocatalysis platform for gram-scale oxidative dearomatization of phenols. Chem. Biol. Drug Des. 2019, 93, 1207– 1213,  DOI: 10.1111/cbdd.13443
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    Whole-cell biocatalysis platform for gram-scale oxidative dearomatization of phenols
    Baker Dockrey, Summer A.; Doyon, Tyler J.; Perkins, Jonathan C.; Narayan, Alison R. H.
    Chemical Biology & Drug Design (2019), 93 (6), 1207-1213CODEN: CBDDAL; ISSN:1747-0277. (Wiley-Blackwell)
    Technologies enabling new enzyme discovery and efficient protein engineering have spurred intense interest in the development of biocatalytic reactions. In recent years, whole-cell biocatalysis has received attention as a simple, efficient, and scalable biocatalytic reaction platform. Inspired by these developments, we have established a whole-cell protocol for oxidative dearomatization of phenols using the flavin-dependent monooxygenase, TropB. This approach provides a scalable biocatalytic platform for accessing gram-scale quantities of chiral synthetic building blocks.
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    Bai, Y.; Yang, X.; Yu, H.; Chen, X. Substrate and Process Engineering for Biocatalytic Synthesis and Facile Purification of Human Milk Oligosaccharides. ChemSusChem 2022, 15, e202102539  DOI: 10.1002/cssc.202102539
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    Substrate and Process Engineering for Biocatalytic Synthesis and Facile Purification of Human Milk Oligosaccharides
    Bai, Yuanyuan; Yang, Xiaohong; Yu, Hai; Chen, Xi
    ChemSusChem (2022), 15 (9), e202102539CODEN: CHEMIZ; ISSN:1864-5631. (Wiley-VCH Verlag GmbH & Co. KGaA)
    Innovation in process development is essential for applying biocatalysis in industrial and lab. prodn. of org. compds., including beneficial carbohydrates such as human milk oligosaccharides (HMOs). HMOs have attracted increasing attention for their potential application as key ingredients in products that can improve human health. To efficiently access HMOs through biocatalysis, a combined substrate and process engineering strategy is developed, namely multistep one-pot multienzyme (MSOPME) design. The strategy allows access to a pure tagged HMO in a single reactor with a single C18-cartridge purifn. process, despite the length of the target. Its efficiency is demonstrated in the high-yielding (71-91%) one-pot synthesis of twenty tagged HMOs (83-155 mg), including long-chain oligosaccharides with or without fucosylation or sialylation up to nonaoses from a lactoside without the isolation of the intermediate oligosaccharides. Gram-scale synthesis of an important HMO deriv. - tagged lacto-N-fucopentaose-I (LNFP-I) - proceeds in 84% yield. Tag removal is carried out in high efficiency (94-97%) without the need for column purifn. to produce the desired natural HMOs with a free reducing end. The method can be readily adapted for large-scale synthesis and automation to allow quick access to HMOs, other glycans, and glycoconjugates.
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    Börner, T.; Grey, C.; Adlercreutz, P. Generic HPLC platform for automated enzyme reaction monitoring: Advancing the assay toolbox for transaminases and other PLP-dependent enzymes. Biotechnol. J. 2016, 11, 1025– 1036,  DOI: 10.1002/biot.201500587
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    Generic HPLC platform for automated enzyme reaction monitoring: Advancing the assay toolbox for transaminases and other PLP-dependent enzymes
    Borner Tim; Grey Carl; Adlercreutz Patrick
    Biotechnology journal (2016), 11 (8), 1025-36 ISSN:.
    Methods for rapid and direct quantification of enzyme kinetics independent of the substrate stand in high demand for both fundamental research and bioprocess development. This study addresses the need for a generic method by developing an automated, standardizable HPLC platform monitoring reaction progress in near real-time. The method was applied to amine transaminase (ATA) catalyzed reactions intensifying process development for chiral amine synthesis. Autosampler-assisted pipetting facilitates integrated mixing and sampling under controlled temperature. Crude enzyme formulations in high and low substrate concentrations can be employed. Sequential, small (1 μL) sample injections and immediate detection after separation permits fast reaction monitoring with excellent sensitivity, accuracy and reproducibility. Due to its modular design, different chromatographic techniques, e.g. reverse phase and size exclusion chromatography (SEC) can be employed. A novel assay for pyridoxal 5'-phosphate-dependent enzymes is presented using SEC for direct monitoring of enzyme-bound and free reaction intermediates. Time-resolved changes of the different cofactor states, e.g. pyridoxal 5'-phosphate, pyridoxamine 5'-phosphate and the internal aldimine were traced in both half reactions. The combination of the automated HPLC platform with SEC offers a method for substrate-independent screening, which renders a missing piece in the assay and screening toolbox for ATAs and other PLP-dependent enzymes.
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    Claaßen, C.; Mack, K.; Rother, D. Benchtop NMR for Online Reaction Monitoring of the Biocatalytic Synthesis of Aromatic Amino Alcohols. ChemCatChem. 2020, 12, 1190– 1199,  DOI: 10.1002/cctc.201901910
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    Benchtop NMR for Online Reaction Monitoring of the Biocatalytic Synthesis of Aromatic Amino Alcohols
    Claassen, C.; Mack, K.; Rother, D.
    ChemCatChem (2020), 12 (4), 1190-1199CODEN: CHEMK3; ISSN:1867-3880. (Wiley-VCH Verlag GmbH & Co. KGaA)
    Online analytics provides insights into the progress of an ongoing reaction without the need for extensive sampling and offline anal. In this study, we investigated benchtop NMR as an online reaction monitoring tool for complex enzyme cascade reactions. Online NMR was used to monitor a two-step cascade beginning with an arom. aldehyde and leading to an arom. amino alc. as the final product, applying two different enzymes and a variety of co-substrates and intermediates. Benchtop NMR enabled the concn. of the reaction components to be detected in buffered systems in the single-digit mM range without using deuterated solvent. The concns. detd. via NMR were correlated with offline samples analyzed via uHPLC and displayed a good correlation between the two methods. In summary, benchtop NMR proved to be a sensitive, selective and reliable method for online reaction monitoring in (multi-step) biosynthesis. In future, online analytic systems such as the benchtop NMR devices described might not only enable direct monitoring of the reaction, but may also form the basis for self-regulation in biocatalytic reactions.
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    Bommarius, A. S. Biocatalysis: A Status Report. Annu. Rev. Chem. Biomol. Eng. 2015, 6, 319– 345,  DOI: 10.1146/annurev-chembioeng-061114-123415
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    Biocatalysis: A Status Report
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    Annual Review of Chemical and Biomolecular Engineering (2015), 6 (), 319-345CODEN: ARCBCY; ISSN:1947-5438. (Annual Reviews)
    This review describes the status of the fields of biocatalysts and enzymes, as well as existing drawbacks, and recent advances in the areas deemed to represent drawbacks. Although biocatalysts are often highly active and extremely selective, there are still drawbacks assocd. with biocatalysis as a generally applicable technique: the lack of designability of biocatalysts; their limits of stability; and the insufficient no. of well-characterized, ready-to-use biocatalysts. There has been significant progress on the following fronts: (a) novel protein engineering tools, both exptl. and computational, have significantly enhanced the toolbox for biocatalyst development. (b) The deactivation of biocatalysts under various stresses can be described quant. via rational models. There are several cases of spectacular leaps of stabilization after accumulating all stabilizing mutations found in earlier rounds. The concept that stabilization against one type of stress commonly also stabilizes against other types of stress is now exptl. considerably better founded than a few years ago. (c) A host of developments of novel biocatalysts in the past few years, in part fueled by improved designability and improved methods of stabilization, has considerably broadened the toolbox for synthetic chem.
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    What are the Limitations of Enzymes in Synthetic Organic Chemistry?
    Reetz, Manfred T.
    Chemical Record (2016), 16 (6), 2449-2459CODEN: CRHEAK; ISSN:1528-0691. (Wiley-VCH Verlag GmbH & Co. KGaA)
    Enzymes have been used in org. chem. and biotechnol. for 100 years, but their widespread application has been prevented by a no. of limitations, including the often-obsd. limited thermostability, narrow substrate scope, and low or wrong stereo- and/or regioselectivity. Directed evolution provides a means to address and generally solve these problems, esp. since recent methodol. development has made this protein engineering method faster, more efficient, and more reliable than in the past. This Darwinian approach to asym. catalysis has led to a no. of industrial applications. Metabolic-pathway engineering, mutasynthesis, and fermn. are likewise enzyme-based techniques that enrich chem. This account outlines the scope, and particularly, the limitations, of biocatalysis. The complementary nature of enzymes and man-made catalysts is emphasized.
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    Stepankova, V.; Bidmanova, S.; Koudelakova, T.; Prokop, Z.; Chaloupkova, R.; Damborsky, J. Strategies for Stabilization of Enzymes in Organic Solvents. ACS Catal. 2013, 3, 2823– 2836,  DOI: 10.1021/cs400684x
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    Strategies for Stabilization of Enzymes in Organic Solvents
    Stepankova, Veronika; Bidmanova, Sarka; Koudelakova, Tana; Prokop, Zbynek; Chaloupkova, Radka; Damborsky, Jiri
    ACS Catalysis (2013), 3 (12), 2823-2836CODEN: ACCACS; ISSN:2155-5435. (American Chemical Society)
    A review. One of the major barriers to the use of enzymes in industrial biotechnol. is their insufficient stability under processing conditions. The use of org. solvent systems instead of aq. media for enzymic reactions offers numerous advantages, such as increased soly. of hydrophobic substrates or suppression of water-dependent side reactions. For example, reverse hydrolysis reactions that form esters from acids and alcs. become thermodynamically favorable. However, org. solvents often inactivate enzymes. Industry and academia have devoted considerable effort into developing effective strategies to enhance the lifetime of enzymes in the presence of org. solvents. The strategies can be grouped into three main categories: (i) isolation of novel enzymes functioning under extreme conditions, (ii) modification of enzyme structures to increase their resistance toward nonconventional media, and (iii) modification of the solvent environment to decrease its denaturing effect on enzymes. Here we discuss successful examples representing each of these categories and summarize their advantages and disadvantages. Finally, we highlight some potential future research directions in the field, such as investigation of novel nanomaterials for immobilization, wider application of computational tools for semirational prediction of stabilizing mutations, knowledge-driven modification of key structural elements learned from successfully engineered proteins, and replacement of volatile org. solvents by ionic liqs. and deep eutectic solvents.
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    Guzik, U.; Hupert-Kocurek, K.; Wojcieszyńska, D. Immobilization as a Strategy for Improving Enzyme Properties-Application to Oxidoreductases. Molecules 2014, 19, 8995– 9018,  DOI: 10.3390/molecules19078995
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    Guzik, Urszula; Hupert-Kocurek, Katarzyna; Wojcieszynska, Danuta
    Molecules (2014), 19 (7), 8995-9018, 24 pp.CODEN: MOLEFW; ISSN:1420-3049. (MDPI AG)
    A review. The main objective of the immobilization of enzymes is to enhance the economics of biocatalytic processes. Immobilization allows one to re-use the enzyme for an extended period of time and enables easier sepn. of the catalyst from the product. Addnl., immobilization improves many properties of enzymes such as performance in org. solvents, pH tolerance, heat stability or the functional stability. It can also increase the structural rigidity of the protein and stabilize multimeric enzymes which prevents dissocn.-related inactivation. In the last decade, several papers about immobilization methods have been published. In our work, we present a relation between the influence of immobilization on the improvement of the properties of selected oxidoreductases and their com. value. We also present our view on the role that different immobilization methods play in the redn. of enzyme inhibition during biotechnol. processes.
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    De Santis, P.; Meyer, L.-E.; Kara, S. The rise of continuous flow biocatalysis – fundamentals, very recent developments and future perspectives. React. Chem. Eng. 2020, 5, 2155– 2184,  DOI: 10.1039/D0RE00335B
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    The rise of continuous flow biocatalysis - fundamentals, very recent developments and future perspectives
    De Santis, Piera; Meyer, Lars-Erik; Kara, Selin
    Reaction Chemistry & Engineering (2020), 5 (12), 2155-2184CODEN: RCEEBW; ISSN:2058-9883. (Royal Society of Chemistry)
    A review. Biocatalysis community has witnessed a drastic increase in the no. of studies for the use of enzymes in continuously operated flow reactors. This significant interest arose from the possibility of combining the strengths of the two worlds: enhanced mass transfer and resource efficient synthesis achieved in flow chem. at micro-scales and excellent selectivities obtained in biocatalysis. Within this review, we present very recent (from 2018 to Sept. 2020) developments in the field of biocatalysis in continuously operated systems. Briefly, we describe the fundamentals of continuously operated reactors with a special focus on enzyme-catalyzed reactions. We devoted special attention on future perspectives in this key emerging technol. area ranging from process anal. technologies to digitalization.
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    France, S. P.; Lewis, R. D.; Martinez, C. A. The Evolving Nature of Biocatalysis in Pharmaceutical Research and Development. JACS Au 2023, 3, 715– 735,  DOI: 10.1021/jacsau.2c00712
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    The Evolving Nature of Biocatalysis in Pharmaceutical Research and Development
    France, Scott P.; Lewis, Russell D.; Martinez, Carlos A.
    JACS Au (2023), 3 (3), 715-735CODEN: JAAUCR; ISSN:2691-3704. (American Chemical Society)
    Biocatalysis is a highly valued enabling technol. for pharmaceutical research and development as it can unlock synthetic routes to complex chiral motifs with unparalleled selectivity and efficiency. This perspective aims to review recent advances in the pharmaceutical implementation of biocatalysis across early and late-stage development with a focus on the implementation of processes for preparative-scale syntheses.
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    Zhang, Y.; Xia, B.; Li, Y.; Lin, X.; Wu, Q. Substrate Engineering in Lipase-Catalyzed Selective Polymerization of d-/l-Aspartates and Diols to Prepare Helical Chiral Polyester. Biomacromolecules 2021, 22, 918– 926,  DOI: 10.1021/acs.biomac.0c01605
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    Substrate Engineering in Lipase-Catalyzed Selective Polymerization of D-/L-Aspartates and Diols to Prepare Helical Chiral Polyester
    Zhang, Yu; Xia, Bo; Li, Yanyan; Lin, Xianfu; Wu, Qi
    Biomacromolecules (2021), 22 (2), 918-926CODEN: BOMAF6; ISSN:1525-7797. (American Chemical Society)
    The synthesis of optically pure polymers is one of the most challenging tasks in polymer chem. Herein, Novozym 435 (Lipase B from Candida antarctica, immobilized on Lewatit VP OC 1600)-catalyzed polycondensation between D-/L-aspartic acid (Asp) diester and diols for the prepn. of helical chiral polyesters was reported. Compared with D-Asp diesters, the fast-reacting L-Asp diesters easily reacted with diols to provide a series of chiral polyesters contg. N-substitutional L-Asp repeating units. Besides amino acid configuration, N-substituent side chains and the chain length of diols were also investigated and optimized. It was found that bulky acyl N-substitutional groups like N-Boc and N-Cbz were more favorable for this polymn. than small ones probably due to competitively binding of these small acyl groups into the active site of Novozym 435. The highest mol. wt. can reach up to 39.5 x 103 g/mol (Mw,D = 1.64). Moreover, the slow-reacting D-Asp diesters were also successfully polymd. by modifying the substrate structure to create a "nonchiral" condensation environment artificially. These enantiocomplementary chiral polyesters are thermally stable and have specific helical structures, which was confirmed by CD (CD) spectra, scanning electron microscope (SEM), and mol. calcn.
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    Turner, N. J. Directed evolution drives the next generation of biocatalysts. Nat. Chem. Biol. 2009, 5, 567– 573,  DOI: 10.1038/nchembio.203
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    Directed evolution drives the next generation of biocatalysts
    Turner, Nicholas J.
    Nature Chemical Biology (2009), 5 (8), 567-573CODEN: NCBABT; ISSN:1552-4450. (Nature Publishing Group)
    A review. Enzymes are increasingly being used as biocatalysts in the generation of products that have until now been derived using traditional chem. processes. Such products range from pharmaceutical and agrochem. building blocks to fine and bulk chems. and, more recently, components of biofuels. For a biocatalyst to be effective in an industrial process, it must be subjected to improvement and optimization, and in this respect the directed evolution of enzymes has emerged as a powerful enabling technol. Directed evolution involves repeated rounds of (1) random gene library generation, (2) expression of genes in a suitable host, and (3) screening of libraries of variant enzymes for the property of interest. Both in vitro screening-based methods and in vivo selection-based methods have been applied to the evolution of enzyme function and properties. Significant developments have occurred recently, particularly with respect to library design, screening methodol., applications in synthetic transformations, and strategies for the generation of new enzyme function.
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    Cobb, R. E.; Chao, R.; Zhao, H. Directed evolution: Past, present, and future. AIChE J. 2013, 59, 1432– 1440,  DOI: 10.1002/aic.13995
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    Directed evolution: Past, present, and future
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    AIChE Journal (2013), 59 (5), 1432-1440CODEN: AICEAC; ISSN:0001-1541. (John Wiley & Sons, Inc.)
    Directed evolution, the lab. process by which biol. entities with desired traits are created through iterative rounds of genetic diversification and library screening or selection, has become one of the most useful and widespread tools in basic and applied biol. From its roots in classical strain engineering and adaptive evolution, modern directed evolution came of age 20 years ago with the demonstration of repeated rounds of polymerase chain reaction (PCR)-driven random mutagenesis and activity screening to improve protein properties. Since then, numerous techniques have been developed that have enabled the evolution of virtually any protein, pathway, network, or entire organism of interest. Here, we recount some of the major milestones in the history of directed evolution, highlight the most promising recent developments in the field, and discuss the future challenges and opportunities that lie ahead. © 2013 American Institute of Chem. Engineers AIChE J, 2013.
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    Fernandes, P. Miniaturization in Biocatalysis. Int. J. Mol. Sci. 2010, 11, 858– 879,  DOI: 10.3390/ijms11030858
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    Miniaturization in biocatalysis
    Fernandes, Pedro
    International Journal of Molecular Sciences (2010), 11 (), 858-879CODEN: IJMCFK; ISSN:1422-0067. (Molecular Diversity Preservation International)
    A review. The use of biocatalysts for the prodn. of both consumer goods and building blocks for chem. synthesis is consistently gaining relevance. A significant contribution for recent advances towards further implementation of enzymes and whole cells is related to the developments in miniature reactor technol. and insights into flow behavior. Due to the high level of parallelization and reduced requirements of chems., intensive screening of biocatalysts and process variables has become more feasible and reproducibility of the bioconversion processes has been substantially improved. The present work aims to provide an overview of the applications of miniaturized reactors in bioconversion processes, considering multi-well plates and microfluidic devices, update information on the engineering characterization of the hardware used, and present perspective developments in this area of research.
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    Bell, E. L.; Finnigan, W.; France, S. P.; Green, A. P.; Hayes, M. A.; Hepworth, L. J.; Lovelock, S. L.; Niikura, H.; Osuna, S.; Romero, E.; Ryan, K. S.; Turner, N. J.; Flitsch, S. L. Biocatalysis. Nat. Rev. Methods Primers 2021, 1, 46,  DOI: 10.1038/s43586-021-00044-z
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    Biocatalysis
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    Nature Reviews Methods Primers (2021), 1 (1), 46CODEN: NRMPAT; ISSN:2662-8449. (Nature Portfolio)
    A review. Biocatalysis has become an important aspect of modern org. synthesis, both in academia and across the chem. and pharmaceutical industries. Its success has been largely due to a rapid expansion of the range of chem. reactions accessible, made possible by advanced tools for enzyme discovery coupled with high-throughput lab. evolution techniques for biocatalyst optimization. A wide range of tailor-made enzymes with high efficiencies and selectivities can now be produced quickly and on a gram to kilogram scale, with dedicated databases and search tools aimed at making these biocatalysts accessible to a broader scientific community. This Primer discusses the current state-of-the-art methodol. in the field, including route design, enzyme discovery, protein engineering and the implementation of biocatalysis in industry. We highlight recent advances, such as de novo design and directed evolution, and discuss parameters that make a good reproducible biocatalytic process for industry. The general concepts will be illustrated by recent examples of applications in academia and industry, including the development of multistep enzyme cascades.
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    Duetz, W. A. Microtiter plates as mini-bioreactors: miniaturization of fermentation methods. Trends Microbiol. 2007, 15, 469– 475,  DOI: 10.1016/j.tim.2007.09.004
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    Microtiter plates as mini-bioreactors: miniaturization of fermentation methods
    Duetz, Wouter A.
    Trends in Microbiology (2007), 15 (10), 469-475CODEN: TRMIEA; ISSN:0966-842X. (Elsevier B.V.)
    A review. In the past decade, the use of microtiter plates for microbial growth has become widespread, particularly in industry. In parallel, research in academia has provided a thorough insight into the complex relation between well dimensions, culture vols., orbital shaking conditions and surface tension on the one hand, and oxygen-transfer rates and degrees of mixing on the other. In this review, I will discuss these fundamental issues and describe the current applications of microtiter plates in microbiol. Microtiter plates can now be considered a mature alternative to Erlenmeyer shake flasks.
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    Diefenbach, X. W.; Farasat, I.; Guetschow, E. D.; Welch, C. J.; Kennedy, R. T.; Sun, S.; Moore, J. C. Enabling Biocatalysis by High-Throughput Protein Engineering Using Droplet Microfluidics Coupled to Mass Spectrometry. ACS Omega 2018, 3, 1498– 1508,  DOI: 10.1021/acsomega.7b01973
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    Enabling Biocatalysis by High-Throughput Protein Engineering Using Droplet Microfluidics Coupled to Mass Spectrometry
    Diefenbach, Xue W.; Farasat, Iman; Guetschow, Erik D.; Welch, Christopher J.; Kennedy, Robert T.; Sun, Shuwen; Moore, Jeffrey C.
    ACS Omega (2018), 3 (2), 1498-1508CODEN: ACSODF; ISSN:2470-1343. (American Chemical Society)
    Directed Evolution is a key technol. driving the utility of biocatalysis in pharmaceutical synthesis. Conventional approaches to Directed Evolution are conducted using bacterial cells expressing enzymes in microplates, with catalyzed reactions measured by HPLC, high-performance liq. chromatog.-mass spectrometry (HPLC-MS), or optical detectors, which require either long cycle times or tailor-made substrates. To better fit modern, fast-paced process chem. development where solns. are rapidly needed for new substrates, droplet microfluidics interfaced with electrospray ionization (ESI)-MS provides a label-free high-throughput screening platform. To apply this method to industrial enzyme screening and to explore potential approaches that may further improve the overall throughput, we optimized the existing droplet-MS methods. Carryover between droplets, traditionally a significant issue, was reduced to undetectable level by replacing the stainless steel ESI needle with a Teflon needle within a capillary electrophoresis (CE)-MS source. Throughput was improved to 3 Hz with a wide range of droplet sizes (10-50 nL) by tuning the sheath flow within the CE-MS source. The optimized method was demonstrated by screening reactions using two different transaminase libraries. Good correlations (r2 ∼ 0.95) were found between the droplet-MS and LC-MS methods, with 100% match on hit variants. We further explored the capability of the system by performing in vitro transcription-translation inside the droplets and directly analyzing the intact reaction mixt. droplets by MS. The synthesized protein attained comparable activity to the protein std., and the complex samples appeared well tolerated by the MS. The success of the above applications indicates that the MS anal. of the microfluidic droplets is an available option for considerably accelerating the screening of enzyme evolution libraries.
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    Finnigan, W.; Hepworth, L. J.; Flitsch, S. L.; Turner, N. J. RetroBioCat as a computer-aided synthesis planning tool for biocatalytic reactions and cascades. Nat. Catal. 2021, 4, 98– 104,  DOI: 10.1038/s41929-020-00556-z
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    RetroBioCat as a computer-aided synthesis planning tool for biocatalytic reactions and cascades
    Finnigan, William; Hepworth, Lorna J.; Flitsch, Sabine L.; Turner, Nicholas J.
    Nature Catalysis (2021), 4 (2), 98-104CODEN: NCAACP; ISSN:2520-1158. (Nature Research)
    Abstr.: As the enzyme toolbox for biocatalysis has expanded, so has the potential for the construction of powerful enzymic cascades for efficient and selective synthesis of target mols. Addnl., recent advances in computer-aided synthesis planning are revolutionizing synthesis design in both synthetic biol. and org. chem. However, the potential for biocatalysis is not well captured by tools currently available in either field. Here we present RetroBioCat, an intuitive and accessible tool for computer-aided design of biocatalytic cascades, freely available at retrobiocat.com. Our approach uses a set of expertly encoded reaction rules encompassing the enzyme toolbox for biocatalysis, and a system for identifying literature precedent for enzymes with the correct substrate specificity where this is available. Applying these rules for automated biocatalytic retrosynthesis, we show our tool to be capable of identifying promising biocatalytic pathways to target mols., validated using a test set of recent cascades described in the literature. [graphic not available: see fulltext].
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    Altschul, S. F.; Gish, W.; Miller, W.; Myers, E. W.; Lipman, D. J. Basic local alignment search tool. J. Mol. Biol. 1990, 215, 403– 410,  DOI: 10.1016/S0022-2836(05)80360-2
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    Basic local alignment search tool
    Altschul, Stephen F.; Gish, Warren; Miller, Webb; Myers, Eugene W.; Lipman, David J.
    Journal of Molecular Biology (1990), 215 (3), 403-10CODEN: JMOBAK; ISSN:0022-2836.
    A new approach to rapid sequence comparison, basic local alignment search tool (BLAST), directly approximates alignments that optimize a measure of local similarity, the maximal segment pair (MSP) score. Recent math. results on the stochastic properties of MSP scores allow an anal. of the performance of this method as well as the statistical significance of alignments it generates. The basic algorithm is simple and robust; it can be implemented in a no. of ways and applied in a variety of contexts including straightforward DNA and protein sequence database searches, motif searches, gene identification searches, and in the anal. of multiple regions of similarity in long DNA sequences. In addn. to its flexibility and tractability to math. anal., BLAST is an order of magnitude faster than existing sequence comparison tools of comparable sensitivity.
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    Cai, X.-H.; Jaroszewski, L.; Wooley, J.; Godzik, A. Internal organization of large protein families: Relationship between the sequence, structure, and function-based clustering. Proteins: Struct., Funct., Bioinf. 2011, 79, 2389– 2402,  DOI: 10.1002/prot.23049
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    Internal organization of large protein families: Relationship between the sequence, structure, and function-based clustering
    Cai, Xiao-Hui; Jaroszewski, Lukasz; Wooley, John; Godzik, Adam
    Proteins: Structure, Function, and Bioinformatics (2011), 79 (8), 2389-2402CODEN: PSFBAF ISSN:. (Wiley-Liss, Inc.)
    The protein universe can be organized in families that group proteins sharing common ancestry. Such families display variable levels of structural and functional divergence, from homogeneous families, where all members have the same function and very similar structure, to very divergent families, where large variations in function and structure are obsd. For practical purposes of structure and function prediction, it would be beneficial to identify sub-groups of proteins with highly similar structures (iso-structural) and/or functions (iso-functional) within divergent protein families. Three algorithms were compared in their ability to cluster large protein families and it was discussed whether any of these methods could reliably identify such iso-structural or iso-functional groups. It was shown that clustering using profile-sequence and profile-profile comparison methods closely reproduces clusters based on similarities between 3D structures or clusters of proteins with similar biol. functions. In contrast, the still commonly used sequence-based methods with fixed thresholds result in vast over-ests. of structural and functional diversity in protein families. As a result, these methods also over-est. the no. of protein structures that have to be detd. to fully characterize structural space of such families. The fact that one can build reliable models based on apparently distantly related templates is crucial for extg. maximal amt. of information from new sequencing projects.
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    Sirota, F. L.; Maurer-Stroh, S.; Li, Z.; Eisenhaber, F.; Eisenhaber, B. Functional Classification of Super-Large Families of Enzymes Based on Substrate Binding Pocket Residues for Biocatalysis and Enzyme Engineering Applications. Front. Bioeng. Biotechnol. 2021, 9, 701120,  DOI: 10.3389/fbioe.2021.701120
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    Functional Classification of Super-Large Families of Enzymes Based on Substrate Binding Pocket Residues for Biocatalysis and Enzyme Engineering Applications
    Sirota Fernanda L; Maurer-Stroh Sebastian; Eisenhaber Frank; Eisenhaber Birgit; Maurer-Stroh Sebastian; Li Zhi; Eisenhaber Frank; Eisenhaber Birgit; Eisenhaber Frank
    Frontiers in bioengineering and biotechnology (2021), 9 (), 701120 ISSN:2296-4185.
    Large enzyme families such as the groups of zinc-dependent alcohol dehydrogenases (ADHs), long chain alcohol oxidases (AOxs) or amine dehydrogenases (AmDHs) with, sometimes, more than one million sequences in the non-redundant protein database and hundreds of experimentally characterized enzymes are excellent cases for protein engineering efforts aimed at refining and modifying substrate specificity. Yet, the backside of this wealth of information is that it becomes technically difficult to rationally select optimal sequence targets as well as sequence positions for mutagenesis studies. In all three cases, we approach the problem by starting with a group of experimentally well studied family members (including those with available 3D structures) and creating a structure-guided multiple sequence alignment and a modified phylogenetic tree (aka binding site tree) based just on a selection of potential substrate binding residue positions derived from experimental information (not from the full-length sequence alignment). Hereupon, the remaining, mostly uncharacterized enzyme sequences can be mapped; as a trend, sequence grouping in the tree branches follows substrate specificity. We show that this information can be used in the target selection for protein engineering work to narrow down to single suitable sequences and just a few relevant candidate positions for directed evolution towards activity for desired organic compound substrates. We also demonstrate how to find the closest thermophile example in the dataset if the engineering is aimed at achieving most robust enzymes.
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    Wilding, M.; Peat, T. S.; Kalyaanamoorthy, S.; Newman, J.; Scott, C.; Jermiin, L. S. Reverse engineering: transaminase biocatalyst development using ancestral sequence reconstruction. Green Chem. 2017, 19, 5375– 5380,  DOI: 10.1039/C7GC02343J
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    Reverse engineering: transaminase biocatalyst development using ancestral sequence reconstruction
    Wilding, Matthew; Peat, Thomas S.; Kalyaanamoorthy, Subha; Newman, Janet; Scott, Colin; Jermiin, Lars S.
    Green Chemistry (2017), 19 (22), 5375-5380CODEN: GRCHFJ; ISSN:1463-9262. (Royal Society of Chemistry)
    The development of new biocatalysts using ancestral sequence reconstruction is reported. When applied to an ω-transaminase, the ancestral proteins demonstrated novel and superior activities with eighty percent of the forty compds. tested compared to the modern day protein, and improvements in activity of up to twenty fold. These included a range of compds. pertinent as feedstocks in polyamide manuf.
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    Gumulya, Y.; Baek, J.-M.; Wun, S.-J.; Thomson, R. E. S.; Harris, K. L.; Hunter, D. J. B.; Behrendorff, J. B. Y. H.; Kulig, J.; Zheng, S.; Wu, X.; Wu, B.; Stok, J. E.; De Voss, J. J.; Schenk, G.; Jurva, U.; Andersson, S.; Isin, E. M.; Bodén, M.; Guddat, L.; Gillam, E. M. J. Engineering highly functional thermostable proteins using ancestral sequence reconstruction. Nat. Catal. 2018, 1, 878– 888,  DOI: 10.1038/s41929-018-0159-5
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    Engineering highly functional thermostable proteins using ancestral sequence reconstruction
    Gumulya, Yosephin; Baek, Jong-Min; Wun, Shun-Jie; Thomson, Raine E. S.; Harris, Kurt L.; Hunter, Dominic J. B.; Behrendorff, James B. Y. H.; Kulig, Justyna; Zheng, Shan; Wu, Xueming; Wu, Bin; Stok, Jeanette E.; De Voss, James J.; Schenk, Gerhard; Jurva, Ulrik; Andersson, Shalini; Isin, Emre M.; Boden, Mikael; Guddat, Luke; Gillam, Elizabeth M. J.
    Nature Catalysis (2018), 1 (11), 878-888CODEN: NCAACP; ISSN:2520-1158. (Nature Research)
    Com. biocatalysis requires robust enzymes that can withstand elevated temps. and long incubations. Ancestral reconstruction has shown that pre-Cambrian enzymes were often much more thermostable than extant forms. Here, we resurrect ancestral enzymes that withstand ∼30 °C higher temps. and ≥100 times longer incubations than their extant forms. This is demonstrated on animal cytochromes P 450 that stereo- and regioselectively functionalize unactivated C-H bonds for the synthesis of valuable chems., and bacterial ketol-acid reductoisomerases that are used to make butanol-based biofuels. The vertebrate CYP3 P 450 ancestor showed a 60T50 of 66 °C and enhanced solvent tolerance compared with the human drug-metabolizing CYP3A4, yet comparable activity towards a similarly broad range of substrates. The ancestral ketol-acid reductoisomerase showed an eight-fold higher specific activity than the cognate Escherichia coli form at 25 °C, which increased 3.5-fold at 50 °C. Thus, thermostable proteins can be devised using sequence data alone from even recent ancestors.
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    Tamura, K.; Peterson, D.; Peterson, N.; Stecher, G.; Nei, M.; Kumar, S. MEGA5: Molecular Evolutionary Genetics Analysis Using Maximum Likelihood, Evolutionary Distance, and Maximum Parsimony Methods. Mol. Biol. Evol. 2011, 28, 2731– 2739,  DOI: 10.1093/molbev/msr121
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    MEGA5: Molecular Evolutionary Genetics Analysis Using Maximum Likelihood, Evolutionary Distance, and Maximum Parsimony Methods
    Tamura, Koichiro; Peterson, Daniel; Peterson, Nicholas; Stecher, Glen; Nei, Masatoshi; Kumar, Sudhir
    Molecular Biology and Evolution (2011), 28 (10), 2731-2739CODEN: MBEVEO; ISSN:0737-4038. (Oxford University Press)
    Comparative anal. of mol. sequence data is essential for reconstructing the evolutionary histories of species and inferring the nature and extent of selective forces shaping the evolution of genes and species. Here, we announce the release of Mol. Evolutionary Genetics Anal. version 5 (MEGA5), which is a user-friendly software for mining online databases, building sequence alignments and phylogenetic trees, and using methods of evolutionary bioinformatics in basic biol., biomedicine, and evolution. The newest addn. in MEGA5 is a collection of max. likelihood (ML) analyses for inferring evolutionary trees, selecting best-fit substitution models (nucleotide or amino acid), inferring ancestral states and sequences (along with probabilities), and estg. evolutionary rates site-by-site. In computer simulation analyses, ML tree inference algorithms in MEGA5 compared favorably with other software packages in terms of computational efficiency and the accuracy of the ests. of phylogenetic trees, substitution parameters, and rate variation among sites. The MEGA user interface has now been enhanced to be activity driven to make it easier for the use of both beginners and experienced scientists. This version of MEGA is intended for the Windows platform, and it has been configured for effective use on Mac OS X and Linux desktops. It is available free of charge from http://www.megasoftware.net.
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  82. 82
    Hall, B. G. Building phylogenetic trees from molecular data with MEGA. Mol. Biol. Evol. 2013, 30, 1229– 1235,  DOI: 10.1093/molbev/mst012
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    82
    Building Phylogenetic Trees from Molecular Data with MEGA
    Hall, Barry G.
    Molecular Biology and Evolution (2013), 30 (5), 1229-1235CODEN: MBEVEO; ISSN:0737-4038. (Oxford University Press)
    Phylogenetic anal. is sometimes regarded as being an intimidating, complex process that requires expertise and years of experience. In fact, it is a fairly straightforward process that can be learned quickly and applied effectively. This Protocol describes the several steps required to produce a phylogenetic tree from mol. data for novices. In the example illustrated here, the program MEGA is used to implement all those steps, thereby eliminating the need to learn several programs, and to deal with multiple file formats from one step to another (Tamura K, Peterson D, Peterson N, Stecher G, Nei M, Kumar S. 2011. MEGA5: mol. evolutionary genetics anal. using max. likelihood, evolutionary distance, and max. parsimony methods.Mol Biol Evol. 28:2731-2739). The first step, identification of a set of homologous sequences and downloading those sequences, is implemented by MEGA's own browser built on top of the Google Chrome toolkit. For the second step, alignment of those sequences, MEGA offers two different algorithms: ClustalW and MUSCLE. For the third step, construction of a phylogenetic tree from the aligned sequences, MEGA offers many different methods. Here we illustrate the max. likelihood method, beginning with MEGA's Models feature, which permits selecting the most suitable substitution model. Finally, MEGA provides a powerful and flexible interface for the final step, actually drawing the tree for publication. Here a step-by-step protocol is presented in sufficient detail to allow a novice to start with a sequence of interest and to build a publication-quality tree illustrating the evolution of an appropriate set of homologs of that sequence. MEGA is available for use on PCs and Macs from www.megasoftware.net.
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  83. 83
    Yates, A. D.; Achuthan, P.; Akanni, W.; Allen, J.; Allen, J.; Alvarez-Jarreta, J.; Amode, M. R.; Armean, I. M.; Azov, A. G.; Bennett, R.; Bhai, J.; Billis, K.; Boddu, S.; Marugán, J. C.; Cummins, C.; Davidson, C.; Dodiya, K.; Fatima, R.; Gall, A.; Giron, C. G.; Gil, L.; Grego, T.; Haggerty, L.; Haskell, E.; Hourlier, T.; Izuogu, O. G.; Janacek, S. H.; Juettemann, T.; Kay, M.; Lavidas, I.; Le, T.; Lemos, D.; Martinez, J. G.; Maurel, T.; McDowall, M.; McMahon, A.; Mohanan, S.; Moore, B.; Nuhn, M.; Oheh, D. N.; Parker, A.; Parton, A.; Patricio, M.; Sakthivel, M. P.; Abdul Salam, A. I.; Schmitt, B. M.; Schuilenburg, H.; Sheppard, D.; Sycheva, M.; Szuba, M.; Taylor, K.; Thormann, A.; Threadgold, G.; Vullo, A.; Walts, B.; Winterbottom, A.; Zadissa, A.; Chakiachvili, M.; Flint, B.; Frankish, A.; Hunt, S. E.; IIsley, G.; Kostadima, M.; Langridge, N.; Loveland, J. E.; Martin, F. J.; Morales, J.; Mudge, J. M.; Muffato, M.; Perry, E.; Ruffier, M.; Trevanion, S. J.; Cunningham, F.; Howe, K. L.; Zerbino, D. R.; Flicek, P. Ensembl 2020. Nucleic Acids Res. 2020, 48, D682– D688,  DOI: 10.1093/nar/gkz966
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    83
    Ensembl 2020
    Yates, Andrew D.; Achuthan, Premanand; Akanni, Wasiu; Allen, James; Allen, Jamie; Alvarez-Jarreta, Jorge; Amode, M. Ridwan; Armean, Irina M.; Azov, Andrey G.; Bennett, Ruth; Bhai, Jyothish; Billis, Konstantinos; Boddu, Sanjay; Marugan, Jose Carlos; Cummins, Carla; Davidson, Claire; Dodiya, Kamalkumar; Fatima, Reham; Gall, Astrid; Giron, Carlos Garcia; Gil, Laurent; Grego, Tiago; Haggerty, Leanne; Haskell, Erin; Hourlier, Thibaut; Izuogu, Osagie G.; Janacek, Sophie H.; Juettemann, Thomas; Kay, Mike; Lavidas, Ilias; Le, Tuan; Lemos, Diana; Martinez, Jose Gonzalez; Maurel, Thomas; Mcdowall, Mark; Mcmahon, Aoife; Mohanan, Shamika; Moore, Benjamin; Nuhn, Michael; Oheh, Denye N.; Parker, Anne; Parton, Andrew; Patricio, Mateus; Sakthivel, Manoj Pandian; Abdul Salam, Ahamed Imran; Schmitt, Bianca M.; Schuilenburg, Helen; Sheppard, Dan; Sycheva, Mira; Szuba, Marek; Taylor, Kieron; Thormann, Anja; Threadgold, Glen; Vullo, Alessandro; Walts, Brandon; Winterbottom, Andrea; Zadissa, Amonida; Chakiachvili, Marc; Flint, Bethany; Frankish, Adam; Hunt, Sarah E.; Iisley, Garth; Kostadima, Myrto; Langridge, Nick; Loveland, Jane E.; Martin, Fergal J.; Morales, Joannella; Mudge, Jonathan M.; Muffato, Matthieu; Perry, Emily; Ruffier, Magali; Trevanion, Stephen J.; Cunningham, Fiona; Howe, Kevin L.; Zerbino, Daniel R.; Flicek, Paul
    Nucleic Acids Research (2020), 48 (D1), D682-D688CODEN: NARHAD; ISSN:1362-4962. (Oxford University Press)
    A review. The Ensembl is a system for generating and distributing genome annotation such as genes, variation, regulation and comparative genomics across the vertebrate subphylum and key model organisms. The Ensembl annotation pipeline is capable of integrating exptl. and ref. data from multiple providers into a single integrated resource. Here, we present 94 newly annotated and re-annotated genomes, bringing the total no. of genomes offered by Ensembl to 227. This represents the single largest expansion of the resource since its inception. We also detail our continued efforts to improve human annotation, developments in our epigenome anal. and display, a new tool for imputing causal genes from genome-wide assocn. studies and visualization of variation within a 3D protein model. Finally, we present information on our new website. Both software and data are made available without restriction via our website, online tools platform and programmatic interfaces (available under an Apache 2.0 license) and data updates made available four times a year.
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    Atkinson, H. J.; Morris, J. H.; Ferrin, T. E.; Babbitt, P. C. Using Sequence Similarity Networks for Visualization of Relationships Across Diverse Protein Superfamilies. PloS One 2009, 4, e4345  DOI: 10.1371/journal.pone.0004345
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    84
    Using sequence similarity networks for visualization of relationships across diverse protein superfamilies
    Atkinson Holly J; Morris John H; Ferrin Thomas E; Babbitt Patricia C
    PloS one (2009), 4 (2), e4345 ISSN:.
    The dramatic increase in heterogeneous types of biological data--in particular, the abundance of new protein sequences--requires fast and user-friendly methods for organizing this information in a way that enables functional inference. The most widely used strategy to link sequence or structure to function, homology-based function prediction, relies on the fundamental assumption that sequence or structural similarity implies functional similarity. New tools that extend this approach are still urgently needed to associate sequence data with biological information in ways that accommodate the real complexity of the problem, while being accessible to experimental as well as computational biologists. To address this, we have examined the application of sequence similarity networks for visualizing functional trends across protein superfamilies from the context of sequence similarity. Using three large groups of homologous proteins of varying types of structural and functional diversity--GPCRs and kinases from humans, and the crotonase superfamily of enzymes--we show that overlaying networks with orthogonal information is a powerful approach for observing functional themes and revealing outliers. In comparison to other primary methods, networks provide both a good representation of group-wise sequence similarity relationships and a strong visual and quantitative correlation with phylogenetic trees, while enabling analysis and visualization of much larger sets of sequences than trees or multiple sequence alignments can easily accommodate. We also define important limitations and caveats in the application of these networks. As a broadly accessible and effective tool for the exploration of protein superfamilies, sequence similarity networks show great potential for generating testable hypotheses about protein structure-function relationships.
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  85. 85
    Zallot, R.; Oberg, N.; Gerlt, J. A. The EFI Web Resource for Genomic Enzymology Tools: Leveraging Protein, Genome, and Metagenome Databases to Discover Novel Enzymes and Metabolic Pathways. Biochemistry 2019, 58, 4169– 4182,  DOI: 10.1021/acs.biochem.9b00735
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    85
    The EFI Web Resource for Genomic Enzymology Tools: Leveraging Protein, Genome, and Metagenome Databases to Discover Novel Enzymes and Metabolic Pathways
    Zallot, Remi; Oberg, Nils; Gerlt, John A.
    Biochemistry (2019), 58 (41), 4169-4182CODEN: BICHAW; ISSN:0006-2960. (American Chemical Society)
    The assignment of functions to uncharacterized proteins discovered in genome projects requires easily accessible tools and computational resources for large-scale, user-friendly leveraging of the protein, genome, and metagenome databases by experimentalists. This article describes the web resource developed by the Enzyme Function Initiative (EFI; accessed at https://efi.igb.illinois.edu/) that provides "genomic enzymol." tools ("web tools") for (1) generating sequence similarity networks (SSNs) for protein families (EFI-EST); (2) analyzing and visualizing genome context of the proteins in clusters in SSNs (in genome neighborhood networks, GNNs, and genome neighborhood diagrams, GNDs) (EFI-GNT); and (3) prioritizing uncharacterized SSN clusters for functional assignment based on metagenome abundance (chem. guided functional profiling, CGFP) (EFI-CGFP). The SSNs generated by EFI-EST are used as the input for EFI-GNT and EFI-CGFP, enabling easy transfer of information among the tools. The networks are visualized and analyzed using Cytoscape, a widely used desktop application; GNDs and CGFP heatmaps summarizing metagenome abundance are viewed within the tools. We provide a detailed example of the integrated use of the tools with an anal. of glycyl radical enzyme superfamily (IPR004184) found in the human gut microbiome. This anal. demonstrates that (1) SwissProt annotations are not always correct, (2) large-scale genome context analyses allow the prediction of novel metabolic pathways, and (3) metagenome abundance can be used to identify/prioritize uncharacterized proteins for functional investigation.
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    Doyon, T. J.; Perkins, J. C.; Baker Dockrey, S. A.; Romero, E. O.; Skinner, K. C.; Zimmerman, P. M.; Narayan, A. R. H. Chemoenzymatic o-Quinone Methide Formation. J. Am. Chem. Soc. 2019, 141, 20269– 20277,  DOI: 10.1021/jacs.9b10474
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    Chemoenzymatic o-quinone methide formation
    Doyon, Tyler J.; Perkins, Jonathan C.; Baker Dockrey, Summer A.; Romero, Evan O.; Skinner, Kevin C.; Zimmerman, Paul M.; Narayan, Alison R. H.
    Journal of the American Chemical Society (2019), 141 (51), 20269-20277CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)
    Generation of reactive intermediates and interception of these fleeting species under physiol. conditions is a common strategy employed by Nature to build mol. complexity. However, selective formation of these species under mild conditions using classical synthetic techniques is an outstanding challenge. Here, we demonstrate the utility of biocatalysis in generating o-quinone methide intermediates with precise chemoselectivity under mild, aq. conditions. Specifically, α-ketoglutarate-dependent non-heme iron enzymes, CitB and ClaD, are employed to selectively modify benzylic C-H bonds of o-cresol substrates. In this transformation, biocatalytic hydroxylation of a benzylic C-H bond affords a benzylic alc. product which, under the aq. reaction conditions, is in equil. with the corresponding o-quinone methide. O-Quinone methide interception by a nucleophile or a dienophile allows for one-pot conversion of benzylic C-H bonds into C-C, C-N, C-O, and C-S bonds in chemoenzymic cascades on preparative scale. The chemoselectivity and mild nature of this platform is showcased here by the selective modification of peptides and chemoenzymic synthesis of the chroman natural product (-)-xyloketal D.
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    Rodriguez Benitez, A.; Narayan, A. R. H. Frontiers in Biocatalysis: Profiling Function across Sequence Space. ACS Cent. Sci. 2019, 5, 1747– 1749,  DOI: 10.1021/acscentsci.9b01112
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    Frontiers in Biocatalysis: Profiling Function across Sequence Space
    Rodriguez Benitez Attabey; Narayan Alison R H
    ACS central science (2019), 5 (11), 1747-1749 ISSN:2374-7943.
    There is no expanded citation for this reference.
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    Fisher, B. F.; Snodgrass, H. M.; Jones, K. A.; Andorfer, M. C.; Lewis, J. C. Site-Selective C–H Halogenation Using Flavin-Dependent Halogenases Identified via Family-Wide Activity Profiling. ACS Cent. Sci. 2019, 5, 1844– 1856,  DOI: 10.1021/acscentsci.9b00835
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    Site-Selective C-H Halogenation Using Flavin-Dependent Halogenases Identified via Family-Wide Activity Profiling
    Fisher, Brian F.; Snodgrass, Harrison M.; Jones, Krysten A.; Andorfer, Mary C.; Lewis, Jared C.
    ACS Central Science (2019), 5 (11), 1844-1856CODEN: ACSCII; ISSN:2374-7951. (American Chemical Society)
    Enzymes are powerful catalysts for site-selective C-H bond functionalization. Identifying suitable enzymes for this task and for biocatalysis in general remains challenging, however, due to the fundamental difficulty of predicting catalytic activity from sequence information. In this study, family-wide activity profiling was used to obtain sequence-function information on flavin-dependent halogenases (FDHs). This broad survey provided a no. of insights into FDH activity, including halide specificity and substrate preference, that were not apparent from the more focused studies reported to date. Regions of FDH sequence space that are most likely to contain enzymes suitable for halogenating small-mol. substrates were also identified. FDHs with novel substrate scope and complementary regioselectivity on large, three-dimensionally complex compds. were characterized and used for preparative-scale late-stage C-H functionalization. In many cases, these enzymes provide activities that required several rounds of directed evolution to accomplish in previous efforts, highlighting that this approach can achieve significant time savings for biocatalyst identification and provide advanced starting points for further evolution. High-throughput screening of >20 000 reactions catalyzed by 87 sol. genome-mined halogenases on 62 substrates found 39 new active halogenases for selective late-stage C-H functionalization.
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    Schülke, K. H.; Ospina, F.; Hörnschemeyer, K.; Gergel, S.; Hammer, S. C. Substrate Profiling of Anion Methyltransferases for Promiscuous Synthesis of S-Adenosylmethionine Analogs from Haloalkanes. ChemBioChem. 2022, 23, e202100632  DOI: 10.1002/cbic.202100632
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    Substrate Profiling of Anion Methyltransferases for Promiscuous Synthesis of S-Adenosylmethionine Analogs from Haloalkanes
    Schuelke, Kai H.; Ospina, Felipe; Hornschemeyer, Kathrin; Gergel, Sebastian; Hammer, Stephan C.
    ChemBioChem (2022), 23 (4), e202100632CODEN: CBCHFX; ISSN:1439-4227. (Wiley-VCH Verlag GmbH & Co. KGaA)
    Biocatalytic alkylation reactions can be performed with high chemo-, regio- and stereoselectivity using S-adenosyl-L-methionine (SAM)-dependent methyltransferases (MTs) and SAM analogs. Currently, however, this methodol. is limited in application due to the rather laborious protocols to access SAM analogs. It has recently been shown that halide methyltransferases (HMTs) enable synthesis and recycling of SAM analogs with readily available haloalkanes as starting material. Here we expand this work by using substrate profiling of the anion MT enzyme family to explore promiscuous SAM analog synthesis. Our study shows that anion MTs are in general very promiscuous with respect to the alkyl chain as well as the halide leaving group. Substrate profiling further suggests that promiscuous anion MTs cluster in sequence space. Next to iodoalkanes, cheaper, less toxic, and more available bromoalkanes have been converted and several haloalkanes bearing short alkyl groups, alkyl rings, and functional groups such as alkene, alkyne and arom. moieties are accepted as substrates. Further, we applied the SAM analogs as electrophiles in enzyme-catalyzed regioselective pyrazole allylation with 3-bromopropene as starting material.
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    Lachowicz, J. C.; Gizzi, A. S.; Almo, S. C.; Grove, T. L. Structural Insight into the Substrate Scope of Viperin and Viperin-like Enzymes from Three Domains of Life. Biochemistry 2021, 60, 2116– 2129,  DOI: 10.1021/acs.biochem.0c00958
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    Structural Insight into the Substrate Scope of Viperin and Viperin-like Enzymes from Three Domains of Life
    Lachowicz, Jake C.; Gizzi, Anthony S.; Almo, Steven C.; Grove, Tyler L.
    Biochemistry (2021), 60 (26), 2116-2129CODEN: BICHAW; ISSN:0006-2960. (American Chemical Society)
    Viperin is a member of the radical S-adenosylmethionine superfamily and was shown to restrict the replication of a wide range of RNA and DNA viruses. The authors recently demonstrated that human viperin (HsVip) catalyzes the conversion of CTP to 3'-deoxy-3',4'-didehydro-CTP (ddhCTP or ddh-synthase), which acts as a chain terminator for virally encoded RNA-dependent RNA polymerases from several flaviviruses. Viperin homologs also exist in non-chordate eukaryotes (e.g., Cnidaria and Mollusca), numerous fungi, and members of the archaeal and eubacterial domains. Recently, it is reported that non-chordate and non-eukaryotic viperin-like homologs are also ddh-synthases and generate a diverse range of ddhNTPs, including the newly discovered ddhUTP and ddhGTP. Herein, the authors expand on the catalytic mechanism of mammalian, fungal, bacterial, and archaeal viperin-like enzymes with a combination of x-ray crystallog. and enzymol. Like mammalian viperins, these recently discovered viperin-like enzymes operate through the same mechanism and can be classified as ddh-synthases. Furthermore, the authors define the unique chem. and phys. determinants supporting ddh-synthase activity and nucleotide selectivity, including the crystallog. characterization of a fungal viperin-like enzyme that uses UTP as a substrate and a cnidaria viperin-like enzyme that uses CTP as a substrate. Together, these results support the evolutionary conservation of the ddh-synthase activity and its broad phylogenetic role in innate antiviral immunity.
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    Tararina, M. A.; Allen, K. N. Bioinformatic Analysis of the Flavin-Dependent Amine Oxidase Superfamily: Adaptations for Substrate Specificity and Catalytic Diversity. J. Mol. Biol. 2020, 432, 3269– 3288,  DOI: 10.1016/j.jmb.2020.03.007
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    Bioinformatic Analysis of the Flavin-Dependent Amine Oxidase Superfamily: Adaptations for Substrate Specificity and Catalytic Diversity
    Tararina, Margarita A.; Allen, Karen N.
    Journal of Molecular Biology (2020), 432 (10), 3269-3288CODEN: JMOBAK; ISSN:0022-2836. (Elsevier Ltd.)
    The flavin-dependent amine oxidase (FAO) superfamily consists of over 9000 nonredundant sequences represented in all domains of life. Of the thousands of members identified, only 214 have been functionally annotated to date, and 40 unique structures are represented in the Protein Data Bank. The few functionally characterized members share a catalytic mechanism involving the oxidn. of an amine substrate through transfer of a hydride to the FAD cofactor, with differences obsd. in substrate specificities. Previous studies have focused on comparing a subset of superfamily members. Here, we present a comprehensive anal. of the FAO superfamily based on reaction mechanism and substrate recognition. Using a dataset of 9192 sequences, a sequence similarity network, and subsequently, a genome neighborhood network were constructed, organizing the superfamily into eight subgroups that accord with substrate type. Likewise, through phylogenetic anal., the evolutionary relationship of subgroups was detd., delineating the divergence between enzymes based on organism, substrate, and mechanism. In addn., using sequences and at. coordinates of 22 structures from the Protein Data Bank to perform sequence and structural alignments, active-site elements were identified, showing divergence from the canonical arom.-cage residues to accommodate large substrates. These specificity determinants are held in a structural framework comprising a core domain catalyzing the oxidn. of amines with an auxiliary domain for substrate recognition. Overall, anal. of the FAO superfamily reveals a modular fold with cofactor and substrate-binding domains allowing for diversity of recognition via insertion/deletions. This flexibility allows facile evolution of new activities, as shown by reinvention of function between subfamilies.
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    Gerlt, J. A.; Bouvier, J. T.; Davidson, D. B.; Imker, H. J.; Sadkhin, B.; Slater, D. R.; Whalen, K. L. Enzyme Function Initiative-Enzyme Similarity Tool (EFI-EST): A web tool for generating protein sequence similarity networks. Biochim. Biophys. Acta, Proteins Proteomics 2015, 1854, 1019– 1037,  DOI: 10.1016/j.bbapap.2015.04.015
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    Enzyme function initiative-enzyme similarity tool (EFI-EST): A web tool for generating protein sequence similarity networks
    Gerlt, John A.; Bouvier, Jason T.; Davidson, Daniel B.; Imker, Heidi J.; Sadkhin, Boris; Slater, David R.; Whalen, Katie L.
    Biochimica et Biophysica Acta, Proteins and Proteomics (2015), 1854 (8), 1019-1037CODEN: BBAPBW; ISSN:1570-9639. (Elsevier B. V.)
    A review. The Enzyme Function Initiative, an NIH/NIGMS-supported Large-Scale Collaborative Project (EFI; U54GM093342; http://enzymefunction.org/), is focused on devising and disseminating bioinformatics and computational tools as well as exptl. strategies for the prediction and assignment of functions (in vitro activities and in vivo physiol./metabolic roles) to uncharacterized enzymes discovered in genome projects. Protein sequence similarity networks (SSNs) are visually powerful tools for analyzing sequence relationships in protein families (H.J. Atkinson, J.H. Morris, T.E. Ferrin, and P.C. Babbitt, PLoS One 2009, 4, e4345). However, the members of the biol./biomedical community have not had access to the capability to generate SSNs for their "favorite" protein families. In this article we announce the EFI-EST (Enzyme Function Initiative-Enzyme Similarity Tool) web tool (http://efi.igb.illinois.edu/efi-est/) that is available without cost for the automated generation of SSNs by the community. The tool can create SSNs for the "closest neighbors" of a user-supplied protein sequence from the UniProt database (Option A) or of members of any user-supplied Pfam and/or InterPro family (Option B). We provide an introduction to SSNs, a description of EFI-EST, and a demonstration of the use of EFI-EST to explore sequence-function space in the OMP decarboxylase superfamily (PF00215). This article is designed as a tutorial that will allow members of the community to use the EFI-EST web tool for exploring sequence/function space in protein families.
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    Kohl, M.; Wiese, S.; Warscheid, B. Cytoscape: Software for Visualization and Analysis of Biological Networks. In Data Mining in Proteomics: From Standards to Applications; Hamacher, M., Eisenacher, M., Stephan, C., Eds.; Methods in Molecular Biology; Humana Press: Totowa, NJ, 2011; pp 291– 303.
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    Burns, M.; Martinez, C. A.; Vanderplas, B.; Wisdom, R.; Yu, S.; Singer, R. A. A Chemoenzymatic Route to Chiral Intermediates Used in the Multikilogram Synthesis of a Gamma Secretase Inhibitor. Org. Process Res. Dev. 2017, 21, 871– 877,  DOI: 10.1021/acs.oprd.7b00096
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    A Chemoenzymatic Route to Chiral Intermediates Used in the Multikilogram Synthesis of a Gamma Secretase Inhibitor
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    Organic Process Research & Development (2017), 21 (6), 871-877CODEN: OPRDFK; ISSN:1083-6160. (American Chemical Society)
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    Alc. dehydrogenases are able to catalyze the conversion of alcs. to aldehydes or ketones, simultaneously reducing the cofactor NAD+ or NADP+ to NAD(P)H. Because of the high costs of these pyridine cofactors, in situ cofactor regeneration is required for preparative applications in order to reach turnover nos. that are sufficient for economically viable processes. Here we present the development of a process for the enantioselective oxidn. of rac-1-phenylethanol to acetophenone, applying an alc. dehydrogenase coupled with an NAD(P)H oxidase for the enzymic cofactor regeneration, which is active towards NADH as well as NADPH. The reaction system was investigated in view of various influential parameters with main focus on the external oxygen supply. We could show that a gassed stirred tank reactor is a promising reactor concept to run NAD(P)H oxidase-coupled alc. dehydrogenase oxidns., including the possibility to scale-up the system.
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    α-2H Amino acids are valuable precursors toward labeled pharmaceutical agents and tools for studying biol. systems; however, these mols. are costly to purchase and challenging to synthesize in a site- and stereoselective manner. Here, we show that an α-oxoamine synthase that evolved for saxitoxin biosynthesis, SxtA AONS, is capable of producing a range of α-2H amino acids and esters site- and stereoselectively using D2O as the deuterium source. Addnl., we demonstrate the utility of this operationally simple reaction on preparative-scale in the stereoselective chemoenzymic synthesis of a deuterated analog of safinamide, a drug used to treat Parkinson's disease.
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    ACS Catalysis (2020), 10 (19), 11438-11447CODEN: ACCACS; ISSN:2155-5435. (American Chemical Society)
    A strategy for the mild generation of synthetically valuable α-amino radicals from robust tertiary amide building blocks has been developed. By combining Vaska's complex-catalyzed tertiary amide reductive activation and photochem. single electron redn. into a streamlined tandem process, metastable hemiaminal intermediates were successfully transformed into nucleophilic α-amino free radical species. This umpolung approach to such reactive intermediates was exemplified through coupling with an electrophilic dehydroalanine acceptor, resulting in the synthesis of an array of α-functionalized tertiary amine derivs., previously inaccessible from the amide starting materials. The utility of the strategy was expanded to include secondary amide substrates, intramol. variants, and late-stage functionalization of an active pharmaceutical ingredient. D. functional theory analyses were used to establish the reaction mechanism and elements of the chem. system that were responsible for the reaction's efficiency. Safety: CO gas alarm must be worn when prepg. precursor to Vaska-II complex.
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    DeHovitz, J. S.; Loh, Y. Y.; Kautzky, J. A.; Nagao, K.; Meichan, A. J.; Yamauchi, M.; MacMillan, D. W. C.; Hyster, T. K. Static to inducibly dynamic stereocontrol: The convergent use of racemic β-substituted ketones. Science 2020, 369, 1113– 1118,  DOI: 10.1126/science.abc9909
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    Science (Washington, DC, United States) (2020), 369 (6507), 1113-1118CODEN: SCIEAS; ISSN:1095-9203. (American Association for the Advancement of Science)
    The synthesis of stereochem. complex mols. in the pharmaceutical and agrochem. industries requires precise control over each distinct stereocenter, a feat that can be challenging and time consuming using traditional asym. synthesis. Although stereoconvergent processes have the potential to streamline and simplify synthetic routes, they are currently limited by a narrow scope of inducibly dynamic stereocenters that can be readily epimerized. Here, we report the use of photoredox catalysis to enable the racemization of traditionally static, unreactive stereocenters through the intermediacy of prochiral radical species. This technol. was applied in conjunction with biocatalysts such as ketoreductases and aminotransferases to realize stereoconvergent syntheses of stereodefined β-substituted alcs. and amines from β-substituted ketones.
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    Generation, Characterization, and Tunable Reactivity of Organometallic Fragments Bound to a Protein Ligand
    Key, Hanna M.; Clark, Douglas S.; Hartwig, John F.
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    Organotransition metal complexes catalyze important synthetic transformations, and the development of these systems has rested on the detailed understanding of the structures and elementary reactions of discrete organometallic complexes bound to org. ligands. One strategy for the creation of new organometallic systems is to exploit the intricate and highly structured ligands found in natural metalloproteins. The authors report the prepn. and characterization of discrete rhodium and iridium fragments bound site-specifically in a κ2-fashion to the protein carbonic anhydrase (CA) as a ligand. The reactions of apo human carbonic anhydrase with [Rh(nbd)2]BF4 or [M(CO)2(acac)] (M = Rh, Ir) form proteins contg. Rh or Ir with organometallic ligands. A colorimetric assay was developed to quantify rapidly the metal occupancy at the native metal-binding site, and 15N-1H NMR spectroscopy was used to establish the amino acids to which the metal is bound. IR spectroscopy and EXAFS revealed the presence and no. of carbonyl ligands and the no. total ligands, while UV-vis spectroscopy provided a signature to readily identify species that had been fully characterized. Exploiting these methods, the authors obsd. fundamental stoichiometric reactions of the artificial organometallic site of this protein, including reactions that simultaneously form and cleave metal-carbon bonds. The authors found that the discrete organometallic protein complexes, Rh(cod)-CA, Rh(nbd)-CA and Rh(CO)2-CA do not catalyze the hydrogenation or hydroformylation of a range of potential substrates of these reactions. These findings suggest that the active catalyst of the previously reported systems was not a Rh center ligated at the native Zn site of CA; instead, it is more likely that these reactions are catalyzed by a dissocd. Rh fragment or fragment assocd. with a different site on the protein.
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    Huang, J.; Liu, Z.; Bloomer, B. J.; Clark, D. S.; Mukhopadhyay, A.; Keasling, J. D.; Hartwig, J. F. Unnatural biosynthesis by an engineered microorganism with heterologously expressed natural enzymes and an artificial metalloenzyme. Nat. Chem. 2021, 13, 1186– 1191,  DOI: 10.1038/s41557-021-00801-3
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    Unnatural biosynthesis by an engineered microorganism with heterologously expressed natural enzymes and an artificial metalloenzyme
    Huang, Jing; Liu, Zhennan; Bloomer, Brandon J.; Clark, Douglas S.; Mukhopadhyay, Aindrila; Keasling, Jay D.; Hartwig, John F.
    Nature Chemistry (2021), 13 (12), 1186-1191CODEN: NCAHBB; ISSN:1755-4330. (Nature Portfolio)
    Synthetic biol. enables microbial hosts to produce complex mols. from organisms that are rare or difficult to cultivate, but the structures of these mols. are limited to those formed by reactions of natural enzymes. The integration of artificial metalloenzymes (ArMs) that catalyze unnatural reactions into metabolic networks could broaden the cache of mols. produced biosynthetically. Here we report an engineered microbial cell expressing a heterologous biosynthetic pathway, contg. both natural enzymes and ArMs, that produces an unnatural product with high diastereoselectivity. We engineered Escherichia coli with a heterologous terpene biosynthetic pathway and an ArM contg. an iridium-porphyrin complex that was transported into the cell with a heterologous transport system. We improved the diastereoselectivity and product titer of the unnatural product by evolving the ArM and selecting the appropriate gene induction and cultivation conditions. This work shows that synthetic biol. and synthetic chem. can produce, by combining natural and artificial enzymes in whole cells, mols. that were previously inaccessible to nature.
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    Gu, Y.; Natoli, S. N.; Liu, Z.; Clark, D. S.; Hartwig, J. F. Site-Selective Functionalization of (sp3)C–H Bonds Catalyzed by Artificial Metalloenzymes Containing an Iridium-Porphyrin Cofactor. Angew. Chem., Int. Ed. 2019, 58, 13954– 13960,  DOI: 10.1002/anie.201907460
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    Site-Selective Functionalization of (sp3)C-H Bonds Catalyzed by Artificial Metalloenzymes Containing an Iridium-Porphyrin Cofactor
    Gu, Yang; Natoli, Sean N.; Liu, Zhennan; Clark, Douglas S.; Hartwig, John F.
    Angewandte Chemie, International Edition (2019), 58 (39), 13954-13960CODEN: ACIEF5; ISSN:1433-7851. (Wiley-VCH Verlag GmbH & Co. KGaA)
    The selective functionalization of one C-H bond over others in nearly identical steric and electronic environments can facilitate the construction of complex mols. We report site-selective functionalizations of C-H bonds, differentiated solely by remote substituents, catalyzed by artificial metalloenzymes (ArMs) that are generated from the combination of an evolvable P 450 scaffold and an iridium-porphyrin cofactor. The generated systems catalyze the insertion of carbenes into the C-H bonds of a range of phthalan derivs. contg. substituents that render the two methylene positions in each phthalan inequivalent. These reactions occur with site-selectivity ratios of up to 17.8:1 and, in most cases, with pairs of enzyme mutants that preferentially form each of the two constitutional isomers. This study demonstrates the potential of abiotic reactions catalyzed by metalloenzymes to functionalize C-H bonds with site selectivity that is difficult to achieve with small-mol. catalysts.
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  2. Lanting Xiang, Jennifer Solarczek, Victor Krajka, Hui Liu, Lina Ahlborn, Anett Schallmey, Iordania Constantinou. Evaluating the Potential of Microdroplet Flow in Two-Phase Biocatalysis: A Systematic Study. ACS Applied Materials & Interfaces 2025, 17 (3) , 4776-4787. https://doi.org/10.1021/acsami.4c15647
  3. Di Yang, Chang-Hwa Chiang, Taveechai Wititsuwannakul, Charles L. Brooks III, Paul M. Zimmerman, Alison R. H. Narayan. Engineering the Reaction Pathway of a Non-heme Iron Oxygenase Using Ancestral Sequence Reconstruction. Journal of the American Chemical Society 2024, 146 (50) , 34352-34363. https://doi.org/10.1021/jacs.4c08420
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  6. Shubhanshu Jain, Felipe Ospina, Stephan C. Hammer. A New Age of Biocatalysis Enabled by Generic Activation Modes. JACS Au 2024, 4 (6) , 2068-2080. https://doi.org/10.1021/jacsau.4c00247
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  16. Fengming Shi, Bin Chen, Jinhai Yu, Ruiqi Zhu, Yu Zheng, Xiaoqiang Huang. Enantioselective biosynthesis of vicinal diamines enabled by synergistic photo/biocatalysis consisting of an ene-reductase and a green-light-excited organic dye. Chinese Journal of Catalysis 2025, 68 , 223-229. https://doi.org/10.1016/S1872-2067(24)60168-3
  17. Gaurav P. Kudalkar, Virendra K. Tiwari, David B. Berkowitz. Exploiting Archaeal/Thermostable Enzymes in Synthetic Chemistry: Back to the Future?. ChemCatChem 2024, 16 (21) https://doi.org/10.1002/cctc.202400835
  18. Deepthi Pulivarthi, Ravinder Reddy Patlolla, Joshna Kuni, Raveena Gajjala, Santhosh Nayak Kethavath, Linga Banoth, Subba Reddy B V. Enzymatic kinetic resolution of an intermediate alcohol of the drug Tegoprazan using lipase from Thermomyces lanuginosus. Biocatalysis and Biotransformation 2024, 42 (6) , 653-660. https://doi.org/10.1080/10242422.2024.2326421
  19. Peng Zhang, Shuaiqi Meng, Zhongyu Li, Dennis Hirtz, Lothar Elling, Leilei Zhu, Yu Ji, Ulrich Schwaneberg. A comparative molecular dynamics approach guides the tailoring of glycosyltransferases to meet synthetic applications. Green Chemistry 2024, 26 (16) , 9186-9194. https://doi.org/10.1039/D4GC01508H
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  21. Marianne B. Haarr, Kotchakorn T. Sriwong, Elaine O'Reilly. Commercial Transaminases for the Asymmetric Synthesis of Bulky Amines. European Journal of Organic Chemistry 2024, 27 (23) https://doi.org/10.1002/ejoc.202400257
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  24. Priya Kamboj, Vikas Tyagi. Enzymatic Synthesis of Indole‐Based Imidazopyridine using α‐Amylase. ChemBioChem 2024, 25 (6) https://doi.org/10.1002/cbic.202300824
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  • Abstract

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    Figure 1

    Figure 1. Select examples of chemical structures accessed by using biocatalysis. (A) Compounds formed through C–C bond forming reactions. (B) Compounds accessed using C–H hydroxylation reactions. (C) Hydroxylative dearomatization in the total synthesis of azaphilone natural products. (D) Amino acid C–H hydroxylation in the synthesis of manzacidin C. (E) Multienzyme cascade toward the process-scale total synthesis of islatravir.

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    Figure 2

    Figure 2. (A) Historical access to enzymes and enzyme products was a time-consuming process. The understanding of biological systems and the lack of enabling technologies make it difficult to efficiently develop new biocatalysts. (B) Example of an early application of biocatalysts in the synthesis of d-amino acids. This process required the use of a specific strain of bacteria to complete the transformation.

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    Figure 3

    Figure 3. Accessing biocatalysts with today’s methods. (A) General workflow for producing enzymes from the gene encoding for an enzyme of interest. The various entry points where a scientist could step into the process are highlighted. (B) Enzymes can be used in biocatalytic reactions at various levels of purity.

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    Figure 4

    Figure 4. Chemoenzymatic synthesis of Molnupiravir demonstrated by Merck (right) compared to the previous small-molecule route (left).

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    Figure 5

    Figure 5. Example of a chemoenzymatic synthesis used in an undergraduate chemistry laboratory course.

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    Figure 6

    Figure 6. Examples of chemoenzymatic and enzymatic methods that result from collaborations between organic and biocatalysis research groups.

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  • References

    This article references 112 other publications.

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      Small mols. remain the backbone for modern drug discovery. They are conceived and synthesized by medicinal chemists, many of whom were originally trained as org. chemists. Support from government and industry to provide training and personnel for continued development of this crit. skill set has been declining for many years. This Viewpoint highlights the value of org. chem. and org. medicinal chemists in the complex journey of drug discovery as a reminder that basic science support must be restored.
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      Chemistry - A European Journal (2020), 26 (6), 1196-1237CODEN: CEUJED; ISSN:0947-6539. (Wiley-VCH Verlag GmbH & Co. KGaA)
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      >> More from SciFinder ®
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      A review. The total synthesis of structurally complex natural products has challenged and inspired generations of chemists and remains an exciting area of active research. Despite their history as privileged bioactivity-rich scaffolds, the use of natural products in drug discovery has waned. This shift is driven by their relatively low abundance hindering isolation from natural sources and the challenges presented by their synthesis. Recent developments in biocatalysis have resulted in the application of enzymes for the construction of complex mols. From the inception of the Narayan lab in 2015, we have focused on harnessing the exquisite selectivity of enzymes alongside contemporary small mol.-based approaches to enable concise chemoenzymic routes to natural products. We have focused on enzymes from various families that perform selective oxidn. reactions. For example, we have targeted xyloketal natural products through a strategy that relies on a chemo- and site-selective biocatalytic hydroxylation. Members of the xyloketal family are characterized by polycyclic ketal cores and demonstrate potent neurol. activity. We envisioned assembling a representative xyloketal natural product (xyloketal D) involving a biocatalytically generated ortho-quinone methide intermediate. The non-heme iron (NHI) dependent monooxygenase ClaD was used to perform the benzylic hydroxylation of a resorcinol precursor, the product of which can undergo spontaneous loss of water to form an ortho-quinone methide under mild conditions. This intermediate was trapped using a chiral dienophile to complete the total synthesis of xyloketal D. A second class of biocatalytic oxidn. that we have employed in synthesis is the hydroxylative dearomatization of resorcinol compds. using flavin-dependent monooxygenases (FDMOs). We anticipated that the catalyst-controlled site- and stereoselectivity of FDMOs would enable the total synthesis of azaphilone natural products. Azaphilones are bioactive compds. characterized by a pyranoquinone bicyclic core and a fully substituted chiral carbon atom. We leveraged the stereodivergent reactivity of FDMOs AzaH and AfoD to achieve the enantioselective synthesis of trichoflectin enantiomers, deflectin 1a, and lunatoic acid. We also leveraged FDMOs to construct tropolone and sorbicillinoid natural products. Tropolones are a structurally diverse class of bioactive mols. characterized by an arom. cycloheptatriene core bearing an α-hydroxyketone moiety. We developed a two-step biocatalytic cascade to the tropolone natural product stipitatic aldehyde using the FDMO TropB and a NHI monooxygenase TropC. The FDMO SorbC obtained from the sorbicillin biosynthetic pathway was used in the concise total synthesis of a urea sorbicillinoid natural product. Our long-standing interest in using enzymes to carry out C-H hydroxylation reactions has also been channeled for the late-stage diversification of complex scaffolds. For example, we have used Rieske oxygenases to hydroxylate the tricyclic core common to paralytic shellfish toxins. The systemic toxicity of these compds. can be reduced by adding hydroxyl and sulfate groups, which improves their properties and potential as therapeutic agents. The enzymes SxtT, GxtA, SxtN, and SxtSUL were used to carry out selective C-H hydroxylation and O-sulfation in saxitoxin and related structures. We conclude this Account with a discussion of existing challenges in biocatalysis and ways we can currently address them.
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      Biocatalytic oxidative cross-coupling reactions for biaryl bond formation
      Zetzsche, Lara E.; Yazarians, Jessica A.; Chakrabarty, Suman; Hinze, Meagan E.; Murray, Lauren A. M.; Lukowski, April L.; Joyce, Leo A.; Narayan, Alison R. H.
      Nature (London, United Kingdom) (2022), 603 (7899), 79-85CODEN: NATUAS; ISSN:1476-4687. (Nature Portfolio)
      Biaryl compds., with two connected arom. rings, are found across medicine, materials science and asym. catalysis1,2. The necessity of joining arene building blocks to access these valuable compds. has inspired several approaches for biaryl bond formation and challenged chemists to develop increasingly concise and robust methods for this task3. Oxidative coupling of two C-H bonds offers an efficient strategy for the formation of a biaryl C-C bond; however, fundamental challenges remain in controlling the reactivity and selectivity for uniting a given pair of substrates4,5. Biocatalytic oxidative cross-coupling reactions have the potential to overcome limitations inherent to numerous small-mol.-mediated methods by providing a paradigm with catalyst-controlled selectivity6. Here we disclose a strategy for biocatalytic cross-coupling through oxidative C-C bond formation using cytochrome P 450 enzymes. We demonstrate the ability to catalyze cross-coupling reactions on a panel of phenolic substrates using natural P 450 catalysts. Moreover, we engineer a P 450 to possess the desired reactivity, site selectivity and atroposelectivity by transforming a low-yielding, unselective reaction into a highly efficient and selective process. This streamlined method for constructing sterically hindered biaryl bonds provides a programmable platform for assembling mols. with catalyst-controlled reactivity and selectivity.
      >> More from SciFinder ®
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      Chakrabarty, S.; Wang, Y.; Perkins, J. C.; Narayan, A. R. H. Scalable biocatalytic C–H oxyfunctionalization reactions. Chem. Soc. Rev. 2020, 49, 8137– 8155,  DOI: 10.1039/D0CS00440E
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      Chakrabarty, Suman; Wang, Ye; Perkins, Jonathan C.; Narayan, Alison R. H.
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      A review. Catalytic C-H oxyfunctionalization reactions have garnered significant attention in recent years with their ability to streamline synthetic routes toward complex mols. Consequently, there have been significant strides in the design and development of catalysts that enable diversification through C-H functionalization reactions. Enzymic C-H oxygenation reactions are often complementary to small mol. based synthetic approaches, providing a powerful tool when deployable on preparative-scale. This review highlights key advances in scalable biocatalytic C-H oxyfunctionalization reactions developed within the past decade.
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      Expanding the organic toolbox. A guide to integrating biocatalysis in synthesis
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      Chemical Society Reviews (2012), 41 (4), 1585-1605CODEN: CSRVBR; ISSN:0306-0012. (Royal Society of Chemistry)
      A review. This crit. review presented an introduction to biocatalysis for synthetic chemists. Advances in biocatalysis of the past 5 years illustrate the breadth of applications for these powerful and selective catalysts in conducting key reaction steps. Asym. synthesis of value-added targets and other reaction types were covered, with an emphasis on pharmaceutical intermediates and bulk chems. Resources of interest for the non-initiated are provided, including specialized web-sites and service providers to facilitate identification of suitable biocatalysts, as well as refs. to recent vols. and reviews for more detailed biocatalytic procedures. Challenges related to the application of biocatalysts were discussed, including how green a biocatalytic reaction may be, and trends in biocatalyst improvement through enzyme engineering were presented (152 refs.).
      >> More from SciFinder ®
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      Sheldon, R. A.; Brady, D.; Bode, M. L. The Hitchhiker’s guide to biocatalysis: recent advances in the use of enzymes in organic synthesis. Chem. Sci. 2020, 11, 2587– 2605,  DOI: 10.1039/C9SC05746C
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      The Hitchhiker's guide to biocatalysis: recent advances in the use of enzymes in organic synthesis
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      Chemical science (2020), 11 (10), 2587-2605 ISSN:2041-6520.
      Enzymes are excellent catalysts that are increasingly being used in industry and academia. This perspective is primarily aimed at synthetic organic chemists with limited experience using enzymes and provides a general and practical guide to enzymes and their synthetic potential, with particular focus on recent applications.
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      A review. Biocatalysis is one of the most promising technologies for the sustainable synthesis of mols. for pharmaceutical, biotechnol. and industrial purposes. From the gram to the ton scale, biocatalysis is employed with success. This is underpinned by the fact that the global enzyme market is predicted to increase from $7 billion to $10 billion by 2024. This review concs. on showing the strong benefits that biocatalysis and the use of enzymes can provide to synthetic chem. Several examples of successful implementations of enzymes are discussed highlighting not only high-value pharmaceutical processes but also low-cost bulk products. Thus, biocatalytic methods make the chem. more environmentally friendly and product specific.
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      >> More from SciFinder ®
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    14. 14
      Huffman, M. A.; Fryszkowska, A.; Alvizo, O.; Borra-Garske, M.; Campos, K. R.; Canada, K. A.; Devine, P. N.; Duan, D.; Forstater, J. H.; Grosser, S. T.; Halsey, H. M.; Hughes, G. J.; Jo, J.; Joyce, L. A.; Kolev, J. N.; Liang, J.; Maloney, K. M.; Mann, B. F.; Marshall, N. M.; McLaughlin, M.; Moore, J. C.; Murphy, G. S.; Nawrat, C. C.; Nazor, J.; Novick, S.; Patel, N. R.; Rodriguez-Granillo, A.; Robaire, S. A.; Sherer, E. C.; Truppo, M. D.; Whittaker, A. M.; Verma, D.; Xiao, L.; Xu, Y.; Yang, H. Design of an in vitro biocatalytic cascade for the manufacture of islatravir. Science 2019, 366, 1255– 1259,  DOI: 10.1126/science.aay8484
      14
      Design of an in vitro biocatalytic cascade for the manufacture of islatravir
      Huffman, Mark A.; Fryszkowska, Anna; Alvizo, Oscar; Borra-Garske, Margie; Campos, Kevin R.; Canada, Keith A.; Devine, Paul N.; Duan, Da; Forstater, Jacob H.; Grosser, Shane T.; Halsey, Holst M.; Hughes, Gregory J.; Jo, Junyong; Joyce, Leo A.; Kolev, Joshua N.; Liang, Jack; Maloney, Kevin M.; Mann, Benjamin F.; Marshall, Nicholas M.; McLaughlin, Mark; Moore, Jeffrey C.; Murphy, Grant S.; Nawrat, Christopher C.; Nazor, Jovana; Novick, Scott; Patel, Niki R.; Rodriguez-Granillo, Agustina; Robaire, Sandra A.; Sherer, Edward C.; Truppo, Matthew D.; Whittaker, Aaron M.; Verma, Deeptak; Xiao, Li; Xu, Yingju; Yang, Hao
      Science (Washington, DC, United States) (2019), 366 (6470), 1255-1259CODEN: SCIEAS; ISSN:1095-9203. (American Association for the Advancement of Science)
      Enzyme-catalyzed reactions have begun to transform pharmaceutical manufg., offering levels of selectivity and tunability that can dramatically improve chem. synthesis. Combining enzymic reactions into multistep biocatalytic cascades brings addnl. benefits. Cascades avoid the waste generated by purifn. of intermediates. They also allow reactions to be linked together to overcome an unfavorable equil. or avoid the accumulation of unstable or inhibitory intermediates. We report an in vitro biocatalytic cascade synthesis of the investigational HIV treatment islatravir. Five enzymes were engineered through directed evolution to act on non-natural substrates. These were combined with four auxiliary enzymes to construct islatravir from simple building blocks in a three-step biocatalytic cascade. The overall synthesis requires fewer than half the no. of steps of the previously reported routes.
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    15. 15
      Loskot, S. A.; Romney, D. K.; Arnold, F. H.; Stoltz, B. M. Enantioselective Total Synthesis of Nigelladine A via Late-Stage C–H Oxidation Enabled by an Engineered P450 Enzyme. J. Am. Chem. Soc. 2017, 139, 10196– 10199,  DOI: 10.1021/jacs.7b05196
      15
      Enantioselective Total Synthesis of Nigelladine A via Late-Stage C-H Oxidation Enabled by an Engineered P450 Enzyme
      Loskot, Steven A.; Romney, David K.; Arnold, Frances H.; Stoltz, Brian M.
      Journal of the American Chemical Society (2017), 139 (30), 10196-10199CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)
      An enantioselective total synthesis of the norditerpenoid alkaloid nigelladine A is described. Strategically, the synthesis relies on a late-stage C-H oxidn. of an advanced intermediate. While traditional chem. methods failed to deliver the desired outcome, an engineered cytochrome P 450 enzyme was employed to effect a chemo- and regioselective allylic C-H oxidn. in the presence of four oxidizable positions. The enzyme variant was readily identified from a focused library of three enzymes, allowing for completion of the synthesis without the need for extensive screening.
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    16. 16
      Chen, K.; Huang, X.; Kan, S. B. J.; Zhang, R. K.; Arnold, F. H. Enzymatic construction of highly strained carbocycles. Science 2018, 360, 71– 75,  DOI: 10.1126/science.aar4239
      16
      Enzymatic construction of highly strained carbocycles
      Chen, Kai; Huang, Xiongyi; Kan, S. B. Jennifer; Zhang, Ruijie K.; Arnold, Frances H.
      Science (Washington, DC, United States) (2018), 360 (6384), 71-75CODEN: SCIEAS; ISSN:0036-8075. (American Association for the Advancement of Science)
      Small carbocycles are structurally rigid and possess high intrinsic energy due to their ring strain. These features lead to broad applications but also create challenges for their construction. We report the engineering of a cytochrome P 450 variant (designated P411) that catalyze the formation of chiral bicyclobutanes, one of the most strained four-membered systems, via successive carbene addn. to unsatd. carbon-carbon bonds. Enzymes that produce cyclopropenes, putative intermediates to the bicyclobutanes, were also identified. These genetically encoded proteins are readily optimized by directed evolution, function in Escherichia coli, and act on structurally diverse substrates with high efficiency and selectivity, providing an effective route to many chiral strained structures. This biotransformation is easily performed at preparative scale, and the resulting strained carbocycles can be derivatized, opening myriad potential applications.
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    17. 17
      Zhang, X.; King-Smith, E.; Dong, L.-B.; Yang, L.-C.; Rudolf, J. D.; Shen, B.; Renata, H. Divergent synthesis of complex diterpenes through a hybrid oxidative approach. Science 2020, 369, 799– 806,  DOI: 10.1126/science.abb8271
      17
      Divergent synthesis of complex diterpenes through a hybrid oxidative approach
      Zhang, Xiao; King-Smith, Emma; Dong, Liao-Bin; Yang, Li-Cheng; Rudolf, Jeffrey D.; Shen, Ben; Renata, Hans
      Science (Washington, DC, United States) (2020), 369 (6505), 799-806CODEN: SCIEAS; ISSN:1095-9203. (American Association for the Advancement of Science)
      Polycyclic diterpenes exhibit many important biol. activities, but de novo synthetic access to these mols. is highly challenging because of their structural complexity. Semisynthetic access has also been limited by the lack of chem. tools for scaffold modifications. We report a chemoenzymic platform to access highly oxidized diterpenes by a hybrid oxidative approach that strategically combines chem. and enzymic oxidn. methods. This approach allows for selective oxidns. of previously inaccessible sites on the parent carbocycles and enables abiotic skeletal rearrangements to addnl. underlying architectures. We synthesized a total of nine complex natural products with rich oxygenation patterns and skeletal diversity in 10 steps or less from ent-steviol.
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    18. 18
      Nakamura, H.; Schultz, E. E.; Balskus, E. P. A new strategy for aromatic ring alkylation in cylindrocyclophane biosynthesis. Nat. Chem. Biol. 2017, 13, 916– 921,  DOI: 10.1038/nchembio.2421
      18
      A new strategy for aromatic ring alkylation in cylindrocyclophane biosynthesis
      Nakamura, Hitomi; Schultz, Erica E.; Balskus, Emily P.
      Nature Chemical Biology (2017), 13 (8), 916-921CODEN: NCBABT; ISSN:1552-4450. (Nature Publishing Group)
      Alkylation of arom. rings with alkyl halides is an important transformation in org. synthesis, yet an enzymic equiv. is unknown. Here, we report that cylindrocyclophane biosynthesis in Cylindrospermum licheniforme ATCC 29412 involves chlorination of an unactivated carbon center by a novel halogenase, followed by a previously uncharacterized enzymic dimerization reaction featuring sequential, stereospecific alkylations of resorcinol arom. rings. Discovery of the enzymic machinery underlying this unique biosynthetic carbon-carbon bond formation has implications for biocatalysis and metabolic engineering.
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    19. 19
      Schultz, E. E.; Braffman, N. R.; Luescher, M. U.; Hager, H. H.; Balskus, E. P. Biocatalytic Friedel–Crafts Alkylation Using a Promiscuous Biosynthetic Enzyme. Angew. Chem., Int. Ed. 2019, 58, 3151– 3155,  DOI: 10.1002/anie.201814016
      19
      Biocatalytic Friedel-Crafts Alkylation Using a Promiscuous Biosynthetic Enzyme
      Schultz, Erica E.; Braffman, Nathaniel R.; Luescher, Michael U.; Hager, Harry H.; Balskus, Emily P.
      Angewandte Chemie, International Edition (2019), 58 (10), 3151-3155CODEN: ACIEF5; ISSN:1433-7851. (Wiley-VCH Verlag GmbH & Co. KGaA)
      The Friedel-Crafts alkylation is commonly used in org. synthesis to form aryl-alkyl C-C linkages. However, this reaction lacks the stereospecificity and regiocontrol of enzymic catalysis. Here, we describe a stereospecific, biocatalytic Friedel-Crafts alkylation of the 2-position of resorcinol rings using the cylindrocyclophane biosynthetic enzyme CylK. This regioselectivity is distinct from that of the classical Friedel-Crafts reaction. Numerous secondary alkyl halides are accepted by this enzyme, as are resorcinol rings with a variety of substitution patterns. Finally, we have been able to use this transformation to access novel analogs of the clin. drug candidate benvitimod that are challenging to construct with existing synthetic methods. These findings highlight the promise of enzymic catalysis for enabling mild and selective C-C bond-forming synthetic methodol.
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    20. 20
      Lau, W.; Sattely, E. S. Six enzymes from mayapple that complete the biosynthetic pathway to the etoposide aglycone. Science 2015, 349, 1224– 1228,  DOI: 10.1126/science.aac7202
      20
      Six enzymes from mayapple that complete the biosynthetic pathway to the etoposide aglycone
      Lau, Warren; Sattely, Elizabeth S.
      Science (Washington, DC, United States) (2015), 349 (6253), 1224-1228CODEN: SCIEAS; ISSN:0036-8075. (American Association for the Advancement of Science)
      Podophyllotoxin is the natural product precursor of the chemotherapeutic etoposide, yet only part of its biosynthetic pathway is known. We used transcriptome mining in Podophyllum hexandrum (mayapple) to identify biosynthetic genes in the podophyllotoxin pathway. We selected 29 candidate genes to combinatorially express in Nicotiana benthamiana (tobacco) and identified six pathway enzymes, including an oxoglutarate-dependent dioxygenase that closes the core cyclohexane ring of the aryltetralin scaffold. By coexpressing 10 genes in tobacco-these 6 plus 4 previously discovered-we reconstitute the pathway to (-)-4'-desmethylepipodophyllotoxin (the etoposide aglycon), a naturally occurring lignan that is the immediate precursor of etoposide and, unlike podophyllotoxin, a potent topoisomerase inhibitor. Our results enable prodn. of the etoposide aglycon in tobacco and circumvent the need for cultivation of mayapple and semisynthetic epimerization and demethylation of podophyllotoxin.
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    21. 21
      Lowell, A. N.; DeMars, M. D.; Slocum, S. T.; Yu, F.; Anand, K.; Chemler, J. A.; Korakavi, N.; Priessnitz, J. K.; Park, S. R.; Koch, A. A.; Schultz, P. J.; Sherman, D. H. Chemoenzymatic Total Synthesis and Structural Diversification of Tylactone-Based Macrolide Antibiotics through Late-Stage Polyketide Assembly, Tailoring, and C─H Functionalization. J. Am. Chem. Soc. 2017, 139, 7913– 7920,  DOI: 10.1021/jacs.7b02875
      21
      Chemoenzymatic Total Synthesis and Structural Diversification of Tylactone-Based Macrolide Antibiotics through Late-Stage Polyketide Assembly, Tailoring, and C-H Functionalization
      Lowell, Andrew N.; DeMars, Matthew D.; Slocum, Samuel T.; Yu, Fengan; Anand, Krithika; Chemler, Joseph A.; Korakavi, Nisha; Priessnitz, Jennifer K.; Park, Sung Ryeol; Koch, Aaron A.; Schultz, Pamela J.; Sherman, David H.
      Journal of the American Chemical Society (2017), 139 (23), 7913-7920CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)
      Polyketide synthases (PKSs) represent a powerful catalytic platform capable of effecting multiple carbon-carbon bond forming reactions and oxidn. state adjustments. We explored the functionality of two terminal PKS modules that produce the 16-membered tylosin macrocycle, using them as biocatalysts in the chemoenzymic synthesis of tylactone and its subsequent elaboration to complete the first total synthesis of the juvenimicin, M-4365, and rosamicin classes of macrolide antibiotics via late-stage diversification. Synthetic chem. was employed to generate the tylactone hexaketide chain elongation intermediate that was accepted by the juvenimicin (Juv) ketosynthase of the penultimate JuvEIV PKS module. The hexaketide is processed through two complete modules (JuvEIV and JuvEV) in vitro, which catalyze elongation and functionalization of two ketide units followed by cyclization of the resulting octaketide into tylactone. After macrolactonization, a combination of in vivo glycosylation, selective in vitro cytochrome P 450-mediated oxidn., and chem. oxidn. was used to complete the scalable construction of a series of macrolide natural products in as few as 15 linear steps (21 total) with an overall yield of 4.6%.
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    22. 22
      Lukowski, A. L.; Denomme, N.; Hinze, M. E.; Hall, S.; Isom, L. L.; Narayan, A. R. H. Biocatalytic Detoxification of Paralytic Shellfish Toxins. ACS Chem. Biol. 2019, 14, 941– 948,  DOI: 10.1021/acschembio.9b00123
      22
      Biocatalytic Detoxification of Paralytic Shellfish Toxins
      Lukowski, April L.; Denomme, Nicholas; Hinze, Meagan E.; Hall, Sherwood; Isom, Lori L.; Narayan, Alison R. H.
      ACS Chemical Biology (2019), 14 (5), 941-948CODEN: ACBCCT; ISSN:1554-8929. (American Chemical Society)
      Small mols. that bind to voltage-gated sodium channels (VGSCs) are promising leads in the treatment of numerous neurodegenerative diseases and pain. Nature is a highly skilled medicinal chemist in this regard, designing potent VGSC ligands capable of binding to and blocking the channel, thereby offering compds. of potential therapeutic interest. Paralytic shellfish toxins (PSTs), produced by cyanobacteria and marine dinoflagellates, are examples of these naturally occurring small mol. VGSC blockers that can potentially be leveraged to solve human health concerns. Unfortunately, the remarkable potency of these natural products results in equally exceptional toxicity, presenting a significant challenge for the therapeutic application of these compds. Identifying less potent analogs and convenient methods for accessing them therefore provides an attractive approach to developing mols. with enhanced therapeutic potential. Fortunately, Nature has evolved tools to modulate the toxicity of PSTs through selective hydroxylation, sulfation, and desulfation of the core scaffold. Function of enzymes encoded in cyanobacterial PST biosynthetic gene clusters that have evolved specifically for the sulfation of highly functionalized PSTs, the substrate scope of these enzymes, and elucidate the biosynthetic route from saxitoxin to monosulfated gonyautoxins and disulfated C-toxins. Finally, the binding affinities of the nonsulfated, monosulfated, and disulfated products of these enzymic reactions have been evaluated for VGSC binding affinity using mouse whole brain membrane prepns. to provide an assessment of relative toxicity. These data demonstrate the unique detoxification effect of sulfotransferases in PST biosynthesis, providing a potential mechanism for the development of more attractive PST-derived therapeutic analogs.
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    23. 23
      Wang, J.; Zhang, Y.; Liu, H.; Shang, Y.; Zhou, L.; Wei, P.; Yin, W.-B.; Deng, Z.; Qu, X.; Zhou, Q. A biocatalytic hydroxylation-enabled unified approach to C19-hydroxylated steroids. Nat. Commun. 2019, 10, 3378,  DOI: 10.1038/s41467-019-11344-0
      23
      A biocatalytic hydroxylation-enabled unified approach to C19-hydroxylated steroids
      Wang Junlin; Shang Yong; Zhou Qianghui; Zhang Yanan; Liu Huanhuan; Zhou Linjun; Deng Zixin; Qu Xudong; Wei Penglin; Yin Wen-Bing
      Nature communications (2019), 10 (1), 3378 ISSN:.
      Steroidal C19-hydroxylation is pivotal to the synthesis of naturally occurring bioactive C19-OH steroids and 19-norsteroidal pharmaceuticals. However, realizing this transformation is proved to be challenging through either chemical or biological synthesis. Herein, we report a highly efficient method to synthesize 19-OH-cortexolone in 80% efficiency at the multi-gram scale. The obtained C19-OH-cortexolone can be readily transformed to various synthetically useful intermediates including the industrially valuable 19-OH-androstenedione, which can serve as a basis for synthesis of C19-functionalized steroids as well as 19-nor steroidal drugs. Using this biocatalytic C19-hydroxylation method, the unified synthesis of six C19-hydroxylated pregnanes is achieved in just 4 to 9 steps. In addition, the structure of sclerosteroid B is revised on the basis of our synthesis.
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    24. 24
      Pyser, J. B.; Baker Dockrey, S. A.; Benítez, A. R.; Joyce, L. A.; Wiscons, R. A.; Smith, J. L.; Narayan, A. R. H. Stereodivergent, Chemoenzymatic Synthesis of Azaphilone Natural Products. J. Am. Chem. Soc. 2019, 141, 18551– 18559,  DOI: 10.1021/jacs.9b09385
      24
      Stereodivergent, Chemoenzymatic Synthesis of Azaphilone Natural Products
      Pyser, Joshua B.; Baker Dockrey, Summer A.; Benitez, Attabey Rodriguez; Joyce, Leo A.; Wiscons, Ren A.; Smith, Janet L.; Narayan, Alison R. H.
      Journal of the American Chemical Society (2019), 141 (46), 18551-18559CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)
      Selective access to a targeted isomer is often crit. in the synthesis of biol. active mols. Whereas small-mol. reagents and catalysts often act with anticipated site- and stereoselectivity, this predictability does not extend to enzymes. Further, the lack of access to catalysts that provide complementary selectivity creates a challenge in the application of biocatalysis in synthesis. Here, we report an approach for accessing biocatalysts with complementary selectivity that is orthogonal to protein engineering. Through the use of a sequence similarity network (SSN), a no. of sequences were selected, and the corresponding biocatalysts were evaluated for reactivity and selectivity. With a no. of biocatalysts identified that operate with complementary site- and stereoselectivity, these catalysts were employed in the stereodivergent, chemoenzymic synthesis of azaphilone natural products. Specifically, the first syntheses of trichoflectin, deflectin-1a, and lunatoic acid A were achieved. In addn., chemoenzymic syntheses of these azaphilones supplied enantioenriched material for reassignment of the abs. configuration of trichoflectin and deflectin-1a based on optical rotation, CD spectra, and X-ray crystallog.
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    25. 25
      Zwick, C. R.; Renata, H. Remote C–H Hydroxylation by an α-Ketoglutarate-Dependent Dioxygenase Enables Efficient Chemoenzymatic Synthesis of Manzacidin C and Proline Analogs. J. Am. Chem. Soc. 2018, 140, 1165– 1169,  DOI: 10.1021/jacs.7b12918
      25
      Remote C-H Hydroxylation by an α-Ketoglutarate-Dependent Dioxygenase Enables Efficient Chemoenzymatic Synthesis of Manzacidin C and Proline Analogs
      Zwick, Christian R.; Renata, Hans
      Journal of the American Chemical Society (2018), 140 (3), 1165-1169CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)
      Selective C-H functionalization at distal positions remains a highly challenging problem in org. synthesis. Though Nature has evolved a myriad of enzymes capable of such feat, their synthetic utility has largely been overlooked. Here, we functionally characterize an α-ketoglutarate-dependent dioxygenase (Fe/αKG) that selectively hydroxylates the δ position of various aliph. amino acids. Kinetic anal. and substrate profiling of the enzyme show superior catalytic efficiency and substrate promiscuity relative to other Fe/αKGs that catalyze similar reactions. We demonstrate the practical utility of this transformation in the concise syntheses of a rare alkaloid, manzacidin C, and densely substituted amino acid derivs. with remarkable step efficiency. This work provides a blueprint for future applications of Fe/αKG hydroxylation in complex mol. synthesis and the development of powerful synthetic paradigms centered on enzymic C-H functionalization logic.
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    26. 26
      Lukowski, A. L.; Liu, J.; Bridwell-Rabb, J.; Narayan, A. R. H. Structural basis for divergent C–H hydroxylation selectivity in two Rieske oxygenases. Nat. Commun. 2020, 11, 2991,  DOI: 10.1038/s41467-020-16729-0
      26
      Structural basis for divergent C-H hydroxylation selectivity in two Rieske oxygenases
      Lukowski, April L.; Liu, Jianxin; Bridwell-Rabb, Jennifer; Narayan, Alison R. H.
      Nature Communications (2020), 11 (1), 2991CODEN: NCAOBW; ISSN:2041-1723. (Nature Research)
      Abstr.: Biocatalysts that perform C-H hydroxylation exhibit exceptional substrate specificity and site-selectivity, often through the use of high valent oxidants to activate these inert bonds. Rieske oxygenases are examples of enzymes with the ability to perform precise mono- or dioxygenation reactions on a variety of substrates. Understanding the structural features of Rieske oxygenases responsible for control over selectivity is essential to enable the development of this class of enzymes for biocatalytic applications. Decades of research has illuminated the crit. features common to Rieske oxygenases, however, structural information for enzymes that functionalize diverse scaffolds is limited. Here, we report the structures of two Rieske monooxygenases involved in the biosynthesis of paralytic shellfish toxins (PSTs), SxtT and GxtA, adding to the short list of structurally characterized Rieske oxygenases. Based on these structures, substrate-bound structures, and mutagenesis expts., we implicate specific residues in substrate positioning and the divergent reaction selectivity obsd. in these two enzymes.
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    27. 27
      Wu, S.; Snajdrova, R.; Moore, J. C.; Baldenius, K.; Bornscheuer, U. T. Biocatalysis: Enzymatic Synthesis for Industrial Applications. Angew. Chem., Int. Ed. 2021, 60, 88– 119,  DOI: 10.1002/anie.202006648
      27
      Biocatalysis: Enzymatic Synthesis for Industrial Applications
      Wu, Shuke; Snajdrova, Radka; Moore, Jeffrey C.; Baldenius, Kai; Bornscheuer, Uwe T.
      Angewandte Chemie, International Edition (2021), 60 (1), 88-119CODEN: ACIEF5; ISSN:1433-7851. (Wiley-VCH Verlag GmbH & Co. KGaA)
      A review. Biocatalysis has found numerous applications in various fields as an alternative to chem. catalysis. The use of enzymes in org. synthesis, esp. to make chiral compds. for pharmaceuticals as well for the flavors and fragrance industry, are the most prominent examples. In addn., biocatalysts are used on a large scale to make specialty and even bulk chems. This review intends to give illustrative examples in this field with a special focus on scalable chem. prodn. using enzymes. It also discusses the opportunities and limitations of enzymic syntheses using distinct examples and provides an outlook on emerging enzyme classes.
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    28. 28
      McIntosh, J. A.; Benkovics, T.; Silverman, S. M.; Huffman, M. A.; Kong, J.; Maligres, P. E.; Itoh, T.; Yang, H.; Verma, D.; Pan, W.; Ho, H.-I.; Vroom, J.; Knight, A. M.; Hurtak, J. A.; Klapars, A.; Fryszkowska, A.; Morris, W. J.; Strotman, N. A.; Murphy, G. S.; Maloney, K. M.; Fier, P. S. Engineered Ribosyl-1-Kinase Enables Concise Synthesis of Molnupiravir, an Antiviral for COVID-19. ACS Cent. Sci. 2021, 7, 1980– 1985,  DOI: 10.1021/acscentsci.1c00608
      28
      Engineered ribosyl-1-kinase enables concise synthesis of molnupiravir, an antiviral for COVID-19
      McIntosh, John A.; Benkovics, Tamas; Silverman, Steven M.; Huffman, Mark A.; Kong, Jongrock; Maligres, Peter E.; Itoh, Tetsuji; Yang, Hao; Verma, Deeptak; Pan, Weilan; Ho, Hsing-I.; Vroom, Jonathan; Knight, Anders M.; Hurtak, Jessica A.; Klapars, Artis; Fryszkowska, Anna; Morris, William J.; Strotman, Neil A.; Murphy, Grant S.; Maloney, Kevin M.; Fier, Patrick S.
      ACS Central Science (2021), 7 (12), 1980-1985CODEN: ACSCII; ISSN:2374-7951. (American Chemical Society)
      Molnupiravir (MK-4482) is an investigational antiviral agent that is under development for the treatment of COVID-19. Given the potential high demand and urgency for this compd., it was crit. to develop a short and sustainable synthesis from simple raw materials that would minimize the time needed to manuf. and supply molnupiravir. The route reported here is enabled through the invention of a novel biocatalytic cascade featuring an engineered ribosyl-1-kinase and uridine phosphorylase. These engineered enzymes were deployed with a pyruvate-oxidase-enabled phosphate recycling strategy. Compared to the initial route, this synthesis of molnupiravir is 70% shorter and approx. 7-fold higher yielding. Looking forward, the biocatalytic approach to molnupiravir outlined here is anticipated to have broad applications for streamlining the synthesis of nucleosides in general.
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    29. 29
      Bornscheuer, U. T.; Buchholz, K. Highlights in Biocatalysis – Historical Landmarks and Current Trends. Eng. Life Sci. 2005, 5, 309– 323,  DOI: 10.1002/elsc.200520089
      29
      Highlights in biocatalysis - historical landmarks and current trends
      Bornscheuer, U. T.; Buchholz, K.
      Engineering in Life Sciences (2005), 5 (4), 309-323CODEN: ELSNAE; ISSN:1618-0240. (Wiley-VCH Verlag GmbH & Co. KGaA)
      A review. Biocatalysis has ancient roots, yet it is developing into a key tool for synthesis in a wide range of applications. Important events in the history of enzyme technol. from the 19th century onwards are highlighted. Considering the most relevant progress steps, the prodn. of penicillanic acid and the impact of genetic engineering are traced in more detail. Applied biocatalysis has been defined as the application of a biocatalyst to achieve a desired conversion selectively, under controlled, mild conditions in a bioreactor. Biocatalysts are currently used to produce a wide range of products in the fields of food manuf. (such as bread, cheese, beer), fine chems. (e.g., amino acids, vitamins), and pharmaceuticals (e.g., derivs. of antibiotics). They not only provide access to innovative products and processes, but also meet criteria of sustainability. In org. synthesis, recombinant technologies and biocatalysts have greatly widened the scope of application. Examples of current applications and processes are given. Recent developments and trends are presented as a survey, covering new methods for accessing biodiversity with new enzymes, directed evolution for improving enzymes, designed cells, and integrated downstream processing.
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    30. 30
      Buchner, E. Alkoholische Gährung ohne Hefezellen. Berichte der deutschen chemischen Gesellschaft 1897, 30, 117– 124,  DOI: 10.1002/cber.18970300121
      30
      Alcoholic fermentation without yeast cells
      Buchner, Eduard
      Berichte der Deutschen Chemischen Gesellschaft (1897), 30 (), 117-24CODEN: BDCGAS ISSN:.
      When brewery yeast, to which no starch has been added, is ground with quartz sand and kieselguhr, moistened with water and pressed, the liquid which is obtained has the power of producing the fermentation of sugar, although it appears to be quite free from yeast cells. It has a sp. gr. of 1.0416, contains about 10 per cent. of residue, and gelatinises when boiled. This liquid produces alcoholic fermentation in solutions of cane-sugar, maltose, glucose, and fructose, but does not ferment either lactose or mannitol. Fermentation continued in many cases for two weeks, even at the temperature of 0°, and was not stopped by nitration of the liquid through a Berkefeldt filter. Plate cultures showed that in some cases small numbers of micro-organisms were present, but yeast cells were in no case detected. The author gives the name zymase to the substance which produces the fermentation. This appears to be a proteid, since the fermentative power of the solution is practically destroyed when it is heated for an hour at 40-50° and the coagulated albumin filtered off. The dried precipitate produced by alcohol does not yield any ferment to water.
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    31. 31
      Heckmann, C. M.; Paradisi, F. Looking Back: A Short History of the Discovery of Enzymes and How They Became Powerful Chemical Tools. ChemCatChem. 2020, 12, 6082– 6102,  DOI: 10.1002/cctc.202001107
      31
      Looking Back: A Short History of the Discovery of Enzymes and How They Became Powerful Chemical Tools
      Heckmann, Christian M.; Paradisi, Francesca
      ChemCatChem (2020), 12 (24), 6082-6102CODEN: CHEMK3; ISSN:1867-3880. (Wiley-VCH Verlag GmbH & Co. KGaA)
      A review. Enzymic approaches to challenges in chem. synthesis are increasingly popular and very attractive to industry given their green nature and high efficiency compared to traditional methods. In this historical review the authors highlight the developments across several fields that were necessary to create the modern field of biocatalysis, with enzyme engineering and directed evolution at its core. The authors exemplify the modular, incremental, and highly unpredictable nature of scientific discovery, driven by curiosity, and showcase the resulting examples of cutting-edge enzymic applications in industry.
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    32. 32
      Whitesides, G. M. Applications of Cell-Free Enzymes in Organic Synthesis. In Ciba Foundation Symposium 111 - Enzymes in Organic Synthesis; Pitman: London, 1985; pp 76– 96.
      There is no corresponding record for this reference.
    33. 33
      Olivieri, R.; Fascetti, E.; Angelini, L.; Degen, L. Microbial transformation of racemic hydantoins to d-amino acids. Biotechnol. Bioeng. 1981, 23, 2173– 2183,  DOI: 10.1002/bit.260231002
      33
      Microbial transformation of racemic hydantoins to D-amino acids
      Olivieri, R.; Fascetti, E.; Angelini, L.; Degen, L.
      Biotechnology and Bioengineering (1981), 23 (10), 2173-83CODEN: BIBIAU; ISSN:0006-3592.
      Resting cells of Agrobacterium radiobacter catalyze a sequence of 2 stereospecific hydrolytic reactions leading to the complete transformation of racemic hydantoins to D-amino acids. These hydantoinase [9030-74-4] and N-carbamoyl-D-amino acid amidohydrolase [71768-08-6] activities and their potential application for the prodn. of some D-amino acids, which are used as intermediates in the prepn. of semisynthetic penicillins and cephalosporins, are described.
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    34. 34
      Liu, Y.; Zhu, L.; Qi, W.; Yu, B. Biocatalytic production of D-p-hydroxyphenylglycine by optimizing protein expression and cell wall engineering in Escherichia coli. Appl. Microbiol. Biotechnol. 2019, 103, 8839– 8851,  DOI: 10.1007/s00253-019-10155-z
      34
      Biocatalytic production of D-p-hydroxyphenylglycine by optimizing protein expression and cell wall engineering in Escherichia coli
      Liu, Yang; Zhu, Lingfeng; Qi, Wenpeng; Yu, Bo
      Applied Microbiology and Biotechnology (2019), 103 (21-22), 8839-8851CODEN: AMBIDG; ISSN:0175-7598. (Springer)
      D-p-hydroxyphenylglycine (D-HPG) functions as an intermediate and has important value in antibiotic industries. The high pollution and costs from chem. processes make biotechnol. route for D-HPG highly desirable. Here, a whole-cell transformation process by D-hydantoinase(Hase) and D-carbamoylase(Case) was developed to produce D-HPG from DL-hydroxyphenylhydantoin(DL-HPH) in Escherichia coli. The artificially designed ribosome binding site with strong intensity significantly facilitated the protein expression of limiting step enzyme Case. Next, the cell wall permeability was improved by disturbing the peptidoglycan structure by overprodn. of D,D-carboxypeptidases without obviously affecting cell growth, to increase the bioavailability of low sol. hydantoin substrate. By fine-tuning regulation of expression level of D,D-carboxypeptidase DacB, the final prodn. yield of D-HPG increased to 100% with 140 mM DL-HPH substrate under the optimized transformation conditions. This is the first example to enhance bio-productivity of chems. by cell wall engineering and creates a new vision on biotransformation of sparingly sol. substrates. Addnl., the newly demonstrated 'hydroxyl occupancy' phenomenon when Case reacts with hydroxyl substrates provides a referential information for the enzyme engineering in future.
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    35. 35
      Buchholz, K. A breakthrough in enzyme technology to fight penicillin resistance─industrial application of penicillin amidase. Appl. Microbiol. Biotechnol. 2016, 100, 3825– 3839,  DOI: 10.1007/s00253-016-7399-6
      35
      A breakthrough in enzyme technology to fight penicillin resistance-industrial application of penicillin amidase
      Buchholz, Klaus
      Applied Microbiology and Biotechnology (2016), 100 (9), 3825-3839CODEN: AMBIDG; ISSN:0175-7598. (Springer)
      A review. Enzymic penicillin hydrolysis by penicillin amidase (also penicillin acylase, PA) represents a Landmark: the first industrially and economically highly important process using an immobilized biocatalyst. Resistance of infective bacteria to antibiotics had become a major topic of research and industrial activities. Solns. to this problem, the antibiotics resistance of infective microorganisms, required the search for new antibiotics, but also the development of derivs., notably penicillin derivs., that overcame resistance. An obvious route was to hydrolyze penicillin to 6-aminopenicillanic acid (6-APA), as a first step, for the introduction via chem. synthesis of various different side chains. Hydrolysis via chem. reaction sequences was tedious requiring large amts. of toxic chems., and they were cost intensive. Enzymic hydrolysis using penicillin amidase represented a much more elegant route. The basis for such a soln. was the development of techniques for enzyme immobilization, a highly difficult task with respect to industrial application. Two pioneer groups started to develop solns. to this problem in the late 1960s and 1970s: that of Gunter Schmidt-Kastner at Bayer AG (Germany) and that of Malcolm Lilly of Imperial College London. Here, one example of this development, that at Bayer, will be presented in more detail since it illustrates well the achievement of a soln. to the problems of industrial application of enzymic processes, notably development of an immobilization method for penicillin amidase suitable for scale up to application in industrial reactors under economic conditions. A range of bottlenecks and tech. problems of large-scale application had to be overcome. Data giving an inside view of this pioneer achievement in the early phase of the new field of biocatalysis are presented. The development finally resulted in a highly innovative and com. important enzymic process to produce 6-APA that created a new antibiotics industry and that opened the way for the establishment of over 100 industrial processes with immobilized biocatalysts worldwide today.
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    36. 36
      Wicks, C.; Hudlicky, T.; Rinner, U. Morphine alkaloids: History, biology, and synthesis. In The Alkaloids: Chemistry and Biology; Knölker, H.-J., Ed.; Academic Press: 2021; Vol. 86, Ch. 2, pp 145– 342.
      There is no corresponding record for this reference.
    37. 37
      Gulland, J. M.; Robinson, R. Constitution of codeine and thebaine. Mem. Proc. Manchester Lit. Philos. Soc. 1925, 69, 79– 86
      37
      Constitution of codeine and thebaine
      Gulland, J. M.; Robinson, R.
      (1925), 69 (), 79-86 ISSN:.
      Hydroxycodeinone (I) and its dihydro deriv. (II) do not reduce Fehling soln. or NH4OH-Ag2O, even on warming; both bases are recovered largely unchanged from solns. in 30% aq. H2SO4 after boiling 2.5 h. These facts argue against the formulation of I as an α-hydroxy ketone. Bromocodeinone and I yield the same I oxime, m. 279°. I is considered 14-hydroxycodeinone. Structural formulas are suggested for these compds. II condenses with piperonal to form an amorphous yellow powder, C26H25O6N, giving a red soln. in concd. HCl and a purplish red color in H2SO4. The corresponding solns. of the benzylidene deriv. are colorless and red, resp. I gives similar derivs., showing the same color reactions, but analyses indicate the occurrence of redn. as well as condensation. Both I and II condense with o-HOC6H4CHO, giving the orange-red solns. characteristic of the salts of most salicylidene-ketones. I and 6-aminopiperonal condense with EtONa to give the compd. C26H26O6N2, crystg. with 1C6H6 m. 243-4°; it gives Gadamer's test and thus contains the CH2O2 group. II gives a dianhydro-6-aminopiperonaldihydrohydroxycodeinone, C26H24O6N2, m. 282-3° (decompn.); the colorless H2SO4 soln. does not exhibit fluorescence.
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    38. 38
      Armstrong, E. F. Enzymes: A Discovery and its Consequences. Nature 1933, 131, 535– 537,  DOI: 10.1038/131535a0
      There is no corresponding record for this reference.
    39. 39
      Mohan, R. S.; Mejia, M. P. Environmentally Friendly Organic Chemistry Laboratory Experiments for the Undergraduate Curriculum: A Literature Survey and Assessment. J. Chem. Educ. 2020, 97, 943– 959,  DOI: 10.1021/acs.jchemed.9b00753
      39
      Environmentally Friendly Organic Chemistry Laboratory Experiments for the Undergraduate Curriculum: A Literature Survey and Assessment
      Mohan, Ram S.; Mejia, Maria P.
      Journal of Chemical Education (2020), 97 (4), 943-959CODEN: JCEDA8; ISSN:0021-9584. (American Chemical Society and Division of Chemical Education, Inc.)
      A review. Due to a growing awareness of environmental issues, green chem. concepts are increasingly being incorporated into the undergraduate org. chem. lecture and lab. component. This minireview summarizes environmentally friendly org. chem. expts. suitable for undergraduate labs. Whenever feasible, LD50 values for various chems. are provided to allow readers to det. the suitability of an expt. for their curriculum based on the toxicity of reagents used.
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    40. 40
      Heather, J. M.; Chain, B. The sequence of sequencers: The history of sequencing DNA. Genomics 2016, 107, 1– 8,  DOI: 10.1016/j.ygeno.2015.11.003
      40
      The sequence of sequencers: The history of sequencing DNA
      Heather, James M.; Chain, Benjamin
      Genomics (2016), 107 (1), 1-8CODEN: GNMCEP; ISSN:0888-7543. (Elsevier Inc.)
      A review. Detg. the order of nucleic acid residues in biol. samples is an integral component of a wide variety of research applications. Over the last fifty years large nos. of researchers have applied themselves to the prodn. of techniques and technologies to facilitate this feat, sequencing DNA and RNA mols. This time-scale has witnessed tremendous changes, moving from sequencing short oligonucleotides to millions of bases, from struggling towards the deduction of the coding sequence of a single gene to rapid and widely available whole genome sequencing. This article traverses those years, iterating through the different generations of sequencing technol., highlighting some of the key discoveries, researchers, and sequences along the way.
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    41. 41
      Baxevanis, A. D. Using Genomic Databases for Sequence-Based Biological Discovery. Mol. Med. 2003, 9, 185– 192,  DOI: 10.1007/BF03402130
      41
      Using genomic databases for sequence-based biological discovery
      Baxevanis, Andreas D.
      Molecular Medicine (Manhasset, NY, United States) (2003), 9 (9-12), 185-192CODEN: MOMEF3; ISSN:1076-1551. (North Shore-Long Island Jewish Research Institute)
      A review. The inherent potential underlying the sequence data produced by the International Human Genome Sequencing Consortium and other systematic sequencing projects is, obviously, tremendous. As such, it becomes increasingly important that all biologists have the ability to navigate through and cull important information from key publicly available databases. The continued rapid rise in available sequence information, particularly as model organism data is generated at breakneck speed, also underscores the necessity for all biologists to learn how to effectively make their way through the expanding "sequence information space.". This review discusses some of the more commonly used tools for sequence discovery; tools have been developed for the effective and efficient mining of sequence information. These include LocusLink, which provides a gene-centric view of sequence-based information, as well as the 3 major genome browsers: the National Center for Biotechnol. Information Map Viewer, the University of California Santa Cruz Genome Browser, and the European Bioinformatics Institute's Ensembl system. An overview of the types of information available through each of these front-ends is given, as well as information on tutorials and other documentation intended to increase the reader's familiarity with these tools.
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    42. 42
      GenBank and WGS Statistics. https://www.ncbi.nlm.nih.gov/genbank/statistics/ (accessed 2023-02–01).
      There is no corresponding record for this reference.
    43. 43
      The UniProt Consortium UniProt: the universal protein knowledgebase. Nucleic Acids Res. 2017, 45, D158– D169,  DOI: 10.1093/nar/gkw1099
      There is no corresponding record for this reference.
    44. 44
      LeProust, E. M.; Peck, B. J.; Spirin, K.; McCuen, H. B.; Moore, B.; Namsaraev, E.; Caruthers, M. H. Synthesis of high-quality libraries of long (150mer) oligonucleotides by a novel depurination controlled process. Nucleic Acids Res. 2010, 38, 2522– 2540,  DOI: 10.1093/nar/gkq163
      44
      Synthesis of high-quality libraries of long (150mer) oligonucleotides by a novel depurination controlled process
      LeProust Emily M; Peck Bill J; Spirin Konstantin; McCuen Heather Brummel; Moore Bridget; Namsaraev Eugeni; Caruthers Marvin H
      Nucleic acids research (2010), 38 (8), 2522-40 ISSN:.
      We have achieved the ability to synthesize thousands of unique, long oligonucleotides (150mers) in fmol amounts using parallel synthesis of DNA on microarrays. The sequence accuracy of the oligonucleotides in such large-scale syntheses has been limited by the yields and side reactions of the DNA synthesis process used. While there has been significant demand for libraries of long oligos (150mer and more), the yields in conventional DNA synthesis and the associated side reactions have previously limited the availability of oligonucleotide pools to lengths <100 nt. Using novel array based depurination assays, we show that the depurination side reaction is the limiting factor for the synthesis of libraries of long oligonucleotides on Agilent Technologies' SurePrint DNA microarray platform. We also demonstrate how depurination can be controlled and reduced by a novel detritylation process to enable the synthesis of high quality, long (150mer) oligonucleotide libraries and we report the characterization of synthesis efficiency for such libraries. Oligonucleotide libraries prepared with this method have changed the economics and availability of several existing applications (e.g. targeted resequencing, preparation of shRNA libraries, site-directed mutagenesis), and have the potential to enable even more novel applications (e.g. high-complexity synthetic biology).
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    45. 45
      Gibson, D. G.; Young, L.; Chuang, R.-Y.; Venter, J. C.; Hutchison, C. A.; Smith, H. O. Enzymatic assembly of DNA molecules up to several hundred kilobases. Nat. Methods 2009, 6, 343– 345,  DOI: 10.1038/nmeth.1318
      45
      Enzymatic assembly of DNA molecules up to several hundred kilobases
      Gibson, Daniel G.; Young, Lei; Chuang, Ray-Yuan; Venter, J. Craig; Hutchison, Clyde A.; Smith, Hamilton O.
      Nature Methods (2009), 6 (5), 343-345CODEN: NMAEA3; ISSN:1548-7091. (Nature Publishing Group)
      The authors describe an isothermal, single-reaction method for assembling multiple overlapping DNA mols. by the concerted action of a 5' exonuclease, a DNA polymerase and a DNA ligase. First they recessed DNA fragments, yielding single-stranded DNA overhangs that specifically annealed, and then covalently joined them. This assembly method can be used to seamlessly construct synthetic and natural genes, genetic pathways and entire genomes, and could be a useful mol. engineering tool.
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    46. 46
      Loftie-Eaton, W.; Heinisch, T.; Soskine, M.; Champion, E.; Godron, X.; Ybert, T. Novel Variants of Endonuclease V and Uses Thereof. WO2022/090057, 2022.
      There is no corresponding record for this reference.
    47. 47
      Moustafa, K.; Makhzoum, A.; Trémouillaux-Guiller, J. Molecular farming on rescue of pharma industry for next generations. Crit. Rev. Biotechnol. 2016, 36, 840– 850,  DOI: 10.3109/07388551.2015.1049934
      47
      Molecular farming on rescue of pharma industry for next generations
      Moustafa, Khaled; Makhzoum, Abdullah; Tremouillaux-Guiller, Jocelyne
      Critical Reviews in Biotechnology (2016), 36 (5), 840-850CODEN: CRBTE5; ISSN:0738-8551. (Taylor & Francis Ltd.)
      A review. Recombinant proteins expressed in plants have been emerged as a novel branch of the biopharmaceutical industry, offering practical and safety advantages over traditional approaches. Cultivable in various platforms (i.e. open field, greenhouses or bioreactors), plants hold great potential to produce different types of therapeutic proteins with reduced risks of contamination with human and animal pathogens. To maximize the yield and quality of plant-made pharmaceuticals, crucial factors should be taken into account, including host plants, expression cassettes, subcellular localization, post-translational modifications, and protein extn. and purifn. methods. DNA technol. and genetic transformation methods have also contributed to great parts with substantial improvements. To play their proper function and stability, proteins require multiple post-translational modifications such as glycosylation. Intensive glycoengineering research has been performed to reduce the immunogenicity of recombinant proteins produced in plants. Important strategies have also been developed to minimize the proteolysis effects and enhance protein accumulation. With growing human population and new epidemic threats, the need for new medications will be paramount so that the traditional pharmaceutical industry will not be alone to answer medication demands for upcoming generations. Here, we review several aspects of plant mol. pharming and outline some important challenges that hamper these ambitious biotechnol. developments.
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    48. 48
      Swartz, J. R. Advances in Escherichia coli production of therapeutic proteins. Curr. Opin. Biotechnol. 2001, 12, 195– 201,  DOI: 10.1016/S0958-1669(00)00199-3
      48
      Advances in Escherichia coli production of therapeutic proteins
      Swartz, James R.
      Current Opinion in Biotechnology (2001), 12 (2), 195-201CODEN: CUOBE3; ISSN:0958-1669. (Elsevier Science Ltd.)
      A review with 59 refs. Escherichia coli offers a means for the rapid and economical prodn. of recombinant proteins. These advantages, coupled with a wealth of biochem. and genetic knowledge, have enabled the prodn. of such economically sensitive products as insulin and bovine growth hormone. Although significant progress has been made in transcription, translation and secretion, one of the major challenges is obtaining the product in a sol. and bioactive form. Recent progress in oxidative cytoplasmic folding and cell-free protein synthesis offers attractive alternatives to std. expression methods.
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    49. 49
      Karbalaei, M.; Rezaee, S. A.; Farsiani, H. Pichia pastoris: A highly successful expression system for optimal synthesis of heterologous proteins. J. Cell. Physiol. 2020, 235, 5867– 5881,  DOI: 10.1002/jcp.29583
      49
      Pichia pastoris: A highly successful expression system for optimal synthesis of heterologous proteins
      Karbalaei, Mohsen; Rezaee, Seyed A.; Farsiani, Hadi
      Journal of Cellular Physiology (2020), 235 (9), 5867-5881CODEN: JCLLAX; ISSN:0021-9541. (Wiley-Blackwell)
      One of the most important branches of genetic engineering is the expression of recombinant proteins using biol. expression systems. Nowadays, different expression systems are used for the prodn. of recombinant proteins including bacteria, yeasts, molds, mammals, plants, and insects. Yeast expression systems such as Saccharomyces cerevisiae (S. cerevisiae) and Pichia pastoris (P. pastoris) are more popular. P. pastoris expression system is one of the most popular and std. tools for the prodn. of recombinant protein in mol. biol. Overall, the benefits of protein prodn. by P. pastoris system include appropriate folding (in the endoplasmic reticulum) and secretion (by Kex2 as signal peptidase) of recombinant proteins to the external environment of the cell. Moreover, in the P. pastoris expression system due to its limited prodn. of endogenous secretory proteins, the purifn. of recombinant protein is easy. It is also considered a unique host for the expression of subunit vaccines which could significantly affect the growing market of medical biotechnol. Although P. pastoris expression systems are impressive and easy to use with well-defined process protocols, some degree of process optimization is required to achieve max. prodn. of the target proteins. Methanol and sorbitol concn., Mut forms, temp. and incubation time have to be adjusted to obtain optimal conditions, which might vary among different strains and externally expressed protein. Eventually, optimal conditions for the prodn. of a recombinant protein in P. pastoris expression system differ according to the target protein.
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    50. 50
      Hunter, M.; Yuan, P.; Vavilala, D.; Fox, M. Optimization of Protein Expression in Mammalian Cells. Curr. Protoc. Protein Sci. 2019, 95, e77  DOI: 10.1002/cpps.77
      There is no corresponding record for this reference.
    51. 51
      Fox, B. G.; Blommel, P. G. Autoinduction of Protein Expression. Curr. Protoc. Protein Sci. 2009, 56, 5.23.1– 5.23.18,  DOI: 10.1002/0471140864.ps0523s56
      There is no corresponding record for this reference.
    52. 52
      Silverman, 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/s41576-019-0186-3
      52
      Cell-free gene expression: an expanded repertoire of applications
      Silverman, 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.
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    53. 53
      de Carvalho, C. C. C. R. Whole cell biocatalysts: essential workers from Nature to the industry. Microb. Biotechnol. 2017, 10, 250– 263,  DOI: 10.1111/1751-7915.12363
      53
      Whole cell biocatalysts: essential workers from Nature to the industry
      de Carvalho Carla C C R
      Microbial biotechnology (2017), 10 (2), 250-263 ISSN:.
      Microorganisms have been exposed to a myriad of substrates and environmental conditions throughout evolution resulting in countless metabolites and enzymatic activities. Although mankind have been using these properties for centuries, we have only recently learned to control their production, to develop new biocatalysts with high stability and productivity and to improve their yields under new operational conditions. However, microbial cells still provide the best known environment for enzymes, preventing conformational changes in the protein structure in non-conventional medium and under harsh reaction conditions, while being able to efficiently regenerate necessary cofactors and to carry out cascades of reactions. Besides, a still unknown microbe is probably already producing a compound that will cure cancer, Alzeihmer's disease or kill the most resistant pathogen. In this review, the latest developments in screening desirable activities and improving production yields are discussed.
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    54. 54
      Alissandratos, A. In vitro multi-enzymatic cascades using recombinant lysates of E. coli: an emerging biocatalysis platform. Biophys. Rev. 2020, 12, 175– 182,  DOI: 10.1007/s12551-020-00618-3
      54
      In vitro multi-enzymatic cascades using recombinant lysates of E. coli: an emerging biocatalysis platform
      Alissandratos, Apostolos
      Biophysical Reviews (2020), 12 (1), 175-182CODEN: BRIECG; ISSN:1867-2450. (Springer)
      A review. Abstr.: In recent years, cell-free exts. (or lysates) have (re-)emerged as a third route to the traditional options of isolated or whole-cell biocatalysts. Advances in mol. biol. and genetic engineering enable facile prodn. of recombinant cell-free exts., where endogenous enzymes are enriched with heterologous activities. These inexpensive prepns. may be used to catalyze multistep enzymic reactions without the constraints of cell toxicity and the cell membrane or the cost and complexity assocd. with prodn. of isolated biocatalysts. Herein, we present an overview of the key advancements in cell-free synthetic biol. that have led to the emergence of cell-free exts. as a promising biocatalysis platform.
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    55. 55
      Gräslund, S.; Nordlund, P.; Weigelt, J.; Hallberg, B. M.; Bray, J.; Gileadi, O.; Knapp, S.; Oppermann, U.; Arrowsmith, C.; Hui, R.; Ming, J.; dhe-Paganon, S.; Park, H.-w.; Savchenko, A.; Yee, A.; Edwards, A.; Vincentelli, R.; Cambillau, C.; Kim, R.; Kim, S.-H.; Rao, Z.; Shi, Y.; Terwilliger, T. C.; Kim, C.-Y.; Hung, L.-W.; Waldo, G. S.; Peleg, Y.; Albeck, S.; Unger, T.; Dym, O.; Prilusky, J.; Sussman, J. L.; Stevens, R. C.; Lesley, S. A.; Wilson, I. A.; Joachimiak, A.; Collart, F.; Dementieva, I.; Donnelly, M. I.; Eschenfeldt, W. H.; Kim, Y.; Stols, L.; Wu, R.; Zhou, M.; Burley, S. K.; Emtage, J. S.; Sauder, J. M.; Thompson, D.; Bain, K.; Luz, J.; Gheyi, T.; Zhang, F.; Atwell, S.; Almo, S. C.; Bonanno, J. B.; Fiser, A.; Swaminathan, S.; Studier, F. W.; Chance, M. R.; Sali, A.; Acton, T. B.; Xiao, R.; Zhao, L.; Ma, L. C.; Hunt, J. F.; Tong, L.; Cunningham, K.; Inouye, M.; Anderson, S.; Janjua, H.; Shastry, R.; Ho, C. K.; Wang, D.; Wang, H.; Jiang, M.; Montelione, G. T.; Stuart, D. I.; Owens, R. J.; Daenke, S.; Schütz, A.; Heinemann, U.; Yokoyama, S.; Büssow, K.; Gunsalus, K. C.; Structural Genomics, C.; Architecture et Fonction des Macromolécules, B.; Berkeley Structural Genomics, C.; China Structural Genomics, C.; Integrated Center for, S.; Function, I.; Israel Structural Proteomics, C.; Joint Center for Structural, G.; Midwest Center for Structural, G.; New York Structural Genomi, X. R. C. f. S. G.; Northeast Structural Genomics, C.; Oxford Protein Production, F.; Protein Sample Production Facility, M. D. C. f. M. M.; Initiative, R. S. G. P.; Complexes, S. Protein production and purification. Nat. Methods 2008, 5, 135– 146,  DOI: 10.1038/nmeth.f.202
      55
      Protein production and purification
      Graslund Susanne; Nordlund Par; Weigelt Johan; Hallberg B Martin; Bray James; Gileadi Opher; Knapp Stefan; Oppermann Udo; Arrowsmith Cheryl; Hui Raymond; Ming Jinrong; dhe-Paganon Sirano; Park Hee-won; Savchenko Alexei; Yee Adelinda; Edwards Aled; Vincentelli Renaud; Cambillau Christian; Kim Rosalind; Kim Sung-Hou; Rao Zihe; Shi Yunyu; Terwilliger Thomas C; Kim Chang-Yub; Hung Li-Wei; Waldo Geoffrey S; Peleg Yoav; Albeck Shira; Unger Tamar; Dym Orly; Prilusky Jaime; Sussman Joel L; Stevens Ray C; Lesley Scott A; Wilson Ian A; Joachimiak Andrzej; Collart Frank; Dementieva Irina; Donnelly Mark I; Eschenfeldt William H; Kim Youngchang; Stols Lucy; Wu Ruying; Zhou Min; Burley Stephen K; Emtage J Spencer; Sauder J Michael; Thompson Devon; Bain Kevin; Luz John; Gheyi Tarun; Zhang Fred; Atwell Shane; Almo Steven C; Bonanno Jeffrey B; Fiser Andras; Swaminathan Sivasubramanian; Studier F William; Chance Mark R; Sali Andrej; Acton Thomas B; Xiao Rong; Zhao Li; Ma Li Chung; Hunt John F; Tong Liang; Cunningham Kellie; Inouye Masayori; Anderson Stephen; Janjua Heleema; Shastry Ritu; Ho Chi Kent; Wang Dongyan; Wang Huang; Jiang Mei; Montelione Gaetano T; Stuart David I; Owens Raymond J; Daenke Susan; Schutz Anja; Heinemann Udo; Yokoyama Shigeyuki; Bussow Konrad; Gunsalus Kristin C
      Nature methods (2008), 5 (2), 135-46 ISSN:.
      In selecting a method to produce a recombinant protein, a researcher is faced with a bewildering array of choices as to where to start. To facilitate decision-making, we describe a consensus 'what to try first' strategy based on our collective analysis of the expression and purification of over 10,000 different proteins. This review presents methods that could be applied at the outset of any project, a prioritized list of alternate strategies and a list of pitfalls that trip many new investigators.
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    56. 56
      Hughes, R. A.; Ellington, A. D. Synthetic DNA Synthesis and Assembly: Putting the Synthetic in Synthetic Biology. Cold Spring Harbor Perspect. Biol. 2017, 9, a023812,  DOI: 10.1101/cshperspect.a023812
      56
      Synthetic DNA synthesis and assembly: putting the synthetic in synthetic biology
      Hughes, Randall A.; Ellington, Andrew D.
      Cold Spring Harbor Perspectives in Biology (2017), 9 (1), a023812/1-a023812/18CODEN: CSHPEU; ISSN:1943-0264. (Cold Spring Harbor Laboratory Press)
      The chem. synthesis of DNA oligonucleotides and their assembly into synthons, genes, circuits, and even entire genomes by gene synthesis methods has become an enabling technol. for modern mol. biol. and enables the design, build, test, learn, and repeat cycle underpinning innovations in synthetic biol. In this perspective, we briefly review the techniques and technologies that enable the synthesis of DNA oligonucleotides and their assembly into larger DNA constructs with a focus on recent advancements that have sought to reduce synthesis cost and increase sequence fidelity. The development of lower-cost methods to produce high-quality synthetic DNA will allow for the exploration of larger biol. hypotheses by lowering the cost of use and help to close the DNA read -write cost gap.
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    57. 57
      Baker Dockrey, S. A.; Doyon, T. J.; Perkins, J. C.; Narayan, A. R. H. Whole-cell biocatalysis platform for gram-scale oxidative dearomatization of phenols. Chem. Biol. Drug Des. 2019, 93, 1207– 1213,  DOI: 10.1111/cbdd.13443
      57
      Whole-cell biocatalysis platform for gram-scale oxidative dearomatization of phenols
      Baker Dockrey, Summer A.; Doyon, Tyler J.; Perkins, Jonathan C.; Narayan, Alison R. H.
      Chemical Biology & Drug Design (2019), 93 (6), 1207-1213CODEN: CBDDAL; ISSN:1747-0277. (Wiley-Blackwell)
      Technologies enabling new enzyme discovery and efficient protein engineering have spurred intense interest in the development of biocatalytic reactions. In recent years, whole-cell biocatalysis has received attention as a simple, efficient, and scalable biocatalytic reaction platform. Inspired by these developments, we have established a whole-cell protocol for oxidative dearomatization of phenols using the flavin-dependent monooxygenase, TropB. This approach provides a scalable biocatalytic platform for accessing gram-scale quantities of chiral synthetic building blocks.
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    58. 58
      Bai, Y.; Yang, X.; Yu, H.; Chen, X. Substrate and Process Engineering for Biocatalytic Synthesis and Facile Purification of Human Milk Oligosaccharides. ChemSusChem 2022, 15, e202102539  DOI: 10.1002/cssc.202102539
      58
      Substrate and Process Engineering for Biocatalytic Synthesis and Facile Purification of Human Milk Oligosaccharides
      Bai, Yuanyuan; Yang, Xiaohong; Yu, Hai; Chen, Xi
      ChemSusChem (2022), 15 (9), e202102539CODEN: CHEMIZ; ISSN:1864-5631. (Wiley-VCH Verlag GmbH & Co. KGaA)
      Innovation in process development is essential for applying biocatalysis in industrial and lab. prodn. of org. compds., including beneficial carbohydrates such as human milk oligosaccharides (HMOs). HMOs have attracted increasing attention for their potential application as key ingredients in products that can improve human health. To efficiently access HMOs through biocatalysis, a combined substrate and process engineering strategy is developed, namely multistep one-pot multienzyme (MSOPME) design. The strategy allows access to a pure tagged HMO in a single reactor with a single C18-cartridge purifn. process, despite the length of the target. Its efficiency is demonstrated in the high-yielding (71-91%) one-pot synthesis of twenty tagged HMOs (83-155 mg), including long-chain oligosaccharides with or without fucosylation or sialylation up to nonaoses from a lactoside without the isolation of the intermediate oligosaccharides. Gram-scale synthesis of an important HMO deriv. - tagged lacto-N-fucopentaose-I (LNFP-I) - proceeds in 84% yield. Tag removal is carried out in high efficiency (94-97%) without the need for column purifn. to produce the desired natural HMOs with a free reducing end. The method can be readily adapted for large-scale synthesis and automation to allow quick access to HMOs, other glycans, and glycoconjugates.
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    59. 59
      Börner, T.; Grey, C.; Adlercreutz, P. Generic HPLC platform for automated enzyme reaction monitoring: Advancing the assay toolbox for transaminases and other PLP-dependent enzymes. Biotechnol. J. 2016, 11, 1025– 1036,  DOI: 10.1002/biot.201500587
      59
      Generic HPLC platform for automated enzyme reaction monitoring: Advancing the assay toolbox for transaminases and other PLP-dependent enzymes
      Borner Tim; Grey Carl; Adlercreutz Patrick
      Biotechnology journal (2016), 11 (8), 1025-36 ISSN:.
      Methods for rapid and direct quantification of enzyme kinetics independent of the substrate stand in high demand for both fundamental research and bioprocess development. This study addresses the need for a generic method by developing an automated, standardizable HPLC platform monitoring reaction progress in near real-time. The method was applied to amine transaminase (ATA) catalyzed reactions intensifying process development for chiral amine synthesis. Autosampler-assisted pipetting facilitates integrated mixing and sampling under controlled temperature. Crude enzyme formulations in high and low substrate concentrations can be employed. Sequential, small (1 μL) sample injections and immediate detection after separation permits fast reaction monitoring with excellent sensitivity, accuracy and reproducibility. Due to its modular design, different chromatographic techniques, e.g. reverse phase and size exclusion chromatography (SEC) can be employed. A novel assay for pyridoxal 5'-phosphate-dependent enzymes is presented using SEC for direct monitoring of enzyme-bound and free reaction intermediates. Time-resolved changes of the different cofactor states, e.g. pyridoxal 5'-phosphate, pyridoxamine 5'-phosphate and the internal aldimine were traced in both half reactions. The combination of the automated HPLC platform with SEC offers a method for substrate-independent screening, which renders a missing piece in the assay and screening toolbox for ATAs and other PLP-dependent enzymes.
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    60. 60
      Claaßen, C.; Mack, K.; Rother, D. Benchtop NMR for Online Reaction Monitoring of the Biocatalytic Synthesis of Aromatic Amino Alcohols. ChemCatChem. 2020, 12, 1190– 1199,  DOI: 10.1002/cctc.201901910
      60
      Benchtop NMR for Online Reaction Monitoring of the Biocatalytic Synthesis of Aromatic Amino Alcohols
      Claassen, C.; Mack, K.; Rother, D.
      ChemCatChem (2020), 12 (4), 1190-1199CODEN: CHEMK3; ISSN:1867-3880. (Wiley-VCH Verlag GmbH & Co. KGaA)
      Online analytics provides insights into the progress of an ongoing reaction without the need for extensive sampling and offline anal. In this study, we investigated benchtop NMR as an online reaction monitoring tool for complex enzyme cascade reactions. Online NMR was used to monitor a two-step cascade beginning with an arom. aldehyde and leading to an arom. amino alc. as the final product, applying two different enzymes and a variety of co-substrates and intermediates. Benchtop NMR enabled the concn. of the reaction components to be detected in buffered systems in the single-digit mM range without using deuterated solvent. The concns. detd. via NMR were correlated with offline samples analyzed via uHPLC and displayed a good correlation between the two methods. In summary, benchtop NMR proved to be a sensitive, selective and reliable method for online reaction monitoring in (multi-step) biosynthesis. In future, online analytic systems such as the benchtop NMR devices described might not only enable direct monitoring of the reaction, but may also form the basis for self-regulation in biocatalytic reactions.
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    61. 61
      Bommarius, A. S. Biocatalysis: A Status Report. Annu. Rev. Chem. Biomol. Eng. 2015, 6, 319– 345,  DOI: 10.1146/annurev-chembioeng-061114-123415
      61
      Biocatalysis: A Status Report
      Bommarius, Andreas S.
      Annual Review of Chemical and Biomolecular Engineering (2015), 6 (), 319-345CODEN: ARCBCY; ISSN:1947-5438. (Annual Reviews)
      This review describes the status of the fields of biocatalysts and enzymes, as well as existing drawbacks, and recent advances in the areas deemed to represent drawbacks. Although biocatalysts are often highly active and extremely selective, there are still drawbacks assocd. with biocatalysis as a generally applicable technique: the lack of designability of biocatalysts; their limits of stability; and the insufficient no. of well-characterized, ready-to-use biocatalysts. There has been significant progress on the following fronts: (a) novel protein engineering tools, both exptl. and computational, have significantly enhanced the toolbox for biocatalyst development. (b) The deactivation of biocatalysts under various stresses can be described quant. via rational models. There are several cases of spectacular leaps of stabilization after accumulating all stabilizing mutations found in earlier rounds. The concept that stabilization against one type of stress commonly also stabilizes against other types of stress is now exptl. considerably better founded than a few years ago. (c) A host of developments of novel biocatalysts in the past few years, in part fueled by improved designability and improved methods of stabilization, has considerably broadened the toolbox for synthetic chem.
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    62. 62
      Reetz, M. T. What are the Limitations of Enzymes in Synthetic Organic Chemistry?. Chem. Rec. 2016, 16, 2449– 2459,  DOI: 10.1002/tcr.201600040
      62
      What are the Limitations of Enzymes in Synthetic Organic Chemistry?
      Reetz, Manfred T.
      Chemical Record (2016), 16 (6), 2449-2459CODEN: CRHEAK; ISSN:1528-0691. (Wiley-VCH Verlag GmbH & Co. KGaA)
      Enzymes have been used in org. chem. and biotechnol. for 100 years, but their widespread application has been prevented by a no. of limitations, including the often-obsd. limited thermostability, narrow substrate scope, and low or wrong stereo- and/or regioselectivity. Directed evolution provides a means to address and generally solve these problems, esp. since recent methodol. development has made this protein engineering method faster, more efficient, and more reliable than in the past. This Darwinian approach to asym. catalysis has led to a no. of industrial applications. Metabolic-pathway engineering, mutasynthesis, and fermn. are likewise enzyme-based techniques that enrich chem. This account outlines the scope, and particularly, the limitations, of biocatalysis. The complementary nature of enzymes and man-made catalysts is emphasized.
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    63. 63
      Stepankova, V.; Bidmanova, S.; Koudelakova, T.; Prokop, Z.; Chaloupkova, R.; Damborsky, J. Strategies for Stabilization of Enzymes in Organic Solvents. ACS Catal. 2013, 3, 2823– 2836,  DOI: 10.1021/cs400684x
      63
      Strategies for Stabilization of Enzymes in Organic Solvents
      Stepankova, Veronika; Bidmanova, Sarka; Koudelakova, Tana; Prokop, Zbynek; Chaloupkova, Radka; Damborsky, Jiri
      ACS Catalysis (2013), 3 (12), 2823-2836CODEN: ACCACS; ISSN:2155-5435. (American Chemical Society)
      A review. One of the major barriers to the use of enzymes in industrial biotechnol. is their insufficient stability under processing conditions. The use of org. solvent systems instead of aq. media for enzymic reactions offers numerous advantages, such as increased soly. of hydrophobic substrates or suppression of water-dependent side reactions. For example, reverse hydrolysis reactions that form esters from acids and alcs. become thermodynamically favorable. However, org. solvents often inactivate enzymes. Industry and academia have devoted considerable effort into developing effective strategies to enhance the lifetime of enzymes in the presence of org. solvents. The strategies can be grouped into three main categories: (i) isolation of novel enzymes functioning under extreme conditions, (ii) modification of enzyme structures to increase their resistance toward nonconventional media, and (iii) modification of the solvent environment to decrease its denaturing effect on enzymes. Here we discuss successful examples representing each of these categories and summarize their advantages and disadvantages. Finally, we highlight some potential future research directions in the field, such as investigation of novel nanomaterials for immobilization, wider application of computational tools for semirational prediction of stabilizing mutations, knowledge-driven modification of key structural elements learned from successfully engineered proteins, and replacement of volatile org. solvents by ionic liqs. and deep eutectic solvents.
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    64. 64
      Guzik, U.; Hupert-Kocurek, K.; Wojcieszyńska, D. Immobilization as a Strategy for Improving Enzyme Properties-Application to Oxidoreductases. Molecules 2014, 19, 8995– 9018,  DOI: 10.3390/molecules19078995
      64
      Immobilization as a strategy for improving enzyme properties-Application to oxidoreductase
      Guzik, Urszula; Hupert-Kocurek, Katarzyna; Wojcieszynska, Danuta
      Molecules (2014), 19 (7), 8995-9018, 24 pp.CODEN: MOLEFW; ISSN:1420-3049. (MDPI AG)
      A review. The main objective of the immobilization of enzymes is to enhance the economics of biocatalytic processes. Immobilization allows one to re-use the enzyme for an extended period of time and enables easier sepn. of the catalyst from the product. Addnl., immobilization improves many properties of enzymes such as performance in org. solvents, pH tolerance, heat stability or the functional stability. It can also increase the structural rigidity of the protein and stabilize multimeric enzymes which prevents dissocn.-related inactivation. In the last decade, several papers about immobilization methods have been published. In our work, we present a relation between the influence of immobilization on the improvement of the properties of selected oxidoreductases and their com. value. We also present our view on the role that different immobilization methods play in the redn. of enzyme inhibition during biotechnol. processes.
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    65. 65
      De Santis, P.; Meyer, L.-E.; Kara, S. The rise of continuous flow biocatalysis – fundamentals, very recent developments and future perspectives. React. Chem. Eng. 2020, 5, 2155– 2184,  DOI: 10.1039/D0RE00335B
      65
      The rise of continuous flow biocatalysis - fundamentals, very recent developments and future perspectives
      De Santis, Piera; Meyer, Lars-Erik; Kara, Selin
      Reaction Chemistry & Engineering (2020), 5 (12), 2155-2184CODEN: RCEEBW; ISSN:2058-9883. (Royal Society of Chemistry)
      A review. Biocatalysis community has witnessed a drastic increase in the no. of studies for the use of enzymes in continuously operated flow reactors. This significant interest arose from the possibility of combining the strengths of the two worlds: enhanced mass transfer and resource efficient synthesis achieved in flow chem. at micro-scales and excellent selectivities obtained in biocatalysis. Within this review, we present very recent (from 2018 to Sept. 2020) developments in the field of biocatalysis in continuously operated systems. Briefly, we describe the fundamentals of continuously operated reactors with a special focus on enzyme-catalyzed reactions. We devoted special attention on future perspectives in this key emerging technol. area ranging from process anal. technologies to digitalization.
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      https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB3cXitVyhsr3N&md5=e14444d8aa3bfafe4690096b74affe73
    66. 66
      France, S. P.; Lewis, R. D.; Martinez, C. A. The Evolving Nature of Biocatalysis in Pharmaceutical Research and Development. JACS Au 2023, 3, 715– 735,  DOI: 10.1021/jacsau.2c00712
      66
      The Evolving Nature of Biocatalysis in Pharmaceutical Research and Development
      France, Scott P.; Lewis, Russell D.; Martinez, Carlos A.
      JACS Au (2023), 3 (3), 715-735CODEN: JAAUCR; ISSN:2691-3704. (American Chemical Society)
      Biocatalysis is a highly valued enabling technol. for pharmaceutical research and development as it can unlock synthetic routes to complex chiral motifs with unparalleled selectivity and efficiency. This perspective aims to review recent advances in the pharmaceutical implementation of biocatalysis across early and late-stage development with a focus on the implementation of processes for preparative-scale syntheses.
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      https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB3sXjvVajsbo%253D&md5=71ea4afe11c8a0b80ddcbad75af67a05
    67. 67
      Zhang, Y.; Xia, B.; Li, Y.; Lin, X.; Wu, Q. Substrate Engineering in Lipase-Catalyzed Selective Polymerization of d-/l-Aspartates and Diols to Prepare Helical Chiral Polyester. Biomacromolecules 2021, 22, 918– 926,  DOI: 10.1021/acs.biomac.0c01605
      67
      Substrate Engineering in Lipase-Catalyzed Selective Polymerization of D-/L-Aspartates and Diols to Prepare Helical Chiral Polyester
      Zhang, Yu; Xia, Bo; Li, Yanyan; Lin, Xianfu; Wu, Qi
      Biomacromolecules (2021), 22 (2), 918-926CODEN: BOMAF6; ISSN:1525-7797. (American Chemical Society)
      The synthesis of optically pure polymers is one of the most challenging tasks in polymer chem. Herein, Novozym 435 (Lipase B from Candida antarctica, immobilized on Lewatit VP OC 1600)-catalyzed polycondensation between D-/L-aspartic acid (Asp) diester and diols for the prepn. of helical chiral polyesters was reported. Compared with D-Asp diesters, the fast-reacting L-Asp diesters easily reacted with diols to provide a series of chiral polyesters contg. N-substitutional L-Asp repeating units. Besides amino acid configuration, N-substituent side chains and the chain length of diols were also investigated and optimized. It was found that bulky acyl N-substitutional groups like N-Boc and N-Cbz were more favorable for this polymn. than small ones probably due to competitively binding of these small acyl groups into the active site of Novozym 435. The highest mol. wt. can reach up to 39.5 x 103 g/mol (Mw,D = 1.64). Moreover, the slow-reacting D-Asp diesters were also successfully polymd. by modifying the substrate structure to create a "nonchiral" condensation environment artificially. These enantiocomplementary chiral polyesters are thermally stable and have specific helical structures, which was confirmed by CD (CD) spectra, scanning electron microscope (SEM), and mol. calcn.
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    68. 68
      Turner, N. J. Directed evolution drives the next generation of biocatalysts. Nat. Chem. Biol. 2009, 5, 567– 573,  DOI: 10.1038/nchembio.203
      68
      Directed evolution drives the next generation of biocatalysts
      Turner, Nicholas J.
      Nature Chemical Biology (2009), 5 (8), 567-573CODEN: NCBABT; ISSN:1552-4450. (Nature Publishing Group)
      A review. Enzymes are increasingly being used as biocatalysts in the generation of products that have until now been derived using traditional chem. processes. Such products range from pharmaceutical and agrochem. building blocks to fine and bulk chems. and, more recently, components of biofuels. For a biocatalyst to be effective in an industrial process, it must be subjected to improvement and optimization, and in this respect the directed evolution of enzymes has emerged as a powerful enabling technol. Directed evolution involves repeated rounds of (1) random gene library generation, (2) expression of genes in a suitable host, and (3) screening of libraries of variant enzymes for the property of interest. Both in vitro screening-based methods and in vivo selection-based methods have been applied to the evolution of enzyme function and properties. Significant developments have occurred recently, particularly with respect to library design, screening methodol., applications in synthetic transformations, and strategies for the generation of new enzyme function.
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    69. 69
      Cobb, R. E.; Chao, R.; Zhao, H. Directed evolution: Past, present, and future. AIChE J. 2013, 59, 1432– 1440,  DOI: 10.1002/aic.13995
      69
      Directed evolution: Past, present, and future
      Cobb, Ryan E.; Chao, Ran; Zhao, Huimin
      AIChE Journal (2013), 59 (5), 1432-1440CODEN: AICEAC; ISSN:0001-1541. (John Wiley & Sons, Inc.)
      Directed evolution, the lab. process by which biol. entities with desired traits are created through iterative rounds of genetic diversification and library screening or selection, has become one of the most useful and widespread tools in basic and applied biol. From its roots in classical strain engineering and adaptive evolution, modern directed evolution came of age 20 years ago with the demonstration of repeated rounds of polymerase chain reaction (PCR)-driven random mutagenesis and activity screening to improve protein properties. Since then, numerous techniques have been developed that have enabled the evolution of virtually any protein, pathway, network, or entire organism of interest. Here, we recount some of the major milestones in the history of directed evolution, highlight the most promising recent developments in the field, and discuss the future challenges and opportunities that lie ahead. © 2013 American Institute of Chem. Engineers AIChE J, 2013.
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    70. 70
      Steiner, K.; Schwab, H. Recent advances in rational approaches for enzyme engineering. Comput. Struct. Biotechnol. J. 2012, 2, e201209010  DOI: 10.5936/csbj.201209010
      There is no corresponding record for this reference.
    71. 71
      Fernandes, P. Miniaturization in Biocatalysis. Int. J. Mol. Sci. 2010, 11, 858– 879,  DOI: 10.3390/ijms11030858
      71
      Miniaturization in biocatalysis
      Fernandes, Pedro
      International Journal of Molecular Sciences (2010), 11 (), 858-879CODEN: IJMCFK; ISSN:1422-0067. (Molecular Diversity Preservation International)
      A review. The use of biocatalysts for the prodn. of both consumer goods and building blocks for chem. synthesis is consistently gaining relevance. A significant contribution for recent advances towards further implementation of enzymes and whole cells is related to the developments in miniature reactor technol. and insights into flow behavior. Due to the high level of parallelization and reduced requirements of chems., intensive screening of biocatalysts and process variables has become more feasible and reproducibility of the bioconversion processes has been substantially improved. The present work aims to provide an overview of the applications of miniaturized reactors in bioconversion processes, considering multi-well plates and microfluidic devices, update information on the engineering characterization of the hardware used, and present perspective developments in this area of research.
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    72. 72
      Bell, E. L.; Finnigan, W.; France, S. P.; Green, A. P.; Hayes, M. A.; Hepworth, L. J.; Lovelock, S. L.; Niikura, H.; Osuna, S.; Romero, E.; Ryan, K. S.; Turner, N. J.; Flitsch, S. L. Biocatalysis. Nat. Rev. Methods Primers 2021, 1, 46,  DOI: 10.1038/s43586-021-00044-z
      72
      Biocatalysis
      Bell, Elizabeth L.; Finnigan, William; France, Scott P.; Green, Anthony P.; Hayes, Martin A.; Hepworth, Lorna J.; Lovelock, Sarah L.; Niikura, Haruka; Osuna, Silvia; Romero, Elvira; Ryan, Katherine S.; Turner, Nicholas J.; Flitsch, Sabine L.
      Nature Reviews Methods Primers (2021), 1 (1), 46CODEN: NRMPAT; ISSN:2662-8449. (Nature Portfolio)
      A review. Biocatalysis has become an important aspect of modern org. synthesis, both in academia and across the chem. and pharmaceutical industries. Its success has been largely due to a rapid expansion of the range of chem. reactions accessible, made possible by advanced tools for enzyme discovery coupled with high-throughput lab. evolution techniques for biocatalyst optimization. A wide range of tailor-made enzymes with high efficiencies and selectivities can now be produced quickly and on a gram to kilogram scale, with dedicated databases and search tools aimed at making these biocatalysts accessible to a broader scientific community. This Primer discusses the current state-of-the-art methodol. in the field, including route design, enzyme discovery, protein engineering and the implementation of biocatalysis in industry. We highlight recent advances, such as de novo design and directed evolution, and discuss parameters that make a good reproducible biocatalytic process for industry. The general concepts will be illustrated by recent examples of applications in academia and industry, including the development of multistep enzyme cascades.
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    73. 73
      Duetz, W. A. Microtiter plates as mini-bioreactors: miniaturization of fermentation methods. Trends Microbiol. 2007, 15, 469– 475,  DOI: 10.1016/j.tim.2007.09.004
      73
      Microtiter plates as mini-bioreactors: miniaturization of fermentation methods
      Duetz, Wouter A.
      Trends in Microbiology (2007), 15 (10), 469-475CODEN: TRMIEA; ISSN:0966-842X. (Elsevier B.V.)
      A review. In the past decade, the use of microtiter plates for microbial growth has become widespread, particularly in industry. In parallel, research in academia has provided a thorough insight into the complex relation between well dimensions, culture vols., orbital shaking conditions and surface tension on the one hand, and oxygen-transfer rates and degrees of mixing on the other. In this review, I will discuss these fundamental issues and describe the current applications of microtiter plates in microbiol. Microtiter plates can now be considered a mature alternative to Erlenmeyer shake flasks.
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    74. 74
      Diefenbach, X. W.; Farasat, I.; Guetschow, E. D.; Welch, C. J.; Kennedy, R. T.; Sun, S.; Moore, J. C. Enabling Biocatalysis by High-Throughput Protein Engineering Using Droplet Microfluidics Coupled to Mass Spectrometry. ACS Omega 2018, 3, 1498– 1508,  DOI: 10.1021/acsomega.7b01973
      74
      Enabling Biocatalysis by High-Throughput Protein Engineering Using Droplet Microfluidics Coupled to Mass Spectrometry
      Diefenbach, Xue W.; Farasat, Iman; Guetschow, Erik D.; Welch, Christopher J.; Kennedy, Robert T.; Sun, Shuwen; Moore, Jeffrey C.
      ACS Omega (2018), 3 (2), 1498-1508CODEN: ACSODF; ISSN:2470-1343. (American Chemical Society)
      Directed Evolution is a key technol. driving the utility of biocatalysis in pharmaceutical synthesis. Conventional approaches to Directed Evolution are conducted using bacterial cells expressing enzymes in microplates, with catalyzed reactions measured by HPLC, high-performance liq. chromatog.-mass spectrometry (HPLC-MS), or optical detectors, which require either long cycle times or tailor-made substrates. To better fit modern, fast-paced process chem. development where solns. are rapidly needed for new substrates, droplet microfluidics interfaced with electrospray ionization (ESI)-MS provides a label-free high-throughput screening platform. To apply this method to industrial enzyme screening and to explore potential approaches that may further improve the overall throughput, we optimized the existing droplet-MS methods. Carryover between droplets, traditionally a significant issue, was reduced to undetectable level by replacing the stainless steel ESI needle with a Teflon needle within a capillary electrophoresis (CE)-MS source. Throughput was improved to 3 Hz with a wide range of droplet sizes (10-50 nL) by tuning the sheath flow within the CE-MS source. The optimized method was demonstrated by screening reactions using two different transaminase libraries. Good correlations (r2 ∼ 0.95) were found between the droplet-MS and LC-MS methods, with 100% match on hit variants. We further explored the capability of the system by performing in vitro transcription-translation inside the droplets and directly analyzing the intact reaction mixt. droplets by MS. The synthesized protein attained comparable activity to the protein std., and the complex samples appeared well tolerated by the MS. The success of the above applications indicates that the MS anal. of the microfluidic droplets is an available option for considerably accelerating the screening of enzyme evolution libraries.
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    75. 75
      Finnigan, W.; Hepworth, L. J.; Flitsch, S. L.; Turner, N. J. RetroBioCat as a computer-aided synthesis planning tool for biocatalytic reactions and cascades. Nat. Catal. 2021, 4, 98– 104,  DOI: 10.1038/s41929-020-00556-z
      75
      RetroBioCat as a computer-aided synthesis planning tool for biocatalytic reactions and cascades
      Finnigan, William; Hepworth, Lorna J.; Flitsch, Sabine L.; Turner, Nicholas J.
      Nature Catalysis (2021), 4 (2), 98-104CODEN: NCAACP; ISSN:2520-1158. (Nature Research)
      Abstr.: As the enzyme toolbox for biocatalysis has expanded, so has the potential for the construction of powerful enzymic cascades for efficient and selective synthesis of target mols. Addnl., recent advances in computer-aided synthesis planning are revolutionizing synthesis design in both synthetic biol. and org. chem. However, the potential for biocatalysis is not well captured by tools currently available in either field. Here we present RetroBioCat, an intuitive and accessible tool for computer-aided design of biocatalytic cascades, freely available at retrobiocat.com. Our approach uses a set of expertly encoded reaction rules encompassing the enzyme toolbox for biocatalysis, and a system for identifying literature precedent for enzymes with the correct substrate specificity where this is available. Applying these rules for automated biocatalytic retrosynthesis, we show our tool to be capable of identifying promising biocatalytic pathways to target mols., validated using a test set of recent cascades described in the literature. [graphic not available: see fulltext].
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    76. 76
      Altschul, S. F.; Gish, W.; Miller, W.; Myers, E. W.; Lipman, D. J. Basic local alignment search tool. J. Mol. Biol. 1990, 215, 403– 410,  DOI: 10.1016/S0022-2836(05)80360-2
      76
      Basic local alignment search tool
      Altschul, Stephen F.; Gish, Warren; Miller, Webb; Myers, Eugene W.; Lipman, David J.
      Journal of Molecular Biology (1990), 215 (3), 403-10CODEN: JMOBAK; ISSN:0022-2836.
      A new approach to rapid sequence comparison, basic local alignment search tool (BLAST), directly approximates alignments that optimize a measure of local similarity, the maximal segment pair (MSP) score. Recent math. results on the stochastic properties of MSP scores allow an anal. of the performance of this method as well as the statistical significance of alignments it generates. The basic algorithm is simple and robust; it can be implemented in a no. of ways and applied in a variety of contexts including straightforward DNA and protein sequence database searches, motif searches, gene identification searches, and in the anal. of multiple regions of similarity in long DNA sequences. In addn. to its flexibility and tractability to math. anal., BLAST is an order of magnitude faster than existing sequence comparison tools of comparable sensitivity.
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    77. 77
      Cai, X.-H.; Jaroszewski, L.; Wooley, J.; Godzik, A. Internal organization of large protein families: Relationship between the sequence, structure, and function-based clustering. Proteins: Struct., Funct., Bioinf. 2011, 79, 2389– 2402,  DOI: 10.1002/prot.23049
      77
      Internal organization of large protein families: Relationship between the sequence, structure, and function-based clustering
      Cai, Xiao-Hui; Jaroszewski, Lukasz; Wooley, John; Godzik, Adam
      Proteins: Structure, Function, and Bioinformatics (2011), 79 (8), 2389-2402CODEN: PSFBAF ISSN:. (Wiley-Liss, Inc.)
      The protein universe can be organized in families that group proteins sharing common ancestry. Such families display variable levels of structural and functional divergence, from homogeneous families, where all members have the same function and very similar structure, to very divergent families, where large variations in function and structure are obsd. For practical purposes of structure and function prediction, it would be beneficial to identify sub-groups of proteins with highly similar structures (iso-structural) and/or functions (iso-functional) within divergent protein families. Three algorithms were compared in their ability to cluster large protein families and it was discussed whether any of these methods could reliably identify such iso-structural or iso-functional groups. It was shown that clustering using profile-sequence and profile-profile comparison methods closely reproduces clusters based on similarities between 3D structures or clusters of proteins with similar biol. functions. In contrast, the still commonly used sequence-based methods with fixed thresholds result in vast over-ests. of structural and functional diversity in protein families. As a result, these methods also over-est. the no. of protein structures that have to be detd. to fully characterize structural space of such families. The fact that one can build reliable models based on apparently distantly related templates is crucial for extg. maximal amt. of information from new sequencing projects.
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    78. 78
      Sirota, F. L.; Maurer-Stroh, S.; Li, Z.; Eisenhaber, F.; Eisenhaber, B. Functional Classification of Super-Large Families of Enzymes Based on Substrate Binding Pocket Residues for Biocatalysis and Enzyme Engineering Applications. Front. Bioeng. Biotechnol. 2021, 9, 701120,  DOI: 10.3389/fbioe.2021.701120
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      Functional Classification of Super-Large Families of Enzymes Based on Substrate Binding Pocket Residues for Biocatalysis and Enzyme Engineering Applications
      Sirota Fernanda L; Maurer-Stroh Sebastian; Eisenhaber Frank; Eisenhaber Birgit; Maurer-Stroh Sebastian; Li Zhi; Eisenhaber Frank; Eisenhaber Birgit; Eisenhaber Frank
      Frontiers in bioengineering and biotechnology (2021), 9 (), 701120 ISSN:2296-4185.
      Large enzyme families such as the groups of zinc-dependent alcohol dehydrogenases (ADHs), long chain alcohol oxidases (AOxs) or amine dehydrogenases (AmDHs) with, sometimes, more than one million sequences in the non-redundant protein database and hundreds of experimentally characterized enzymes are excellent cases for protein engineering efforts aimed at refining and modifying substrate specificity. Yet, the backside of this wealth of information is that it becomes technically difficult to rationally select optimal sequence targets as well as sequence positions for mutagenesis studies. In all three cases, we approach the problem by starting with a group of experimentally well studied family members (including those with available 3D structures) and creating a structure-guided multiple sequence alignment and a modified phylogenetic tree (aka binding site tree) based just on a selection of potential substrate binding residue positions derived from experimental information (not from the full-length sequence alignment). Hereupon, the remaining, mostly uncharacterized enzyme sequences can be mapped; as a trend, sequence grouping in the tree branches follows substrate specificity. We show that this information can be used in the target selection for protein engineering work to narrow down to single suitable sequences and just a few relevant candidate positions for directed evolution towards activity for desired organic compound substrates. We also demonstrate how to find the closest thermophile example in the dataset if the engineering is aimed at achieving most robust enzymes.
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    79. 79
      Wilding, M.; Peat, T. S.; Kalyaanamoorthy, S.; Newman, J.; Scott, C.; Jermiin, L. S. Reverse engineering: transaminase biocatalyst development using ancestral sequence reconstruction. Green Chem. 2017, 19, 5375– 5380,  DOI: 10.1039/C7GC02343J
      79
      Reverse engineering: transaminase biocatalyst development using ancestral sequence reconstruction
      Wilding, Matthew; Peat, Thomas S.; Kalyaanamoorthy, Subha; Newman, Janet; Scott, Colin; Jermiin, Lars S.
      Green Chemistry (2017), 19 (22), 5375-5380CODEN: GRCHFJ; ISSN:1463-9262. (Royal Society of Chemistry)
      The development of new biocatalysts using ancestral sequence reconstruction is reported. When applied to an ω-transaminase, the ancestral proteins demonstrated novel and superior activities with eighty percent of the forty compds. tested compared to the modern day protein, and improvements in activity of up to twenty fold. These included a range of compds. pertinent as feedstocks in polyamide manuf.
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    80. 80
      Gumulya, Y.; Baek, J.-M.; Wun, S.-J.; Thomson, R. E. S.; Harris, K. L.; Hunter, D. J. B.; Behrendorff, J. B. Y. H.; Kulig, J.; Zheng, S.; Wu, X.; Wu, B.; Stok, J. E.; De Voss, J. J.; Schenk, G.; Jurva, U.; Andersson, S.; Isin, E. M.; Bodén, M.; Guddat, L.; Gillam, E. M. J. Engineering highly functional thermostable proteins using ancestral sequence reconstruction. Nat. Catal. 2018, 1, 878– 888,  DOI: 10.1038/s41929-018-0159-5
      80
      Engineering highly functional thermostable proteins using ancestral sequence reconstruction
      Gumulya, Yosephin; Baek, Jong-Min; Wun, Shun-Jie; Thomson, Raine E. S.; Harris, Kurt L.; Hunter, Dominic J. B.; Behrendorff, James B. Y. H.; Kulig, Justyna; Zheng, Shan; Wu, Xueming; Wu, Bin; Stok, Jeanette E.; De Voss, James J.; Schenk, Gerhard; Jurva, Ulrik; Andersson, Shalini; Isin, Emre M.; Boden, Mikael; Guddat, Luke; Gillam, Elizabeth M. J.
      Nature Catalysis (2018), 1 (11), 878-888CODEN: NCAACP; ISSN:2520-1158. (Nature Research)
      Com. biocatalysis requires robust enzymes that can withstand elevated temps. and long incubations. Ancestral reconstruction has shown that pre-Cambrian enzymes were often much more thermostable than extant forms. Here, we resurrect ancestral enzymes that withstand ∼30 °C higher temps. and ≥100 times longer incubations than their extant forms. This is demonstrated on animal cytochromes P 450 that stereo- and regioselectively functionalize unactivated C-H bonds for the synthesis of valuable chems., and bacterial ketol-acid reductoisomerases that are used to make butanol-based biofuels. The vertebrate CYP3 P 450 ancestor showed a 60T50 of 66 °C and enhanced solvent tolerance compared with the human drug-metabolizing CYP3A4, yet comparable activity towards a similarly broad range of substrates. The ancestral ketol-acid reductoisomerase showed an eight-fold higher specific activity than the cognate Escherichia coli form at 25 °C, which increased 3.5-fold at 50 °C. Thus, thermostable proteins can be devised using sequence data alone from even recent ancestors.
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    81. 81
      Tamura, K.; Peterson, D.; Peterson, N.; Stecher, G.; Nei, M.; Kumar, S. MEGA5: Molecular Evolutionary Genetics Analysis Using Maximum Likelihood, Evolutionary Distance, and Maximum Parsimony Methods. Mol. Biol. Evol. 2011, 28, 2731– 2739,  DOI: 10.1093/molbev/msr121
      81
      MEGA5: Molecular Evolutionary Genetics Analysis Using Maximum Likelihood, Evolutionary Distance, and Maximum Parsimony Methods
      Tamura, Koichiro; Peterson, Daniel; Peterson, Nicholas; Stecher, Glen; Nei, Masatoshi; Kumar, Sudhir
      Molecular Biology and Evolution (2011), 28 (10), 2731-2739CODEN: MBEVEO; ISSN:0737-4038. (Oxford University Press)
      Comparative anal. of mol. sequence data is essential for reconstructing the evolutionary histories of species and inferring the nature and extent of selective forces shaping the evolution of genes and species. Here, we announce the release of Mol. Evolutionary Genetics Anal. version 5 (MEGA5), which is a user-friendly software for mining online databases, building sequence alignments and phylogenetic trees, and using methods of evolutionary bioinformatics in basic biol., biomedicine, and evolution. The newest addn. in MEGA5 is a collection of max. likelihood (ML) analyses for inferring evolutionary trees, selecting best-fit substitution models (nucleotide or amino acid), inferring ancestral states and sequences (along with probabilities), and estg. evolutionary rates site-by-site. In computer simulation analyses, ML tree inference algorithms in MEGA5 compared favorably with other software packages in terms of computational efficiency and the accuracy of the ests. of phylogenetic trees, substitution parameters, and rate variation among sites. The MEGA user interface has now been enhanced to be activity driven to make it easier for the use of both beginners and experienced scientists. This version of MEGA is intended for the Windows platform, and it has been configured for effective use on Mac OS X and Linux desktops. It is available free of charge from http://www.megasoftware.net.
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    82. 82
      Hall, B. G. Building phylogenetic trees from molecular data with MEGA. Mol. Biol. Evol. 2013, 30, 1229– 1235,  DOI: 10.1093/molbev/mst012
      82
      Building Phylogenetic Trees from Molecular Data with MEGA
      Hall, Barry G.
      Molecular Biology and Evolution (2013), 30 (5), 1229-1235CODEN: MBEVEO; ISSN:0737-4038. (Oxford University Press)
      Phylogenetic anal. is sometimes regarded as being an intimidating, complex process that requires expertise and years of experience. In fact, it is a fairly straightforward process that can be learned quickly and applied effectively. This Protocol describes the several steps required to produce a phylogenetic tree from mol. data for novices. In the example illustrated here, the program MEGA is used to implement all those steps, thereby eliminating the need to learn several programs, and to deal with multiple file formats from one step to another (Tamura K, Peterson D, Peterson N, Stecher G, Nei M, Kumar S. 2011. MEGA5: mol. evolutionary genetics anal. using max. likelihood, evolutionary distance, and max. parsimony methods.Mol Biol Evol. 28:2731-2739). The first step, identification of a set of homologous sequences and downloading those sequences, is implemented by MEGA's own browser built on top of the Google Chrome toolkit. For the second step, alignment of those sequences, MEGA offers two different algorithms: ClustalW and MUSCLE. For the third step, construction of a phylogenetic tree from the aligned sequences, MEGA offers many different methods. Here we illustrate the max. likelihood method, beginning with MEGA's Models feature, which permits selecting the most suitable substitution model. Finally, MEGA provides a powerful and flexible interface for the final step, actually drawing the tree for publication. Here a step-by-step protocol is presented in sufficient detail to allow a novice to start with a sequence of interest and to build a publication-quality tree illustrating the evolution of an appropriate set of homologs of that sequence. MEGA is available for use on PCs and Macs from www.megasoftware.net.
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    83. 83
      Yates, A. D.; Achuthan, P.; Akanni, W.; Allen, J.; Allen, J.; Alvarez-Jarreta, J.; Amode, M. R.; Armean, I. M.; Azov, A. G.; Bennett, R.; Bhai, J.; Billis, K.; Boddu, S.; Marugán, J. C.; Cummins, C.; Davidson, C.; Dodiya, K.; Fatima, R.; Gall, A.; Giron, C. G.; Gil, L.; Grego, T.; Haggerty, L.; Haskell, E.; Hourlier, T.; Izuogu, O. G.; Janacek, S. H.; Juettemann, T.; Kay, M.; Lavidas, I.; Le, T.; Lemos, D.; Martinez, J. G.; Maurel, T.; McDowall, M.; McMahon, A.; Mohanan, S.; Moore, B.; Nuhn, M.; Oheh, D. N.; Parker, A.; Parton, A.; Patricio, M.; Sakthivel, M. P.; Abdul Salam, A. I.; Schmitt, B. M.; Schuilenburg, H.; Sheppard, D.; Sycheva, M.; Szuba, M.; Taylor, K.; Thormann, A.; Threadgold, G.; Vullo, A.; Walts, B.; Winterbottom, A.; Zadissa, A.; Chakiachvili, M.; Flint, B.; Frankish, A.; Hunt, S. E.; IIsley, G.; Kostadima, M.; Langridge, N.; Loveland, J. E.; Martin, F. J.; Morales, J.; Mudge, J. M.; Muffato, M.; Perry, E.; Ruffier, M.; Trevanion, S. J.; Cunningham, F.; Howe, K. L.; Zerbino, D. R.; Flicek, P. Ensembl 2020. Nucleic Acids Res. 2020, 48, D682– D688,  DOI: 10.1093/nar/gkz966
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      Ensembl 2020
      Yates, Andrew D.; Achuthan, Premanand; Akanni, Wasiu; Allen, James; Allen, Jamie; Alvarez-Jarreta, Jorge; Amode, M. Ridwan; Armean, Irina M.; Azov, Andrey G.; Bennett, Ruth; Bhai, Jyothish; Billis, Konstantinos; Boddu, Sanjay; Marugan, Jose Carlos; Cummins, Carla; Davidson, Claire; Dodiya, Kamalkumar; Fatima, Reham; Gall, Astrid; Giron, Carlos Garcia; Gil, Laurent; Grego, Tiago; Haggerty, Leanne; Haskell, Erin; Hourlier, Thibaut; Izuogu, Osagie G.; Janacek, Sophie H.; Juettemann, Thomas; Kay, Mike; Lavidas, Ilias; Le, Tuan; Lemos, Diana; Martinez, Jose Gonzalez; Maurel, Thomas; Mcdowall, Mark; Mcmahon, Aoife; Mohanan, Shamika; Moore, Benjamin; Nuhn, Michael; Oheh, Denye N.; Parker, Anne; Parton, Andrew; Patricio, Mateus; Sakthivel, Manoj Pandian; Abdul Salam, Ahamed Imran; Schmitt, Bianca M.; Schuilenburg, Helen; Sheppard, Dan; Sycheva, Mira; Szuba, Marek; Taylor, Kieron; Thormann, Anja; Threadgold, Glen; Vullo, Alessandro; Walts, Brandon; Winterbottom, Andrea; Zadissa, Amonida; Chakiachvili, Marc; Flint, Bethany; Frankish, Adam; Hunt, Sarah E.; Iisley, Garth; Kostadima, Myrto; Langridge, Nick; Loveland, Jane E.; Martin, Fergal J.; Morales, Joannella; Mudge, Jonathan M.; Muffato, Matthieu; Perry, Emily; Ruffier, Magali; Trevanion, Stephen J.; Cunningham, Fiona; Howe, Kevin L.; Zerbino, Daniel R.; Flicek, Paul
      Nucleic Acids Research (2020), 48 (D1), D682-D688CODEN: NARHAD; ISSN:1362-4962. (Oxford University Press)
      A review. The Ensembl is a system for generating and distributing genome annotation such as genes, variation, regulation and comparative genomics across the vertebrate subphylum and key model organisms. The Ensembl annotation pipeline is capable of integrating exptl. and ref. data from multiple providers into a single integrated resource. Here, we present 94 newly annotated and re-annotated genomes, bringing the total no. of genomes offered by Ensembl to 227. This represents the single largest expansion of the resource since its inception. We also detail our continued efforts to improve human annotation, developments in our epigenome anal. and display, a new tool for imputing causal genes from genome-wide assocn. studies and visualization of variation within a 3D protein model. Finally, we present information on our new website. Both software and data are made available without restriction via our website, online tools platform and programmatic interfaces (available under an Apache 2.0 license) and data updates made available four times a year.
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    84. 84
      Atkinson, H. J.; Morris, J. H.; Ferrin, T. E.; Babbitt, P. C. Using Sequence Similarity Networks for Visualization of Relationships Across Diverse Protein Superfamilies. PloS One 2009, 4, e4345  DOI: 10.1371/journal.pone.0004345
      84
      Using sequence similarity networks for visualization of relationships across diverse protein superfamilies
      Atkinson Holly J; Morris John H; Ferrin Thomas E; Babbitt Patricia C
      PloS one (2009), 4 (2), e4345 ISSN:.
      The dramatic increase in heterogeneous types of biological data--in particular, the abundance of new protein sequences--requires fast and user-friendly methods for organizing this information in a way that enables functional inference. The most widely used strategy to link sequence or structure to function, homology-based function prediction, relies on the fundamental assumption that sequence or structural similarity implies functional similarity. New tools that extend this approach are still urgently needed to associate sequence data with biological information in ways that accommodate the real complexity of the problem, while being accessible to experimental as well as computational biologists. To address this, we have examined the application of sequence similarity networks for visualizing functional trends across protein superfamilies from the context of sequence similarity. Using three large groups of homologous proteins of varying types of structural and functional diversity--GPCRs and kinases from humans, and the crotonase superfamily of enzymes--we show that overlaying networks with orthogonal information is a powerful approach for observing functional themes and revealing outliers. In comparison to other primary methods, networks provide both a good representation of group-wise sequence similarity relationships and a strong visual and quantitative correlation with phylogenetic trees, while enabling analysis and visualization of much larger sets of sequences than trees or multiple sequence alignments can easily accommodate. We also define important limitations and caveats in the application of these networks. As a broadly accessible and effective tool for the exploration of protein superfamilies, sequence similarity networks show great potential for generating testable hypotheses about protein structure-function relationships.
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    85. 85
      Zallot, R.; Oberg, N.; Gerlt, J. A. The EFI Web Resource for Genomic Enzymology Tools: Leveraging Protein, Genome, and Metagenome Databases to Discover Novel Enzymes and Metabolic Pathways. Biochemistry 2019, 58, 4169– 4182,  DOI: 10.1021/acs.biochem.9b00735
      85
      The EFI Web Resource for Genomic Enzymology Tools: Leveraging Protein, Genome, and Metagenome Databases to Discover Novel Enzymes and Metabolic Pathways
      Zallot, Remi; Oberg, Nils; Gerlt, John A.
      Biochemistry (2019), 58 (41), 4169-4182CODEN: BICHAW; ISSN:0006-2960. (American Chemical Society)
      The assignment of functions to uncharacterized proteins discovered in genome projects requires easily accessible tools and computational resources for large-scale, user-friendly leveraging of the protein, genome, and metagenome databases by experimentalists. This article describes the web resource developed by the Enzyme Function Initiative (EFI; accessed at https://efi.igb.illinois.edu/) that provides "genomic enzymol." tools ("web tools") for (1) generating sequence similarity networks (SSNs) for protein families (EFI-EST); (2) analyzing and visualizing genome context of the proteins in clusters in SSNs (in genome neighborhood networks, GNNs, and genome neighborhood diagrams, GNDs) (EFI-GNT); and (3) prioritizing uncharacterized SSN clusters for functional assignment based on metagenome abundance (chem. guided functional profiling, CGFP) (EFI-CGFP). The SSNs generated by EFI-EST are used as the input for EFI-GNT and EFI-CGFP, enabling easy transfer of information among the tools. The networks are visualized and analyzed using Cytoscape, a widely used desktop application; GNDs and CGFP heatmaps summarizing metagenome abundance are viewed within the tools. We provide a detailed example of the integrated use of the tools with an anal. of glycyl radical enzyme superfamily (IPR004184) found in the human gut microbiome. This anal. demonstrates that (1) SwissProt annotations are not always correct, (2) large-scale genome context analyses allow the prediction of novel metabolic pathways, and (3) metagenome abundance can be used to identify/prioritize uncharacterized proteins for functional investigation.
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      https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC1MXhvVent73O&md5=36936982cb5816c67629147348df2c79
    86. 86
      Doyon, T. J.; Perkins, J. C.; Baker Dockrey, S. A.; Romero, E. O.; Skinner, K. C.; Zimmerman, P. M.; Narayan, A. R. H. Chemoenzymatic o-Quinone Methide Formation. J. Am. Chem. Soc. 2019, 141, 20269– 20277,  DOI: 10.1021/jacs.9b10474
      86
      Chemoenzymatic o-quinone methide formation
      Doyon, Tyler J.; Perkins, Jonathan C.; Baker Dockrey, Summer A.; Romero, Evan O.; Skinner, Kevin C.; Zimmerman, Paul M.; Narayan, Alison R. H.
      Journal of the American Chemical Society (2019), 141 (51), 20269-20277CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)
      Generation of reactive intermediates and interception of these fleeting species under physiol. conditions is a common strategy employed by Nature to build mol. complexity. However, selective formation of these species under mild conditions using classical synthetic techniques is an outstanding challenge. Here, we demonstrate the utility of biocatalysis in generating o-quinone methide intermediates with precise chemoselectivity under mild, aq. conditions. Specifically, α-ketoglutarate-dependent non-heme iron enzymes, CitB and ClaD, are employed to selectively modify benzylic C-H bonds of o-cresol substrates. In this transformation, biocatalytic hydroxylation of a benzylic C-H bond affords a benzylic alc. product which, under the aq. reaction conditions, is in equil. with the corresponding o-quinone methide. O-Quinone methide interception by a nucleophile or a dienophile allows for one-pot conversion of benzylic C-H bonds into C-C, C-N, C-O, and C-S bonds in chemoenzymic cascades on preparative scale. The chemoselectivity and mild nature of this platform is showcased here by the selective modification of peptides and chemoenzymic synthesis of the chroman natural product (-)-xyloketal D.
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    87. 87
      Rodriguez Benitez, A.; Narayan, A. R. H. Frontiers in Biocatalysis: Profiling Function across Sequence Space. ACS Cent. Sci. 2019, 5, 1747– 1749,  DOI: 10.1021/acscentsci.9b01112
      87
      Frontiers in Biocatalysis: Profiling Function across Sequence Space
      Rodriguez Benitez Attabey; Narayan Alison R H
      ACS central science (2019), 5 (11), 1747-1749 ISSN:2374-7943.
      There is no expanded citation for this reference.
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    88. 88
      Fisher, B. F.; Snodgrass, H. M.; Jones, K. A.; Andorfer, M. C.; Lewis, J. C. Site-Selective C–H Halogenation Using Flavin-Dependent Halogenases Identified via Family-Wide Activity Profiling. ACS Cent. Sci. 2019, 5, 1844– 1856,  DOI: 10.1021/acscentsci.9b00835
      88
      Site-Selective C-H Halogenation Using Flavin-Dependent Halogenases Identified via Family-Wide Activity Profiling
      Fisher, Brian F.; Snodgrass, Harrison M.; Jones, Krysten A.; Andorfer, Mary C.; Lewis, Jared C.
      ACS Central Science (2019), 5 (11), 1844-1856CODEN: ACSCII; ISSN:2374-7951. (American Chemical Society)
      Enzymes are powerful catalysts for site-selective C-H bond functionalization. Identifying suitable enzymes for this task and for biocatalysis in general remains challenging, however, due to the fundamental difficulty of predicting catalytic activity from sequence information. In this study, family-wide activity profiling was used to obtain sequence-function information on flavin-dependent halogenases (FDHs). This broad survey provided a no. of insights into FDH activity, including halide specificity and substrate preference, that were not apparent from the more focused studies reported to date. Regions of FDH sequence space that are most likely to contain enzymes suitable for halogenating small-mol. substrates were also identified. FDHs with novel substrate scope and complementary regioselectivity on large, three-dimensionally complex compds. were characterized and used for preparative-scale late-stage C-H functionalization. In many cases, these enzymes provide activities that required several rounds of directed evolution to accomplish in previous efforts, highlighting that this approach can achieve significant time savings for biocatalyst identification and provide advanced starting points for further evolution. High-throughput screening of >20 000 reactions catalyzed by 87 sol. genome-mined halogenases on 62 substrates found 39 new active halogenases for selective late-stage C-H functionalization.
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    89. 89
      Schülke, K. H.; Ospina, F.; Hörnschemeyer, K.; Gergel, S.; Hammer, S. C. Substrate Profiling of Anion Methyltransferases for Promiscuous Synthesis of S-Adenosylmethionine Analogs from Haloalkanes. ChemBioChem. 2022, 23, e202100632  DOI: 10.1002/cbic.202100632
      89
      Substrate Profiling of Anion Methyltransferases for Promiscuous Synthesis of S-Adenosylmethionine Analogs from Haloalkanes
      Schuelke, Kai H.; Ospina, Felipe; Hornschemeyer, Kathrin; Gergel, Sebastian; Hammer, Stephan C.
      ChemBioChem (2022), 23 (4), e202100632CODEN: CBCHFX; ISSN:1439-4227. (Wiley-VCH Verlag GmbH & Co. KGaA)
      Biocatalytic alkylation reactions can be performed with high chemo-, regio- and stereoselectivity using S-adenosyl-L-methionine (SAM)-dependent methyltransferases (MTs) and SAM analogs. Currently, however, this methodol. is limited in application due to the rather laborious protocols to access SAM analogs. It has recently been shown that halide methyltransferases (HMTs) enable synthesis and recycling of SAM analogs with readily available haloalkanes as starting material. Here we expand this work by using substrate profiling of the anion MT enzyme family to explore promiscuous SAM analog synthesis. Our study shows that anion MTs are in general very promiscuous with respect to the alkyl chain as well as the halide leaving group. Substrate profiling further suggests that promiscuous anion MTs cluster in sequence space. Next to iodoalkanes, cheaper, less toxic, and more available bromoalkanes have been converted and several haloalkanes bearing short alkyl groups, alkyl rings, and functional groups such as alkene, alkyne and arom. moieties are accepted as substrates. Further, we applied the SAM analogs as electrophiles in enzyme-catalyzed regioselective pyrazole allylation with 3-bromopropene as starting material.
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    90. 90
      Lachowicz, J. C.; Gizzi, A. S.; Almo, S. C.; Grove, T. L. Structural Insight into the Substrate Scope of Viperin and Viperin-like Enzymes from Three Domains of Life. Biochemistry 2021, 60, 2116– 2129,  DOI: 10.1021/acs.biochem.0c00958
      90
      Structural Insight into the Substrate Scope of Viperin and Viperin-like Enzymes from Three Domains of Life
      Lachowicz, Jake C.; Gizzi, Anthony S.; Almo, Steven C.; Grove, Tyler L.
      Biochemistry (2021), 60 (26), 2116-2129CODEN: BICHAW; ISSN:0006-2960. (American Chemical Society)
      Viperin is a member of the radical S-adenosylmethionine superfamily and was shown to restrict the replication of a wide range of RNA and DNA viruses. The authors recently demonstrated that human viperin (HsVip) catalyzes the conversion of CTP to 3'-deoxy-3',4'-didehydro-CTP (ddhCTP or ddh-synthase), which acts as a chain terminator for virally encoded RNA-dependent RNA polymerases from several flaviviruses. Viperin homologs also exist in non-chordate eukaryotes (e.g., Cnidaria and Mollusca), numerous fungi, and members of the archaeal and eubacterial domains. Recently, it is reported that non-chordate and non-eukaryotic viperin-like homologs are also ddh-synthases and generate a diverse range of ddhNTPs, including the newly discovered ddhUTP and ddhGTP. Herein, the authors expand on the catalytic mechanism of mammalian, fungal, bacterial, and archaeal viperin-like enzymes with a combination of x-ray crystallog. and enzymol. Like mammalian viperins, these recently discovered viperin-like enzymes operate through the same mechanism and can be classified as ddh-synthases. Furthermore, the authors define the unique chem. and phys. determinants supporting ddh-synthase activity and nucleotide selectivity, including the crystallog. characterization of a fungal viperin-like enzyme that uses UTP as a substrate and a cnidaria viperin-like enzyme that uses CTP as a substrate. Together, these results support the evolutionary conservation of the ddh-synthase activity and its broad phylogenetic role in innate antiviral immunity.
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    91. 91
      Tararina, M. A.; Allen, K. N. Bioinformatic Analysis of the Flavin-Dependent Amine Oxidase Superfamily: Adaptations for Substrate Specificity and Catalytic Diversity. J. Mol. Biol. 2020, 432, 3269– 3288,  DOI: 10.1016/j.jmb.2020.03.007
      91
      Bioinformatic Analysis of the Flavin-Dependent Amine Oxidase Superfamily: Adaptations for Substrate Specificity and Catalytic Diversity
      Tararina, Margarita A.; Allen, Karen N.
      Journal of Molecular Biology (2020), 432 (10), 3269-3288CODEN: JMOBAK; ISSN:0022-2836. (Elsevier Ltd.)
      The flavin-dependent amine oxidase (FAO) superfamily consists of over 9000 nonredundant sequences represented in all domains of life. Of the thousands of members identified, only 214 have been functionally annotated to date, and 40 unique structures are represented in the Protein Data Bank. The few functionally characterized members share a catalytic mechanism involving the oxidn. of an amine substrate through transfer of a hydride to the FAD cofactor, with differences obsd. in substrate specificities. Previous studies have focused on comparing a subset of superfamily members. Here, we present a comprehensive anal. of the FAO superfamily based on reaction mechanism and substrate recognition. Using a dataset of 9192 sequences, a sequence similarity network, and subsequently, a genome neighborhood network were constructed, organizing the superfamily into eight subgroups that accord with substrate type. Likewise, through phylogenetic anal., the evolutionary relationship of subgroups was detd., delineating the divergence between enzymes based on organism, substrate, and mechanism. In addn., using sequences and at. coordinates of 22 structures from the Protein Data Bank to perform sequence and structural alignments, active-site elements were identified, showing divergence from the canonical arom.-cage residues to accommodate large substrates. These specificity determinants are held in a structural framework comprising a core domain catalyzing the oxidn. of amines with an auxiliary domain for substrate recognition. Overall, anal. of the FAO superfamily reveals a modular fold with cofactor and substrate-binding domains allowing for diversity of recognition via insertion/deletions. This flexibility allows facile evolution of new activities, as shown by reinvention of function between subfamilies.
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    92. 92
      Gerlt, J. A.; Bouvier, J. T.; Davidson, D. B.; Imker, H. J.; Sadkhin, B.; Slater, D. R.; Whalen, K. L. Enzyme Function Initiative-Enzyme Similarity Tool (EFI-EST): A web tool for generating protein sequence similarity networks. Biochim. Biophys. Acta, Proteins Proteomics 2015, 1854, 1019– 1037,  DOI: 10.1016/j.bbapap.2015.04.015
      92
      Enzyme function initiative-enzyme similarity tool (EFI-EST): A web tool for generating protein sequence similarity networks
      Gerlt, John A.; Bouvier, Jason T.; Davidson, Daniel B.; Imker, Heidi J.; Sadkhin, Boris; Slater, David R.; Whalen, Katie L.
      Biochimica et Biophysica Acta, Proteins and Proteomics (2015), 1854 (8), 1019-1037CODEN: BBAPBW; ISSN:1570-9639. (Elsevier B. V.)
      A review. The Enzyme Function Initiative, an NIH/NIGMS-supported Large-Scale Collaborative Project (EFI; U54GM093342; http://enzymefunction.org/), is focused on devising and disseminating bioinformatics and computational tools as well as exptl. strategies for the prediction and assignment of functions (in vitro activities and in vivo physiol./metabolic roles) to uncharacterized enzymes discovered in genome projects. Protein sequence similarity networks (SSNs) are visually powerful tools for analyzing sequence relationships in protein families (H.J. Atkinson, J.H. Morris, T.E. Ferrin, and P.C. Babbitt, PLoS One 2009, 4, e4345). However, the members of the biol./biomedical community have not had access to the capability to generate SSNs for their "favorite" protein families. In this article we announce the EFI-EST (Enzyme Function Initiative-Enzyme Similarity Tool) web tool (http://efi.igb.illinois.edu/efi-est/) that is available without cost for the automated generation of SSNs by the community. The tool can create SSNs for the "closest neighbors" of a user-supplied protein sequence from the UniProt database (Option A) or of members of any user-supplied Pfam and/or InterPro family (Option B). We provide an introduction to SSNs, a description of EFI-EST, and a demonstration of the use of EFI-EST to explore sequence-function space in the OMP decarboxylase superfamily (PF00215). This article is designed as a tutorial that will allow members of the community to use the EFI-EST web tool for exploring sequence/function space in protein families.
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      https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2MXnvFSisb0%253D&md5=4897a23daaf5c2437acb0bed815095f8
    93. 93
      Kohl, M.; Wiese, S.; Warscheid, B. Cytoscape: Software for Visualization and Analysis of Biological Networks. In Data Mining in Proteomics: From Standards to Applications; Hamacher, M., Eisenacher, M., Stephan, C., Eds.; Methods in Molecular Biology; Humana Press: Totowa, NJ, 2011; pp 291– 303.
      There is no corresponding record for this reference.
    94. 94
      E. J. Corey, X.-M. C. The Logic of Chemical Synthesis; Wiley, :New York, 1989.
      There is no corresponding record for this reference.
    95. 95
      Bommarius, A. S.; Riebel Bommarius, B. R. Biocatalysis; Wiley-VCH Verlag: Weinheim, Germany, 2004; p 634.
      There is no corresponding record for this reference.
    96. 96
      Whittall, J. Applied biocatalysis; John Wiley & Sons: Nashville, TN, 2020; p 560.
      There is no corresponding record for this reference.
    97. 97
      Turner, N. J.; Humphreys, L. Biocatalysis in Organic Synthesis: The Retrosynthesis Approach; Royal Society of Chemistry: 2018.
      There is no corresponding record for this reference.
    98. 98
      Burns, M.; Martinez, C. A.; Vanderplas, B.; Wisdom, R.; Yu, S.; Singer, R. A. A Chemoenzymatic Route to Chiral Intermediates Used in the Multikilogram Synthesis of a Gamma Secretase Inhibitor. Org. Process Res. Dev. 2017, 21, 871– 877,  DOI: 10.1021/acs.oprd.7b00096
      98
      A Chemoenzymatic Route to Chiral Intermediates Used in the Multikilogram Synthesis of a Gamma Secretase Inhibitor
      Burns, Michael; Martinez, Carlos A.; Vanderplas, Brian; Wisdom, Richard; Yu, Shu; Singer, Robert A.
      Organic Process Research & Development (2017), 21 (6), 871-877CODEN: OPRDFK; ISSN:1083-6160. (American Chemical Society)
      A chemoenzymic route for the prodn. of an intermediate to a gamma secretase inhibitor is described. The route is robust and was run at multikilogram scale. The process employs both a transaminase catalyzed reductive amination of a substituted tetralone and an alc. dehydrogenase catalyzed redn. of an α-ketoester to generate the two chiral centers in the mol., with nearly perfect stereoselectivity. The process also features simple isolation schemes, including a direct drop isolation of the aminotetralin phosphate salt.
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    99. 99
      Raker, J. R.; Holme, T. A. A Historical Analysis of the Curriculum of Organic Chemistry Using ACS Exams as Artifacts. J. Chem. Educ. 2013, 90, 1437– 1442,  DOI: 10.1021/ed400327b
      99
      A Historical Analysis of the Curriculum of Organic Chemistry Using ACS Exams as Artifacts
      Raker, Jeffrey R.; Holme, Thomas A.
      Journal of Chemical Education (2013), 90 (11), 1437-1442CODEN: JCEDA8; ISSN:0021-9584. (American Chemical Society and Division of Chemical Education, Inc.)
      Standardized examns., such as those developed and disseminated by the ACS Examns. Institute, are artifacts of the teaching of a course and over time may provide a historical perspective on how curricula have changed and evolved. This study investigated changes in org. chem. curricula across a 60-yr period by evaluating 18 ACS Org. Chem. Exams through the lenses of problem-type, visualization use, content covered, and percentile rankings. For all lenses, the early 1970s emerged as a focal point for change and stabilization of the org. chem. curricula.
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    100. 100
      Cooper, M. M.; Stowe, R. L.; Crandell, O. M.; Klymkowsky, M. W. Organic Chemistry, Life, the Universe and Everything (OCLUE): A Transformed Organic Chemistry Curriculum. J. Chem. Educ. 2019, 96, 1858– 1872,  DOI: 10.1021/acs.jchemed.9b00401
      100
      Organic Chemistry, Life, the Universe and Everything (OCLUE): A Transformed Organic Chemistry Curriculum
      Cooper, Melanie M.; Stowe, Ryan L.; Crandell, Olivia M.; Klymkowsky, Michael W.
      Journal of Chemical Education (2019), 96 (9), 1858-1872CODEN: JCEDA8; ISSN:0021-9584. (American Chemical Society and Division of Chemical Education, Inc.)
      The fundamental structure of a typical mainstream two-semester org. chem. course, populated mostly by life science majors and taught at universities throughout the United States, has changed little since the 1970s. However, much of the research on learning in org. chem. has been devoted to characterizing student difficulties of various types, and there is now persuasive evidence that org. chem. as currently taught is neither effective nor relevant for a majority of students. In an attempt to address the problems with traditional approaches to org. chem. instruction, we have developed an approach to the design of a transformed org. chem. course (Org. Chem., Life, the Universe and Everything or OCLUE) suitable for the vast majority of org. chem. students that includes (1) using the Framework of three-dimensional learning (3DL) to support knowledge in use and (2) emphasizing biol. important mechanisms. In this course, topics are connected to core ideas by using scientific practices, such as constructing models and explanations, analyzing and interpreting data, and emphasizing causal mechanistic reasoning. Here we discuss the theory and the decisions that went into the development of the course, including the compromises made and the rationales behind those choices. The outcome is a course that emphasizes causal mechanistic reasoning, has an increased focus on biol. prevalent reactions, and uses spectroscopy early and often to support evidence-based arguments about structure-property relationships. The materials we have developed are freely available to students and to potential users.
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    101. 101
      Raker, J.; Holme, T.; Murphy, K. The ACS Exams Institute Undergraduate Chemistry Anchoring Concepts Content Map II: Organic Chemistry. J. Chem. Educ. 2013, 90, 1443– 1445,  DOI: 10.1021/ed400175w
      101
      The ACS Exams Institute Undergraduate Chemistry Anchoring Concepts Content Map II: Organic Chemistry
      Raker, Jeffrey; Holme, Thomas; Murphy, Kristen
      Journal of Chemical Education (2013), 90 (11), 1443-1445CODEN: JCEDA8; ISSN:0021-9584. (American Chemical Society and Division of Chemical Education, Inc.)
      As a way to assist chem. departments with programmatic assessment of undergraduate chem. curricula, the ACS Examns. Institute is devising a map of the content taught throughout the undergraduate curriculum. The structure of the map is hierarchal, with large grain size at the top and more content detail as one moves "down" the levels of the map, of which there are four levels total. This paper presents these four levels of the map with ref. to second-year, org. chem.
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    102. 102
      Brummund, J.; Sonke, T.; Müller, M. Process Development for Biocatalytic Oxidations Applying Alcohol Dehydrogenases. Org. Process Res. Dev. 2015, 19, 1590– 1595,  DOI: 10.1021/op500307e
      102
      Process Development for Biocatalytic Oxidations Applying Alcohol Dehydrogenases
      Brummund, Jan; Sonke, Theo; Mueller, Monika
      Organic Process Research & Development (2015), 19 (11), 1590-1595CODEN: OPRDFK; ISSN:1083-6160. (American Chemical Society)
      Alc. dehydrogenases are able to catalyze the conversion of alcs. to aldehydes or ketones, simultaneously reducing the cofactor NAD+ or NADP+ to NAD(P)H. Because of the high costs of these pyridine cofactors, in situ cofactor regeneration is required for preparative applications in order to reach turnover nos. that are sufficient for economically viable processes. Here we present the development of a process for the enantioselective oxidn. of rac-1-phenylethanol to acetophenone, applying an alc. dehydrogenase coupled with an NAD(P)H oxidase for the enzymic cofactor regeneration, which is active towards NADH as well as NADPH. The reaction system was investigated in view of various influential parameters with main focus on the external oxygen supply. We could show that a gassed stirred tank reactor is a promising reactor concept to run NAD(P)H oxidase-coupled alc. dehydrogenase oxidns., including the possibility to scale-up the system.
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    103. 103
      Wong, C.-H.; Whitesides, G. M. Enzyme-catalyzed organic synthesis: NAD(P)H cofactor regeneration by using glucose-6-phosphate and the glucose-5-phosphate dehydrogenase from Leuconostoc mesenteroides. J. Am. Chem. Soc. 1981, 103, 4890– 4899,  DOI: 10.1021/ja00406a037
      103
      Enzyme-catalyzed organic synthesis: NAD(P)H cofactor regeneration by using glucose-6-phosphate and the glucose-5-phosphate dehydrogenase from Leuconostoc mesenteroides
      Wong, Chi-Huey; Whitesides, George M.
      Journal of the American Chemical Society (1981), 103 (16), 4890-9CODEN: JACSAT; ISSN:0002-7863.
      Glucose 6-phosphate dehydrogenase from L. mesenteroides and glucose 6-phosphate comprise a useful system for regeneration of reduced nicotinamide nucleotide cofactors for use in enzyme-catalyzed org. synthesis. This enzyme is approx. equally active in redn. of NAD and NADP and it is com. available, inexpensive, stable, and easily immobilized. Glucose 6-phosphate can be prepd. in quantity by hexokinase-catalyzed phosphorylation of glucose by ATP (coupled with ATP regeneration) or by other methods. The operation of this regeneration system is illustrated by syntheses of enantiomerically enriched D-lactic acid (0.4 mol, enantiomeric excess 95%) and (S)-benzyl-α-d1 alc. (0.4 mol, enantiomeric excess 95%), and by a synthesis of threo-Ds-(+)-isocitric acid (0.17 mol). Factors influencing the stability of NAD(P)(H) in soln. were explored.
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      https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaL3MXkslKns78%253D&md5=b8a95a5d9f39cf9f1a743e4a3d216646
    104. 104
      Johnston, M. R.; Makriyannis, A.; Whitten, K. M.; Drew, O. C.; Best, F. A. Biocatalyzed Regioselective Synthesis in Undergraduate Organic Laboratories: Multistep Synthesis of 2-Arachidonoylglycerol. J. Am. Chem. Soc. 2016, 93, 2080– 2083,  DOI: 10.1021/acs.jchemed.6b00225
      There is no corresponding record for this reference.
    105. 105
      Beers, M.; Archer, C.; Feske, B. D.; Mateer, S. C. Using biocatalysis to integrate organic chemistry into a molecular biology laboratory course. Biochem. Mol. Biol. Educ. 2012, 40, 130– 137,  DOI: 10.1002/bmb.20578
      105
      Using biocatalysis to integrate organic chemistry into a molecular biology laboratory course
      Beers, Mande; Archer, Crystal; Feske, Brent D.; Mateer, Scott C.
      Biochemistry and Molecular Biology Education (2012), 40 (2), 130-137CODEN: BMBECE; ISSN:1470-8175. (John Wiley & Sons, Inc.)
      Current cutting-edge biomedical investigation requires that the researcher have an operational understanding of several diverse disciplines. Biocatalysis is a field of science that operates at the crossroads of org. chem., biochem., microbiol., and mol. biol., and provides an excellent model for interdisciplinary research. We have developed an inquiry-based module that uses the mutagenesis of the yeast reductase, YDL124w, to study the bioorg. synthesis of the taxol side-chain, a pharmacol. important mol. Using related structures, students identify regions they think will affect enzyme stereoselective, design and generate site-specific mutants, and then characterize the effect of these changes on enzyme activity. This lab. activity gives our students experience, working in a scientific discipline outside of biol. and exposes them to techniques and equipment they do not normally work with in a mol. biol. course. These inter-disciplinary experiences not only show the relevance of other sciences to biol., but also give our students the ability to communicate more effectively with scientists outside their discipline.
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    106. 106
      Fronier, A. Not Voodoo X.4. http://www.chem.rochester.edu/notvoodoo/ (accessed 2023-02-01).
      There is no corresponding record for this reference.
    107. 107
      Chun, S. W.; Narayan, A. R. H. Biocatalytic, Stereoselective Deuteration of α-Amino Acids and Methyl Esters. ACS Catal. 2020, 10, 7413– 7418,  DOI: 10.1021/acscatal.0c01885
      107
      Biocatalytic, Stereoselective Deuteration of α-Amino Acids and Methyl Esters
      Chun, Stephanie W.; Narayan, Alison R. H.
      ACS Catalysis (2020), 10 (13), 7413-7418CODEN: ACCACS; ISSN:2155-5435. (American Chemical Society)
      α-2H Amino acids are valuable precursors toward labeled pharmaceutical agents and tools for studying biol. systems; however, these mols. are costly to purchase and challenging to synthesize in a site- and stereoselective manner. Here, we show that an α-oxoamine synthase that evolved for saxitoxin biosynthesis, SxtA AONS, is capable of producing a range of α-2H amino acids and esters site- and stereoselectively using D2O as the deuterium source. Addnl., we demonstrate the utility of this operationally simple reaction on preparative-scale in the stereoselective chemoenzymic synthesis of a deuterated analog of safinamide, a drug used to treat Parkinson's disease.
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    108. 108
      Rogova, T.; Gabriel, P.; Zavitsanou, S.; Leitch, J. A.; Duarte, F.; Dixon, D. J. Reverse Polarity Reductive Functionalization of Tertiary Amides via a Dual Iridium-Catalyzed Hydrosilylation and Single Electron Transfer Strategy. ACS Catal. 2020, 10, 11438– 11447,  DOI: 10.1021/acscatal.0c03089
      108
      Reverse Polarity Reductive Functionalization of Tertiary Amides via a Dual Iridium-Catalyzed Hydrosilylation and Single Electron Transfer Strategy
      Rogova, Tatiana; Gabriel, Pablo; Zavitsanou, Stamatia; Leitch, Jamie A.; Duarte, Fernanda; Dixon, Darren J.
      ACS Catalysis (2020), 10 (19), 11438-11447CODEN: ACCACS; ISSN:2155-5435. (American Chemical Society)
      A strategy for the mild generation of synthetically valuable α-amino radicals from robust tertiary amide building blocks has been developed. By combining Vaska's complex-catalyzed tertiary amide reductive activation and photochem. single electron redn. into a streamlined tandem process, metastable hemiaminal intermediates were successfully transformed into nucleophilic α-amino free radical species. This umpolung approach to such reactive intermediates was exemplified through coupling with an electrophilic dehydroalanine acceptor, resulting in the synthesis of an array of α-functionalized tertiary amine derivs., previously inaccessible from the amide starting materials. The utility of the strategy was expanded to include secondary amide substrates, intramol. variants, and late-stage functionalization of an active pharmaceutical ingredient. D. functional theory analyses were used to establish the reaction mechanism and elements of the chem. system that were responsible for the reaction's efficiency. Safety: CO gas alarm must be worn when prepg. precursor to Vaska-II complex.
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    109. 109
      DeHovitz, J. S.; Loh, Y. Y.; Kautzky, J. A.; Nagao, K.; Meichan, A. J.; Yamauchi, M.; MacMillan, D. W. C.; Hyster, T. K. Static to inducibly dynamic stereocontrol: The convergent use of racemic β-substituted ketones. Science 2020, 369, 1113– 1118,  DOI: 10.1126/science.abc9909
      109
      Static to inducibly dynamic stereocontrol: The convergent use of racemic β-substituted ketones
      DeHovitz, Jacob S.; Loh, Yong Yao; Kautzky, Jacob A.; Nagao, Kazunori; Meichan, Andrew J.; Yamauchi, Motoshi; MacMillan, David W. C.; Hyster, Todd K.
      Science (Washington, DC, United States) (2020), 369 (6507), 1113-1118CODEN: SCIEAS; ISSN:1095-9203. (American Association for the Advancement of Science)
      The synthesis of stereochem. complex mols. in the pharmaceutical and agrochem. industries requires precise control over each distinct stereocenter, a feat that can be challenging and time consuming using traditional asym. synthesis. Although stereoconvergent processes have the potential to streamline and simplify synthetic routes, they are currently limited by a narrow scope of inducibly dynamic stereocenters that can be readily epimerized. Here, we report the use of photoredox catalysis to enable the racemization of traditionally static, unreactive stereocenters through the intermediacy of prochiral radical species. This technol. was applied in conjunction with biocatalysts such as ketoreductases and aminotransferases to realize stereoconvergent syntheses of stereodefined β-substituted alcs. and amines from β-substituted ketones.
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    110. 110
      Key, H. M.; Clark, D. S.; Hartwig, J. F. Generation, Characterization, and Tunable Reactivity of Organometallic Fragments Bound to a Protein Ligand. J. Am. Chem. Soc. 2015, 137, 8261– 8268,  DOI: 10.1021/jacs.5b04431
      110
      Generation, Characterization, and Tunable Reactivity of Organometallic Fragments Bound to a Protein Ligand
      Key, Hanna M.; Clark, Douglas S.; Hartwig, John F.
      Journal of the American Chemical Society (2015), 137 (25), 8261-8268CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)
      Organotransition metal complexes catalyze important synthetic transformations, and the development of these systems has rested on the detailed understanding of the structures and elementary reactions of discrete organometallic complexes bound to org. ligands. One strategy for the creation of new organometallic systems is to exploit the intricate and highly structured ligands found in natural metalloproteins. The authors report the prepn. and characterization of discrete rhodium and iridium fragments bound site-specifically in a κ2-fashion to the protein carbonic anhydrase (CA) as a ligand. The reactions of apo human carbonic anhydrase with [Rh(nbd)2]BF4 or [M(CO)2(acac)] (M = Rh, Ir) form proteins contg. Rh or Ir with organometallic ligands. A colorimetric assay was developed to quantify rapidly the metal occupancy at the native metal-binding site, and 15N-1H NMR spectroscopy was used to establish the amino acids to which the metal is bound. IR spectroscopy and EXAFS revealed the presence and no. of carbonyl ligands and the no. total ligands, while UV-vis spectroscopy provided a signature to readily identify species that had been fully characterized. Exploiting these methods, the authors obsd. fundamental stoichiometric reactions of the artificial organometallic site of this protein, including reactions that simultaneously form and cleave metal-carbon bonds. The authors found that the discrete organometallic protein complexes, Rh(cod)-CA, Rh(nbd)-CA and Rh(CO)2-CA do not catalyze the hydrogenation or hydroformylation of a range of potential substrates of these reactions. These findings suggest that the active catalyst of the previously reported systems was not a Rh center ligated at the native Zn site of CA; instead, it is more likely that these reactions are catalyzed by a dissocd. Rh fragment or fragment assocd. with a different site on the protein.
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    111. 111
      Huang, J.; Liu, Z.; Bloomer, B. J.; Clark, D. S.; Mukhopadhyay, A.; Keasling, J. D.; Hartwig, J. F. Unnatural biosynthesis by an engineered microorganism with heterologously expressed natural enzymes and an artificial metalloenzyme. Nat. Chem. 2021, 13, 1186– 1191,  DOI: 10.1038/s41557-021-00801-3
      111
      Unnatural biosynthesis by an engineered microorganism with heterologously expressed natural enzymes and an artificial metalloenzyme
      Huang, Jing; Liu, Zhennan; Bloomer, Brandon J.; Clark, Douglas S.; Mukhopadhyay, Aindrila; Keasling, Jay D.; Hartwig, John F.
      Nature Chemistry (2021), 13 (12), 1186-1191CODEN: NCAHBB; ISSN:1755-4330. (Nature Portfolio)
      Synthetic biol. enables microbial hosts to produce complex mols. from organisms that are rare or difficult to cultivate, but the structures of these mols. are limited to those formed by reactions of natural enzymes. The integration of artificial metalloenzymes (ArMs) that catalyze unnatural reactions into metabolic networks could broaden the cache of mols. produced biosynthetically. Here we report an engineered microbial cell expressing a heterologous biosynthetic pathway, contg. both natural enzymes and ArMs, that produces an unnatural product with high diastereoselectivity. We engineered Escherichia coli with a heterologous terpene biosynthetic pathway and an ArM contg. an iridium-porphyrin complex that was transported into the cell with a heterologous transport system. We improved the diastereoselectivity and product titer of the unnatural product by evolving the ArM and selecting the appropriate gene induction and cultivation conditions. This work shows that synthetic biol. and synthetic chem. can produce, by combining natural and artificial enzymes in whole cells, mols. that were previously inaccessible to nature.
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    112. 112
      Gu, Y.; Natoli, S. N.; Liu, Z.; Clark, D. S.; Hartwig, J. F. Site-Selective Functionalization of (sp3)C–H Bonds Catalyzed by Artificial Metalloenzymes Containing an Iridium-Porphyrin Cofactor. Angew. Chem., Int. Ed. 2019, 58, 13954– 13960,  DOI: 10.1002/anie.201907460
      112
      Site-Selective Functionalization of (sp3)C-H Bonds Catalyzed by Artificial Metalloenzymes Containing an Iridium-Porphyrin Cofactor
      Gu, Yang; Natoli, Sean N.; Liu, Zhennan; Clark, Douglas S.; Hartwig, John F.
      Angewandte Chemie, International Edition (2019), 58 (39), 13954-13960CODEN: ACIEF5; ISSN:1433-7851. (Wiley-VCH Verlag GmbH & Co. KGaA)
      The selective functionalization of one C-H bond over others in nearly identical steric and electronic environments can facilitate the construction of complex mols. We report site-selective functionalizations of C-H bonds, differentiated solely by remote substituents, catalyzed by artificial metalloenzymes (ArMs) that are generated from the combination of an evolvable P 450 scaffold and an iridium-porphyrin cofactor. The generated systems catalyze the insertion of carbenes into the C-H bonds of a range of phthalan derivs. contg. substituents that render the two methylene positions in each phthalan inequivalent. These reactions occur with site-selectivity ratios of up to 17.8:1 and, in most cases, with pairs of enzyme mutants that preferentially form each of the two constitutional isomers. This study demonstrates the potential of abiotic reactions catalyzed by metalloenzymes to functionalize C-H bonds with site selectivity that is difficult to achieve with small-mol. catalysts.
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