This publication is Open Access under the license indicated. Learn More
Letter
Core Catalysis of the Reductive Glycine Pathway Demonstrated in YeastClick to copy article linkArticle link copied!
- Jorge Gonzalez de la CruzJorge Gonzalez de la CruzMax Planck Institute of Molecular Plant Physiology, Am Mühlenberg 1, 14476 Potsdam, GermanyMore by Jorge Gonzalez de la Cruz
- Fabian MachensFabian MachensDepartment Molecular Biology, University of Potsdam, Karl-Liebknecht-Str. 24/25, 14476 Potsdam, GermanyMore by Fabian Machens
- Katrin MesserschmidtKatrin MesserschmidtUniversity of Potsdam, Cell2Fab Research Unit, Karl-Liebknecht-Str. 24/25, 14476 Potsdam, GermanyMore by Katrin Messerschmidt
- Arren Bar-Even*Arren Bar-Even*Phone: +49-331-567-8910. E-mail: [email protected]Max Planck Institute of Molecular Plant Physiology, Am Mühlenberg 1, 14476 Potsdam, GermanyMore by Arren Bar-Even
Abstract
Click to copy section linkSection link copied!
One-carbon (C1) compounds are attractive microbial feedstocks as they can be efficiently produced from widely available resources. Formate, in particular, represents a promising growth substrate, as it can be generated from electrochemical reduction of CO2 and fed to microorganisms in a soluble form. We previously identified the synthetic reductive glycine pathway as the most efficient route for aerobic growth on formate. We further demonstrated pathway activity in Escherichia coli after expression of both native and foreign genes. Here, we explore whether the reductive glycine pathway could be established in a model microorganism using only native enzymes. We used the yeast Saccharomyces cerevisiae as host and show that overexpression of only endogenous enzymes enables glycine biosynthesis from formate and CO2 in a strain that is otherwise auxotrophic for glycine. We find the pathway to be highly active in this host, where 0.125 mM formate is sufficient to support growth. Notably, the formate-dependent growth rate of the engineered S. cerevisiae strain remained roughly constant over a very wide range of formate concentrations, 1–500 mM, indicating both high affinity for formate use and high tolerance toward elevated concentration of this C1 feedstock. Our results, as well the availability of endogenous NAD-dependent formate dehydrogenase, indicate that yeast might be an especially suitable host for engineering growth on formate.
Copyright © 2019 American Chemical Society
Reduced one-carbon (C1) compounds are abundant in natural habitats (e.g., methanol in phyllosphere, the aerial parts of plants (1)) and prevalent as byproducts of industrial processes (e.g., carbon monoxide in the flue gas of the steel industry (2)). As C1 compounds can also be produced abiotically in an efficient and cost-effective manner—for example, formate from electrochemical reduction of CO2 (3,4)—they could potentially serve as ideal feedstocks for sustainable microbial growth and bioproduction, (5−7) alleviating the problems associated with sugar feedstocks, the use of which erodes food security and biodiversity. (8)
Yet, biological assimilation of C1 compounds is limited to a small number of metabolic pathways and specialized microbial lineages. (9−11) Synthetic biology can prove useful by offering tailor-made solutions that can surpass natural alternatives. (12) In previous studies, we put forward the reductive glycine pathway as the most efficient route for aerobic growth on formate. (7,11,13) In this pathway, formate is first attached to tetrahydrofolate (THF)—the universal C1 carrier—and then reduced to methylene-THF. The glycine cleavage/synthase system (GCS) then condenses the C1-moiety of methylene-THF with CO2 and ammonia to give glycine. Glycine can be further metabolized to biomass and chemical products, e.g., by further condensation with the C1-moiety of methylene-THF to give serine that is deaminated to pyruvate. (11)
Only a small group of anaerobic purine- and amino-acid-degrading microbes are thought to produce glycine from one carbon units. (14,15) In a recently published paper, we demonstrated that the reductive activities of the THF enzymes and GCS can support the net biosynthesis of C2 and C3 compounds from formate and CO2 in E. coli. (16) Yet, as E. coli does not harbor an NAD-dependent formate dehydrogenase (FDH)—which is vital for using formate to supply the cell with reducing power and energy—it might not be an ideal host. Furthermore, the activity of the reductive glycine pathway in Escherichia coli was possible only via overexpression of foreign enzymes (from Methylobacterium extorquens). As the enzymatic components of the reductive glycine pathway are prevalent throughout the tree of life, we wondered whether the pathway could be established using only endogenous enzymes of a model host microbe that also naturally harbors NAD-dependent FDH. This would support the premise that C1 assimilation via the reductive glycine pathway could be a “latent” metabolic capability shared by multiple microorganisms, which could be induced by overexpression of naturally occurring components.
We decided to focus on the model yeast Saccharomyces cerevisiae since it endogenously harbors NAD-dependent FDH as well as all the enzymatic components of the reductive glycine pathway. Furthermore, the GCS of yeast was previously demonstrated to be reversible, such that feeding with 13C-formate resulted in detection of labeled glycine. (17,18) However, net production of glycine from formate and CO2 (Figure 1A)—as to indicate the possibility to support growth on C1 compounds—was never demonstrated in any eukaryotic organism. Here, we show the biosynthesis of glycine in a eukaryotic host solelyvia the reductive glycine pathway upon overexpression of native enzymes. We further demonstrate that yeast can sustain a constant growth rate across almost 3 orders of magnitude of formate concentrations, making it an especially promising host to support the assimilation of this key C1 compound.
Figure 1
Figure 1. Reductive glycine pathway and a selection scheme for its activity in yeast. (A) The “metabolic engine” of the reductive glycine pathway: condensation of C1-moieties into the C2 compound glycine. Substructure of tetrahydrofolate (THF) is shown in brown. Lipoic acid attached to the H-protein of the glycine cleavage/synthase system (GCS) is shown in green. (B) Gene deletions (marked in red) required for the construction of a glycine auxotroph strain, which we used to select for glycine biosynthesis from the activity of the reductive glycine pathway; pathway enzymes are shown in green.
Results
Click to copy section linkSection link copied!
We started with a glycine auxotroph strain—schematically shown in Figure 1B—deleted in the mitochondrial and cytosolic isozymes of serine hydroxymethyltransferase (ΔSHM1 ΔSHM2), as well as in threonine aldolase (ΔGLY1) and alanine:glyoxylate aminotransferase (ΔAGX1). (19) This metabolic background was used to select for the biosynthesis of glycine from formate and CO2. We cultivated the strain under high concentrations of formate (100 mM), CO2 (10%), and ammonia (100 mM), in order to kinetically and thermodynamically push the mitochondrial MIS1 enzyme (trifunctional formyl-THF synthetase, methenyl-THF cyclohydrolase, and methylene-THF dehydrogenase (20)) and the GCS in the reductive direction. Still, we were unable to establish growth without adding glycine to the medium. This indicated that the endogenous activities of MIS1, the GCS, or both are too low to support the required flux.
Next, we used the recently developed AssemblX method (21) to construct plasmids overexpressing the native MIS1 gene (pJGC1), the genes of the GCS (pJGC2), or both (pJGC3). As shown in Figure 2, each gene was regulated by a (different) strong constitutive yeast promoter to ensure high expression levels. These plasmids were transformed into the glycine auxotroph strain. The transformed strains were then cultivated in the presence of formate and high CO2. Growth of the strains harboring pJGC1 or pJGC2 was not observed without glycine supplement, regardless of the concentrations of formate and CO2. However, the strain harboring pJGC3—expressing both MIS1 and the genes of the GCS—was able to grow with formate substituting for glycine in the medium. This growth was dependent on elevated CO2 concentration (10% CO2) that is needed both thermodynamically, pushing the reversible GCS in the reductive direction, and kinetically, due to the relatively low affinity toward inorganic carbon. (22,23)
Figure 2
Figure 2. Three plasmids harboring genes encoding for different subsets of the enzymes of the reductive glycine pathway. pJGC1 harbors only the gene that encodes for MIS1, a trifunctional enzyme that converts formate to methylene-THF. pJGC2 harbors the genes encoding for the subunits of the GCS (the gene encoding for dihydrolipoamide dehydrogenase, LPD1, was not overexpressed since we reasoned its native expression would suffice as it participates in other complexes in the mitochondria, i.e., pyruvate dehydrogenase and 2-ketoglutarate dehydrogenase). pJGC3 harbors the genes encoding for MIS1 and the enzymes of the GCS. Each gene was regulated by a different strong, constitutive promoter as shown in the figure. Each plasmid was based on the pL1A-lc vector backbone as explained in the Methods section.
As shown in Figure 3A and B, formate concentrations below 1 mM sufficed to support growth of the glycine auxotroph strain. Maximal growth rate (or close to it) was observed with 1 mM formate and remained nearly constant up to 500 mM formate. At 750 mM formate, growth was severally inhibited, and at 1000 mM formate, no growth was observed. As formate is added as sodium salt, inhibition at concentrations above 500 mM might be attributed to the accumulation of sodium ions. However, while we did observe growth inhibition with NaCl concentrations above 500 mM, the growth inhibition associated with >500 mM sodium formate was considerably more severe. This indicates that at these high concentrations, formate becomes toxic to yeast.
Figure 3
Figure 3. Formate-dependent growth. (A) Growth of the glycine auxotroph strain harboring the pJGC3 plasmid using different concentrations of formate, 2% glucose and 10% CO2. “No OE” refers to the negative control, i.e., a glycine auxotroph strain without a plasmid, while “No OE + glycine” refers to the positive control, i.e., a glycine auxotroph strain without a plasmid where glycine was added to the medium. Each curve represents the average of three replicates, which were not different by more than 10%. Growth curves were cut after reaching stationary phase. (B) Calculated growth rate as a function of formate concentration. Growth rate increases with increasing formate concentration up to 1 mM, remains rather stable up to 500 mM, and then sharply decreases with higher concentrations. .
To confirm that glycine is indeed produced solely via the reductive activity of MIS1 and the GCS, we conducted several carbon labeling experiments providing (i) 13C-formate and unlabeled CO2, (ii) 13C-CO2 and unlabeled formate, or (iii) 13C-formate and 13C-CO2. As shown in Figure 4, the results match the expected labeling:
Figure 4
Figure 4. 13C-labeling experiments confirm glycine production from formate. Fraction of labeling of different amino acids in different strains and labeled feedstocks is shown. “G” corresponds to glycine, “S” to serine, “A” to alanine, “M” to methionine, and “T” to threonine. Complete labeling of glycine in the glycine auxotroph strain harboring pJGC3 upon feeding with 13C-formate confirms that glycine biosynthesis occurs only via the reductive glycine pathway. Partial labeling of glycine with 13C-CO2 is attributed to the high production rate of unlabeled CO2 in the mitochondria. See main text for a detailed discussion on the labeling pattern of these amino acids.
Threonine was partially labeled when 13C-CO2 was used, as it is derived from carbon-fixing anaplerosis. The structure of methionine corresponds to that of threonine with the addition of a carbon that originates from methyl-THF. The difference between the labeling of methionine and threonine thus represents the labeling of cytoplasmatic C1 units carried by THF. As shown by the labeling pattern observed upon feeding a WT strain with 13C-formate, this C1 moiety is only partially derived from formate, where the rest originates from serine cleavage. On the other hand, in the glycine auxotroph strain, in which serine hydroxymethyltransferase (SHM1, SHM2) is deleted, all cytoplasmic C1 units originate from formate.
Upon feeding with 13C-formate, alomst all glycine was singly labeled in the glycine auxotroph strain expressing MIS1 and genes of the GCS. This confirms the activity of the reductive glycine pathway where glycine is derived from formate. When feeding with 13C-CO2, glycine was only partially labeled, which can be attributed to the high production rate of unlabeled CO2 by mitochondrial pyruvate oxidation as well as acetyl-CoA oxidation via the TCA cycle. Serine was partially labeled in the WT strain upon feeding with 13C-formate, indicating substantial reductive flux of formate toward methylene-THF and the beta-carbon of serine. This labeling was obviously absent in the glycine auxotroph strain in which serine hydroxymethyltransferase is deleted. As a control, we confirmed that alanine was always unlabeled.
Discussion
Click to copy section linkSection link copied!
The results presented here confirm that the “metabolic engine” of the reductive glycine pathway—net production of the C2 compound glycine from the C1 moieties formate and CO2—can be established within a model microbe using only native enzymes. While this activity was made possible only via overexpression of the necessary endogenous genes (using strong endogenous promoters), once established, it was able to support formate utilization with high affinity, as indicated by the fact that 0.125 mM formate sufficed to support growth. Interestingly, growth rate showed little change with formate concentration varying between 1 and 500 mM. This suggests that, beyond the high efficiency of the reductive glycine pathway, yeast is highly tolerant to formate, a compound that is known to inhibit the growth of other microorganisms at a much lower concentration. (24,25) Specifically, many bacteria show severe growth impairment at formate concentrations higher than 100 mM. (26) Yeast high tolerance toward formate is in line with previous reports that formate can serve as an auxiliary substrate enhancing growth by providing further reducing power via the endogenous activity of FDH. (27,28)
Since yeast, as well as many other microorganisms, harbors all the enzymes of the reductive glycine pathway, it is tempting to ask why it cannot support net glycine biosynthesis from formate without the need for gene overexpression. One possible answer is that formate—while being a metabolic intermediate transferred between organelles in eukaryotic organisms (29)—is not a common compound found in the native habitat of this microorganism. Hence, cells were not adapted to incorporate it efficiently. Another barrier relates to the high concentration of CO2 required to thermodynamically and kinetically support pathway activity—a condition that might not be frequently met in the relevant natural environment.
Luckily, sustaining high CO2 concentration is quite straightforward within a biotechnological context, as is the case in multiple fermentation processes, for example, autotrophic cultivation of acetogens. (9) Moreover, in the ultimate yeast strain growing on formate, the oxidation of this compound to CO2 (to provide the cell with reducing power and energy) is expected to surpass CO2 assimilation. Hence, maintaining high CO2 concentration within the bioreactor would be rather straightforward and at most would require the recycling of CO2 from the bioreactor outflow.
In the current study, the dependence of cellular growth on formate is rather low, where only the biosynthesis of glycine and the cellular C1-units requires this C1 feedstock. Confirming this, we did not observe any significant decrease in the concentration of formate in the medium when cultivating our strain for 30 h and up to an OD ∼ 2 (with a starting concentration of 10 mM, see Methods). We speculate, however, that once formate will become a sole carbon source for growth, its consumption rate will become significant.
To conclude, we demonstrate the net production of glycine in a eukaryotic organism. Our findings suggest that S. cerevisiae can become an ideal host for the reductive glycine pathway as it harbors a highly efficient NAD-dependent FDH, requires overexpression of only endogenous enzymes, and supports a rather constant growth rate across ∼3 orders of magnitude of formate concentration. It remains for future studies to engineer the downstream assimilation of glycine to biomass, presumably also via native enzymes, e.g., serine hydroxymethyltransferase and serine deaminase. Beyond yeast, this study suggests that the activity of the reductive glycine pathway might be a “latent” metabolic trait in many microorganisms that endogenously harbor all pathway components, requiring only change in gene expression to support formate assimilation. A recent study that indicates the endogenous activity of the pathway supports this premise. (30)
Methods
Click to copy section linkSection link copied!
Reagents
PCR reactions were done with PrimeSTAR GXL polymerase (BD Clontech GmbH, Heidelberg, Germany) or Phusion High-Fidelity polymerase (Thermo Fisher Scientific GmbH, Dreieich, Germany), following the manufacturer’s recommendations. All primers were synthesized by Eurofins Genomics GmbH (Ebersberg, Germany). All media and media supplements were ordered from Sigma-Aldrich Chemie GmbH (Munich, Germany). Glucose was ordered from Carl Roth GmbH + Co. KG (Karlsruhe, Germany).
Yeast Strains, Media, and Cultivation
The following Saccharomyces cerevisiae strains were used: YUW1 (MATa ura3-1 trp-1 ade2-1 his3-11-15 leu2-3-112 can1-100 shm1::HIS3 shm2::LEU2 glyΔ0 AGX1::KanMX4), (19) and BY4741 (MATa his3Δ1 leu2Δ0 met15Δ0 ura3Δ0). (31) The BY4741 strain was used for in vivo assembly of Level 0 constructs, (21) and the glycine auxotroph strain YUW1 was used for in vivo assembly of the final Level 1 multigene plasmids (21) and as genetic background for all growth experiments described in the main text.
We used a “semirich” synthetic complete (SC) medium (2% w/v glucose, 0.67% w/v yeast nitrogen base without amino acids, and 0.14% w/v of the appropriate amino acid drop-out mix) to select yeast strains harboring the multigene construct after transformation. Synthetic minimal (SM) medium (2% glucose, 0.67% yeast nitrogen base without amino acids and without ammonium sulfate, 100 mM ammonium sulfate, and 0–1000 mM sodium formate) supplemented with tryptophan and adenine (each 0.0076% w/v) was used to test the YUW1 strain carrying plasmid pJGC1, pJGC2, or pJGC3, for its ability to synthesize glycine from formate and CO2. Additional glycine and/or uracil (0.0076% w/v each) were added to test growth of the YUW1 parental strain, not harboring any plasmid. YPAD medium (2% w/v peptone, 1% w/v yeast extract, 2% w/v glucose, and 0.004% w/v of adenine hemisulfate) was used for yeast recovery during the transformation procedure and for propagation of yeast strains requiring no selection, e.g., plasmid free YUW1 and BY4741.
Yeast liquid cultures were cultivated under shaking at 220 rpm and 30 °C. Different CO2 concentrations (atmospheric or 10%) were used as indicated along with each experiment. Agar plates were prepared using liquid media supplemented with 2% w/v agar and incubated at 30 °C at the indicated CO2 concentration.
Plasmid and Genomic DNA Extraction from Yeast
For PCR amplification of yeast genes, genomic DNA was extracted using the SDS/lithium acetate method. (32) In brief, a small amount of a colony was transferred into an SDS/LiAc solution (1% w/v SDS, 200 mM LiAc), incubated for 15 min at 70 °C, and pelleted by centrifugation (21 000g, 2 min). The pellet was subsequently washed with 70% ethanol, dried and resuspended in 10 μL TE buffer. Plasmids from yeast colonies were extracted with the ChargeSwitch Plasmid Yeast Mini Kit (Thermo Fisher Scientific GmbH).
Growth Conditions and Determination of Growth Rate
Growth experiments were performed using a TECAN SPARK 10 M plate reader (Tecan Deutschland GmbH, Crailsheim, Germany) at 30 °C and different CO2 concentrations (atmospheric or 10%, as indicated in the main text). A cycle with 12 individual 60 s shaking steps was programmed with the steps alternating between linear and orbital shaking (2 mm amplitude). To determine the growth rate of yeast cultures, their optical density (OD) at 600 nm was measured immediately after each shaking cycle throughout the complete growth experiment. Growth rate and doubling time were calculated using a custom MATLAB script. Raw data from the plate reader were calibrated to cuvette values according to ODcuvette = ODplate × 3.3. Growth curves were plotted in MATLAB and represent averages of triplicate measurements; in all cases, variability between triplicate measurements was less than 5%.
Yeast Transformation
For plasmid transformation, yeast cells were transformed using the lithium acetate/single-stranded carrier (LiAc/SS) method as described in ref (33). We used 100 ng of each DNA fragment or plasmid to be transformed. For strain YUW1, the cells were heat-shocked at 42 °C for 30 min, recovered in YPDA medium for 4 h at 30 °C, and then plated on appropriate selective SC medium. BY4741 cells were heat-shocked for 40 min at 42 °C and directly plated on appropriate selective SC media without recovery step.
Plasmid Construction
In order to create the different multigene expression plasmids for the enzymes involved in the reductive glycine pathway, we used the AssemblX cloning toolkit, which offers a modular way to create multigene plasmids using a level-based strategy. (21)
To generate Level 0 constructs (see Supplementary Table S1), all necessary promoters and terminators were PCR-amplified from the AssemblX promoter library, while all CDS that participate in the pathway (GCV1–3, LPD1, and MIS1) were amplified directly from the BY4741 yeast genome. All primers used were designed with the AssemblX webtool or the J5 software (34) and contained additional 5′ sequences allowing for homology-directed assemblies. For the list of primers see Supplementary Table S2. For in vivo assembly in yeast BY4741, purified PCR fragments (100 ng per fragment) were mixed with appropriate Level 0 backbone plasmid (linearized with HindIII) according to the assembly protocol—generated by the webtools mentioned above—and transformed into yeast.
Transformants were selected on solid SC medium without uracil and analyzed by colony PCR. Plasmids from potential positive colonies were extracted from yeast with the ChargeSwitch Plasmid Yeast Mini Kit (Thermo Fisher Scientific GmbH), retransformed into E. coli, isolated, and sent for sequencing.
For construction of the final multigene Level 1 plasmids, the Level 0 modules created above were released from their backbones by restriction digestion, according to the AssemblX protocol. During this process, proprietary homology regions, present in the Level 0 backbones, are released along with the previously assembled Level 0 module. These regions overlap between neighboring Level 0 modules and thus allow ordered assembly by in vivo recombination. Following gel purification all Level 0 modules belonging to one intended multigene construct were mixed together with the linearized Level 1 backbone pL1A-lc and transformed directly into yeast YUW1 to allow in vivo assembly.
Selection for successfully assembled plasmids was done on solid SC medium without uracil. Verification of correctly assembled plasmids was done as described above, whereby only the junctions between individual assembly parts were sequenced.
Carbon Labeling
Cells were grown in 3 mL SM media supplemented with adenine, tryptophane and labeled or unlabeled formate (250 mM) in the presence of 10% labeled or unlabeled CO2. After reaching stationary phase, ∼109 cells were harvested by centrifugation for 1 min at 11 500g. The biomass was hydrolyzed by incubation with 1 mL 6 N hydrochloric acid for 24 h at 95 °C. The acid was then evaporated by continued heating at 95 °C and nitrogen streaming. Hydrolyzed amino acids were separated using ultraperformance liquid chromatography (Acquity UPLC, Waters GmbH, Eschborn, Germany) with a C18-reversed-phase column (Waters GmbH). Mass spectra were acquired using an Exactive mass spectrometer (Thermo Fisher Scientific GmbH). Data analysis was performed using Xcalibur software (Thermo Fisher Scientific GmbH). Prior to analysis, amino-acid standards (Sigma-Aldrich Chemie GmbH) were analyzed under the same conditions to determine typical retention times.
Determination of Formate Concentration in Media
The glycine auxotroph strain carrying the pJGC3 plasmid was inoculated, in duplicates, at an OD600 of 0.03 in synthetic minimal medium with 10 mM formate. A sample of the growth medium was taken from each duplicate every ∼2 h during a 34-h fermentation (reaching on OD600 of ∼2). Each sample was centrifuged twice and diluted 1:1000. 500 μL of each diluted sample run in high-performance anion- and cation-exchange chromatography with conductivity detection facilitated by a Dionex ICS-3000 system (Thermo Fisher Scientific GmbH, Dreieich, Germany) with the columns IonPac AS11 Analytical Column 2 × 250 mm (Dionex) and IonPac AG11 Guard Column 2 × 50 mm (Dionex).
Supporting Information
Click to copy section linkSection link copied!
The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acssynbio.8b00464.
Table S1: Genetic constructs used in this study; Table S2: DNA primers used in this study (PDF)
Terms & Conditions
Most electronic Supporting Information files are available without a subscription to ACS Web Editions. Such files may be downloaded by article for research use (if there is a public use license linked to the relevant article, that license may permit other uses). Permission may be obtained from ACS for other uses through requests via the RightsLink permission system: http://pubs.acs.org/page/copyright/permissions.html.
Author Information
Click to copy section linkSection link copied!
- Arren Bar-Even - Max Planck Institute of Molecular Plant Physiology, Am Mühlenberg 1, 14476 Potsdam, Germany;
http://orcid.org/0000-0002-1039-4328;
- Fabian Machens - Department Molecular Biology, University of Potsdam, Karl-Liebknecht-Str. 24/25, 14476 Potsdam, Germany;http://orcid.org/0000-0001-6039-3860
Acknowledgments
Click to copy section linkSection link copied!
The authors thank Charlie Cotton and Hai He for critical reading of the manuscript. This work was funded by the Max Planck Society.
References
Click to copy section linkSection link copied!
This article references 34 other publications.
- 1Vorholt, J. A. (2012) Microbial life in the phyllosphere. Nat. Rev. Microbiol. 10, 828– 840, DOI: 10.1038/nrmicro2910Google Scholar1https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC38Xhs1Ogs7jO&md5=04dcdc5f7e3ba05dda35863a5ea327bfMicrobial life in the phyllosphereVorholt, Julia A.Nature Reviews Microbiology (2012), 10 (12), 828-840CODEN: NRMACK; ISSN:1740-1526. (Nature Publishing Group)A review. Our knowledge of the microbiol. of the phyllosphere, or the aerial parts of plants, has historically lagged behind our knowledge of the microbiol. of the rhizosphere, or the below-ground habitat of plants, particularly with respect to fundamental questions such as which microorganisms are present and what they do there. In recent years, however, this has begun to change. Cultivation-independent studies have revealed that a few bacterial phyla predominate in the phyllosphere of different plants and that plant factors are involved in shaping these phyllosphere communities, which feature specific adaptations and exhibit multipartite relationships both with host plants and among community members. Insights into the underlying structural principles of indigenous microbial phyllosphere populations will help us to develop a deeper understanding of the phyllosphere microbiota and will have applications in the promotion of plant growth and plant protection.
- 2Abubackar, H. N., eiga, M. C., and Kennes, C. (2011) Biological conversion of carbon monoxide: rich syngas or waste gases to bioethanol. Biofuels, Bioprod. Biorefin. 5, 93– 114, DOI: 10.1002/bbb.256Google Scholar2https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3MXnvVaruw%253D%253D&md5=ca9c58a89bbb972d7c61e1b727ea1cbcBiological conversion of carbon monoxide: rich syngas or waste gases to bioethanolAbubackar, Haris Nalakath; Veiga, Maria C.; Kennes, ChristianBiofuels, Bioproducts & Biorefining (2011), 5 (1), 93-114CODEN: BBBICH; ISSN:1932-104X. (John Wiley & Sons Ltd.)A review. Bioconversion of syngas/waste gas components to produce ethanol appears to be a promising alternative compared to the existing chem. techniques. Recently, several lab.-scale studies have demonstrated the use of acetogens that have the ability to convert various syngas components (CO, CO2, and H2) to multicarbon compds., such as acetate, butyrate, butanol, lactate, and ethanol, in which ethanol is often produced as a minor end-product. This bioconversion process has several advantages, such as its high specificity, the fact that it does not require a highly specific H2/CO ratio, and that biocatalysts are less susceptible to metal poisoning. Furthermore, this process occurs under mild temp. and pressure and does not require any costly pre-treatment of the feed gas or costly metal catalysts, making the process superior over the conventional chem. catalytic conversion process. The main challenge faced for commercializing this technol. is the poor aq. soly. of the gaseous substrates (mainly CO and H2). In this paper, a crit. review of CO-rich gas fermn. to produce ethanol has been analyzed systematically and published results have been compared. Special emphasis has been given to understand the microbial aspects of the conversion process, by highlighting the role of different micro-organisms used, pathways, and parameters affecting the bioconversion. An anal. of the process fundamentals of various bioreactors used for the biol. conversion of CO-rich gases, mainly syngas to ethanol, has been made and reported in this paper. Various challenges faced by the syngas fermn. process for commercialization and future research requirements are also discussed.
- 3Kopljar, D., Inan, A., Vindayer, P., Wagner, N., and Klemm, E. (2014) Electrochemical reduction of CO2 to formate at high current density using gas diffusion electrodes. J. Appl. Electrochem. 44, 1107– 1116, DOI: 10.1007/s10800-014-0731-xGoogle Scholar3https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2cXhsVWkt7vF&md5=fb54c80ec5d782ad34f53b4e73d93d22Electrochemical reduction of CO2 to formate at high current density using gas diffusion electrodesKopljar, D.; Inan, A.; Vindayer, P.; Wagner, N.; Klemm, E.Journal of Applied Electrochemistry (2014), 44 (10), 1107-1116CODEN: JAELBJ; ISSN:0021-891X. (Springer)The electrochem. redn. of carbon dioxide into formate was studied using gas diffusion electrodes (GDE) with Sn as electrocatalyst in order to overcome mass transport limitations and to achieve high current densities. For this purpose, a dry pressing method was developed for GDE prepn. and optimized with respect to mech. stability and the performance in the redn. of CO2. Using this approach, GDEs can be obtained with a high reproducibility in a very simple, fast, and straightforward manner. The influence of the metal loading on c.d. and product distribution was investigated. Furthermore, the effect of changing the electrolyte pH was evaluated. Under optimized conditions, the GDE allowed current densities up to 200 mA cm-2 to be achieved with a Faradaic efficiency of around 90 % toward formate and a substantial suppression of hydrogen prodn. (<3 %) at ambient pressure. At higher current densities mass transport issues come into effect and hydrogen is increasingly produced. The corresponding cathode potential was found to be 1.57 V vs. SHE.
- 4Yang, H., Kaczur, J. J., Sajjad, S. D., and Masel, R. I. (2017) Electrochemical conversion of CO2 to formic acid utilizing Sustainion membranes. Journal of CO2 Utilization 20, 208– 217, DOI: 10.1016/j.jcou.2017.04.011Google Scholar4https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2sXpvFeltb4%253D&md5=2c2323d1e4095c98baabe3fe925b7e81Electrochemical conversion of CO2 to formic acid utilizing Sustainion membranesYang, Hongzhou; Kaczur, Jerry J.; Sajjad, Syed Dawar; Masel, Richard I.Journal of CO2 Utilization (2017), 20 (), 208-217CODEN: JCUOAJ; ISSN:2212-9839. (Elsevier Ltd.)Formic acid generated from CO2 has been proposed both as a key intermediate renewable chem. feedstock as well as a potential chem.-based energy storage media for hydrogen. In this paper, we describe a novel three-compartment electrochem. cell configuration with the capability of directly producing a pure formic acid product in the concn. range of 5-20 wt% at high current densities and Faradaic yields. The electrochem. cell employs a Dioxide Materials Sustainion anion exchange membrane and a nanoparticle Sn GDE cathode contg. an imidazole ionomer, allowing for improved CO2 electrochem. redn. performance. Stable electrochem. cell performance for more than 500 h was exptl. demonstrated at a c.d. of 140 mA cm-2 at a cell voltage of only 3.5 V. Future work will include cell scale-up and increasing cell Faradaic performance using selected electrocatalysts and membranes.
- 5Bennett, R. K., Steinberg, L. M., Chen, W., and Papoutsakis, E. T. (2018) Engineering the bioconversion of methane and methanol to fuels and chemicals in native and synthetic methylotrophs. Curr. Opin. Biotechnol. 50, 81– 93, DOI: 10.1016/j.copbio.2017.11.010Google Scholar5https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2sXhvVOrsLvL&md5=e54377475ba45693d57a2a4385b88372Engineering the bioconversion of methane and methanol to fuels and chemicals in native and synthetic methylotrophsBennett, R. Kyle; Steinberg, Lisa M.; Chen, Wilfred; Papoutsakis, Eleftherios T.Current Opinion in Biotechnology (2018), 50 (), 81-93CODEN: CUOBE3; ISSN:0958-1669. (Elsevier B.V.)Methylotrophy describes the ability of organisms to utilize reduced one-carbon compds., notably methane and methanol, as growth and energy sources. Abundant natural gas supplies, composed primarily of methane, have prompted interest in using these compds., which are more reduced than sugars, as substrates to improve product titers and yields of bioprocesses. Engineering native methylotophs or developing synthetic methylotrophs are emerging fields to convert methane and methanol into fuels and chems. under aerobic and anaerobic conditions. This review discusses recent progress made toward engineering native methanotrophs for aerobic and anaerobic methane utilization and synthetic methylotrophs for methanol utilization. Finally, strategies to overcome the limitations involved with synthetic methanol utilization, notably methanol dehydrogenase kinetics and ribulose 5-phosphate regeneration, are discussed.
- 6Bengelsdorf, F. R. and Durre, P. (2017) Gas fermentation for commodity chemicals and fuels. Microb. Biotechnol. 10, 1167– 1170, DOI: 10.1111/1751-7915.12763Google Scholar6https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A280%3ADC%252BC1cjmsVSksg%253D%253D&md5=19d3e29d23f4194e3806cfecece8cdbaGas fermentation for commodity chemicals and fuelsBengelsdorf Frank R; Durre PeterMicrobial biotechnology (2017), 10 (5), 1167-1170 ISSN:.Gas fermentation is a microbial process that contributes to at least four of the sustainable development goals (SDGs) of the United Nations. The process converts waste and greenhouse gases into commodity chemicals and fuels. Thus, world's climate is positively affected. Briefly, we describe the background of the process, some biocatalytic strains, and legal implications.
- 7Yishai, O., Lindner, S. N., Gonzalez de la Cruz, J., Tenenboim, H., and Bar-Even, A. (2016) The formate bio-economy. Curr. Opin. Chem. Biol. 35, 1– 9, DOI: 10.1016/j.cbpa.2016.07.005Google Scholar7https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC28Xht1CksL%252FL&md5=f265a337019047fe6d9233688a70a52bThe formate bio-economyYishai, Oren; Lindner, Steffen N.; Gonzalez de la Cruz, Jorge; Tenenboim, Hezi; Bar-Even, ArrenCurrent Opinion in Chemical Biology (2016), 35 (), 1-9CODEN: COCBF4; ISSN:1367-5931. (Elsevier B.V.)In this review we discuss the concept of the formate bio-economy: formate can be produced efficiently from various available resources and can be consumed by microbes as the sole carbon source for the prodn. of value-added chems., directly addressing major challenges in energy storage and chem. prodn. We show that the formate assimilation pathways utilized by natural formatotrophs are either inefficient or are constrained to organisms that are difficult to cultivate and engineer. Instead, adapting model industrial organisms to formatotrophic growth using synthetic, specially tailored formate-assimilation routes could prove an advantageous strategy. Several studies have started to tackle this challenge, but a fully active synthetic pathway has yet to be established, leaving room for future undertakings.
- 8Naik, S. N., Goud, V. V., Rout, P. K., and Dalai, A. K. (2010) Production of first and second generation biofuels: a comprehensive review. Renewable Sustainable Energy Rev. 14, 578– 597, DOI: 10.1016/j.rser.2009.10.003Google Scholar8https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD1MXhsF2isbvF&md5=5c93f3970ae56e2ee6b7a9763c1677beProduction of first and second generation biofuels: A comprehensive reviewNaik, S. N.; Goud, Vaibhav V.; Rout, Prasant K.; Dalai, Ajay K.Renewable & Sustainable Energy Reviews (2010), 14 (2), 578-597CODEN: RSERFH; ISSN:1364-0321. (Elsevier Ltd.)A review. Sustainable economic and industrial growth requires safe, sustainable resources of energy. For the future re-arrangement of a sustainable economy to biol. raw materials, completely new approaches in research and development, prodn., and economy are necessary. The first-generation' biofuels appear unsustainable because of the potential stress that their prodn. places on food commodities. For org. chems. and materials these needs to follow a biorefinery model under environmentally sustainable conditions. Where these operate at present, their product range is largely limited to simple materials (i.e. cellulose, ethanol, and biofuels). Second generation biorefineries need to build on the need for sustainable chem. products through modern and proven green chem. technologies such as bioprocessing including pyrolysis, Fisher Tropsch, and other catalytic processes in order to make more complex mols. and materials on which a future sustainable society will be based. This review focus on cost effective technologies and the processes to convert biomass into useful liq. biofuels and bioproducts, with particular focus on some biorefinery concepts based on different feedstocks aiming at the integral utilization of these feedstocks for the prodn. of value added chems.
- 9Drake, H. L., Küsel, K., and Matthies, C. (2013) Acetogenic prokaryotes, In The Prokaryotes, pp 3– 60, Springer, Berlin, Heidelberg.Google ScholarThere is no corresponding record for this reference.
- 10Anthony, C. (1982) The Biochemistry of Methylotrophs, Academic Press, London, New York.Google ScholarThere is no corresponding record for this reference.
- 11Bar-Even, A. (2016) Formate Assimilation: The Metabolic Architecture of Natural and Synthetic Pathways. Biochemistry 55, 3851– 3863, DOI: 10.1021/acs.biochem.6b00495Google Scholar11https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC28XhtVGjtLjJ&md5=e866cc5b94b857c1b8038b370c208db3Formate Assimilation: The Metabolic Architecture of Natural and Synthetic PathwaysBar-Even, ArrenBiochemistry (2016), 55 (28), 3851-3863CODEN: BICHAW; ISSN:0006-2960. (American Chemical Society)Formate may become an ideal mediator between the physicochem. and biol. realms, as it can be produced efficiently from multiple available sources, such as electricity and biomass, and serve as one of the simplest org. compds. for providing both carbon and energy to living cells. However, limiting the realization of formate as a microbial feedstock is the low diversity of formate-fixing enzymes and thereby the small no. of naturally occurring formate-assimilation pathways. Here, the natural enzymes and pathways supporting formate assimilation are presented and discussed together with proposed synthetic routes that could permit growth on formate via existing as well as novel formate-fixing reactions. By considering such synthetic routes, the diversity of metabolic solns. for formate assimilation can be expanded dramatically, such that different host organisms, cultivation conditions, and desired products could be matched with the most suitable pathway. Astute application of old and new formate-assimilation pathways may thus become a cornerstone in the development of sustainable strategies for microbial prodn. of value-added chems.
- 12Erb, T. J., Jones, P. R., and Bar-Even, A. (2017) Synthetic metabolism: metabolic engineering meets enzyme design. Curr. Opin. Chem. Biol. 37, 56– 62, DOI: 10.1016/j.cbpa.2016.12.023Google Scholar12https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2sXhvFyrsLs%253D&md5=325943c541fdc461f8d7f2548ee8fe7eSynthetic metabolism: metabolic engineering meets enzyme designErb, Tobias J.; Jones, Patrik R.; Bar-Even, ArrenCurrent Opinion in Chemical Biology (2017), 37 (), 56-62CODEN: COCBF4; ISSN:1367-5931. (Elsevier B.V.)Metabolic engineering aims at modifying the endogenous metabolic network of an organism to harness it for a useful biotechnol. task, for example, prodn. of a value-added compd. Several levels of metabolic engineering can be defined and are the topic of this review. Basic 'copy, paste and fine-tuning' approaches are limited to the structure of naturally existing pathways. 'Mix and match' approaches freely recombine the repertoire of existing enzymes to create synthetic metabolic networks that are able to outcompete naturally evolved pathways or redirect flux toward non-natural products. The space of possible metabolic soln. can be further increased through approaches including 'new enzyme reactions', which are engineered on the basis of known enzyme mechanisms. Finally, by considering completely 'novel enzyme chemistries' with de novo enzyme design, the limits of nature can be breached to derive the most advanced form of synthetic pathways. We discuss the challenges and promises assocd. with these different metabolic engineering approaches and illuminate how enzyme engineering is expected to take a prime role in synthetic metabolic engineering for biotechnol., chem. industry and agriculture of the future.
- 13Bar-Even, A., Noor, E., Flamholz, A., and Milo, R. (2013) Design and analysis of metabolic pathways supporting formatotrophic growth for electricity-dependent cultivation of microbes. Biochim. Biophys. Acta, Bioenerg. 1827, 1039– 1047, DOI: 10.1016/j.bbabio.2012.10.013Google Scholar13https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC38Xhs1KqsrjF&md5=9ae940dc5a31b77357bc6d44ac9fdccbDesign and analysis of metabolic pathways supporting formatotrophic growth for electricity-dependent cultivation of microbesBar-Even, Arren; Noor, Elad; Flamholz, Avi; Milo, RonBiochimica et Biophysica Acta, Bioenergetics (2013), 1827 (8-9), 1039-1047CODEN: BBBEB4; ISSN:0005-2728. (Elsevier B. V.)A review, with commentary. Electrosynthesis is a promising approach that enables the biol. prodn. of commodities, like fuels and fine chems., using renewably produced electricity. Several techniques have been proposed to mediate the transfer of electrons from the cathode to living cells. Of these, the electroprodn. of formate as a mediator seems esp. promising: formate is readily sol., of low toxicity and can be produced at relatively high efficiency and at reasonable c.d. While organisms that are capable of formatotrophic growth, i.e. growth on formate, exist naturally, they are generally less suitable for bulk cultivation and industrial needs. Hence, it may be helpful to engineer a model organism of industrial relevance, such as E. coli, for growth on formate. There are numerous metabolic pathways that can potentially support formatotrophic growth. Here we analyze these diverse pathways according to various criteria including biomass yield, thermodn. favorability, chem. motive force, kinetics and the practical challenges posed by their expression. We find that the reductive glycine pathway, composed of the tetrahydrofolate system, the glycine cleavage system, serine hydroxymethyltransferase and serine deaminase, is a promising candidate to support electrosynthesis in E. coli. The approach presented here exemplifies how combining different computational approaches into a systematic anal. methodol. provides assistance in redesigning metab. This article is part of a Special Issue entitled: Metals in Bioenergetics and Biomimetics Systems.
- 14Fuchs, G. (1986) CO2 fixation in acetogenic bacteria: variations on a theme. FEMS Microbiol. Lett. 39, 181– 213, DOI: 10.1111/j.1574-6968.1986.tb01859.xGoogle ScholarThere is no corresponding record for this reference.
- 15Schneeberger, A., Frings, J., and Schink, B. (1999) Net synthesis of acetate from CO2 by Eubacterium acidaminophilum through the glycine reductase pathway. FEMS Microbiol. Lett. 177, 1, DOI: 10.1111/j.1574-6968.1999.tb13705.xGoogle Scholar15https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaK1MXls1SmsLo%253D&md5=2bf6878a461fe7c70762757287d4145bNet synthesis of acetate from CO2 by Eubacterium acidaminophilum through the glycine reductase pathwaySchneeberger, Anne; Frings, Jochen; Schink, BernhardFEMS Microbiology Letters (1999), 177 (1), 1-6CODEN: FMLED7; ISSN:0378-1097. (Elsevier Science B.V.)Eubacterium acidaminophilum combines the oxidn. of amino acids such as alanine or valine with the redn. of glycine to acetate in a two-substrate fermn. (Stickland reaction). In the absence of glycine, dense cell suspensions oxidized alanine or valine only to a small extent, with limited prodn. of hydrogen and acetate. Expts. with 14C-labeled carbonate revealed that acetate was formed under these conditions by net redn. of CO2/HCO3-; 14C-labeled formate was formed as an intermediate. E. acidaminophilum did not grow with hydrogen plus CO2; dense cell suspensions under H2/CO2 produced only very small amts. (<0.5 mM) of acetate. There was no activity of carbon monoxide dehydrogenase, indicating that the glycine pathway was used for acetate synthesis. The results are explained on the basis of biochem. and energetic considerations.
- 16Yishai, O., Bouzon, M., Doring, V., and Bar-Even, A. (2018) In Vivo Assimilation of One-Carbon via a Synthetic Reductive Glycine Pathway in Escherichia coli. ACS Synth. Biol. 7, 2023, DOI: 10.1021/acssynbio.8b00131Google Scholar16https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC1cXpsVChs7s%253D&md5=5f9029d1c836fcf2c889c15b830dfd04In Vivo Assimilation of One-Carbon via a Synthetic Reductive Glycine Pathway in Escherichia coliYishai, Oren; Bouzon, Madeleine; Doering, Volker; Bar-Even, ArrenACS Synthetic Biology (2018), 7 (9), 2023-2028CODEN: ASBCD6; ISSN:2161-5063. (American Chemical Society)Assimilation of one-carbon compds. presents a key biochem. challenge that limits their use as sustainable feedstocks for microbial growth and prodn. The reductive glycine pathway is a synthetic metabolic route that could provide an optimal way for the aerobic assimilation of reduced C1 compds. Here, we show that a rational integration of native and foreign enzymes enables the tetrahydrofolate and glycine cleavage/synthase systems to operate in the reductive direction, such that Escherichia coli satisfies all of its glycine and serine requirements from the assimilation of formate and CO2. Importantly, the biosynthesis of serine from formate and CO2 does not lower the growth rate, indicating high flux that is able to provide 10% of cellular carbon. Our findings assert that the reductive glycine pathway could support highly efficient aerobic assimilation of C1-feedstocks.
- 17Pasternack, L. B., Laude, D. A., Jr., and Appling, D. R. (1992) 13C NMR detection of folate-mediated serine and glycine synthesis in vivo in Saccharomyces cerevisiae. Biochemistry 31, 8713– 8719, DOI: 10.1021/bi00152a005Google Scholar17https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaK38XlsVKru7s%253D&md5=d0d13ec236e34bf5bc874461c21b980aCarbon-13 NMR detection of folate-mediated serine and glycine synthesis in vivo in Saccharomyces cerevisiaePasternack, Laura B.; Laude, David A., Jr.; Appling, Dean R.Biochemistry (1992), 31 (37), 8713-19CODEN: BICHAW; ISSN:0006-2960.S. cerevisiae has both cytoplasmic and mitochondrial C1-tetrahydrofolate (THF) synthases. These trifunctional isoenzymes are central to C1 metab. and are responsible for interconversion of the THF derivs. in the resp. compartments. 13C-NMR was used to study folate-mediated C1 metab. in these 2 compartments, using glycine and serine synthesis as metabolic endpoints. The availability of yeast strains carrying deletions of cytoplasmic and/or mitochondrial C1-THF synthase allows a dissection of the role each compartment plays in this metab. When yeast are incubated with [13C]formate, 13C-NMR spectra establish that prodn. of [3-13C]serine is dependent on C1-THF synthase and occurs primarily in the cytosol. However, in a strain lacking cytoplasmic C1-THF synthase but possessing the mitochondrial isoenzyme, [13C]formate can be metabolized to [2-13C]glycine and [3-13C]serine. This provides in vivo evidence for the mitochondrial assimilation of formate, activation and conversion to [13C]CH2-THF via mitochondrial C1-THF synthase, and subsequent glycine synthesis via reversal of the glycine cleavage system. Addnl. supporting evidence of reversibility of the glycine cleavage system in vivo is the prodn. of [2-13C]glycine and [2,3-13C]serine in yeast strains grown with [3-13C]serine. This metab. is independent of C1-THF synthase, since these products were obsd. in strains lacking both the cytoplasmic and mitochondrial isoenzymes. These results suggest that when formate is the C1 donor, assimilation is primarily cytoplasmic, whereas when serine serves as C1 donor, considerable metab. occurs via mitochondrial pathways.
- 18Maaheimo, H., Fiaux, J., Cakar, Z. P., Bailey, J. E., Sauer, U., and Szyperski, T. (2001) Central carbon metabolism of Saccharomyces cerevisiae explored by biosynthetic fractional (13)C labeling of common amino acids. Eur. J. Biochem. 268, 2464– 2479, DOI: 10.1046/j.1432-1327.2001.02126.xGoogle Scholar18https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD3MXjt1Wlsrw%253D&md5=122ba33dbef44434483755f2bc398f23Central carbon metabolism of Saccharomyces cerevisiae explored by biosynthetic fractional 13C labeling of common amino acidsMaaheimo, Hannu; Fiaux, Jocelyne; Cakar, Z. Petek; Bailey, James E.; Sauer, Uwe; Szyperski, ThomasEuropean Journal of Biochemistry (2001), 268 (8), 2464-2479CODEN: EJBCAI; ISSN:0014-2956. (Blackwell Science Ltd.)Aerobic and anaerobic central metab. of Saccharomyces cerevisiae cells was explored in batch cultures on a minimal medium contg. glucose as the sole carbon source, using biosynthetic fractional 13C labeling of proteinogenic amino acids. This allowed, firstly, unravelling of the network of active central pathways in cytosol and mitochondria, secondly, detn. of flux ratios characterizing glycolysis, pentose phosphate cycle, tricarboxylic acid cycle and Cl-metab., and thirdly, assessment of intercompartmental transport fluxes of pyruvate, acetyl-CoA, oxaloacetate and glycine. The data also revealed that alanine aminotransferase is located in the mitochondria, and that amino acids are synthesized according to documented pathways. In both the aerobic and the anaerobic regime: (a) the mitochondrial glycine cleavage pathway is active, and efflux of glycine into the cytosol is obsd.; (b) the pentose phosphate pathways serve for biosynthesis only, i.e. phosphoenolpyruvate is entirely generated via glycolysis; (c) the majority of the cytosolic oxaloacetate is synthesized via anaplerotic carboxylation of pyruvate; (d) the malic enzyme plays a key role for mitochondrial pyruvate metab.; (e) the transfer of oxaloacetate from the cytosol to the mitochondria is largely unidirectional, and the activity of the malate-aspartate shuttle and the succinate-fumarate carrier is low; (e) a large fraction of the mitochondrial pyruvate is imported from the cytosol; and (f) the glyoxylate cycle is inactive. In the aerobic regime, 75% of mitochondrial oxaloacetate arises from anaplerotic carboxylation of pyruvate, while in the anaerobic regime, the tricarboxylic acid cycle is operating in a branched fashion to fulfill biosynthetic demands only. The present study shows that fractional 13C labeling of amino acids represents a powerful approach to study compartmented eukaryotic systems.
- 19Schlosser, T., Gatgens, C., Weber, U., and Stahmann, K. P. (2004) Alanine: glyoxylate aminotransferase of Saccharomyces cerevisiae-encoding gene AGX1 and metabolic significance. Yeast 21, 63– 73, DOI: 10.1002/yea.1058Google Scholar19https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A280%3ADC%252BD2c%252FksFWitw%253D%253D&md5=c8543750a284cef4e2ac7fd254c876c7Alanine : glyoxylate aminotransferase of Saccharomyces cerevisiae-encoding gene AGX1 and metabolic significanceSchlosser Thomas; Gatgens Cornelia; Weber Ulrike; Stahmann K-PeterYeast (Chichester, England) (2004), 21 (1), 63-73 ISSN:0749-503X.Alanine : glyoxylate aminotransferase is one of three different enzymes used for glycine synthesis in Saccharomyces cerevisiae. The open reading frame YFL030w (named AGX1 in the following), encoding this enzyme, was identified by comparing enzyme specific activities in knockout strains. While 100% activity was detectable in the parental strain, 2% was found in a YFL030w::kanMX4 strain. The ORF found at that locus was suspected to encode alanine : glyoxylate aminotransferase because its predicted amino acid sequence showed 23% identity to the human homologue. Since the YFL030w::kanMX4 strain showed no glycine auxtrophic phenotype, AGX1 was replaced by KanMX4 in a Delta GLY1 Delta SHM1 Delta SHM2 background. These background mutations, which cause inactivation of threonine aldolase, mitochondrial and cytosolic serine hydroxymethyltransferase, respectively, lead to a conditional glycine auxotrophy. This means that growth is not possible on glucose but on ethanol as the sole carbon source. Additional disruption of AGX1 revealed a complete glycine auxotrophy. Complementation was observed by transformation with a plasmid-encoded AGX1.
- 20Shannon, K. W. and Rabinowitz, J. C. (1988) Isolation and characterization of the Saccharomyces cerevisiae MIS1 gene encoding mitochondrial C1-tetrahydrofolate synthase. J. Biol. Chem. 263, 7717– 7725Google Scholar20https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaL1MXjsVGksA%253D%253D&md5=b0c4885ba2870f799227fe8c39ab9836Isolation and characterization of the Saccharomyces cerevisiae MIS1 gene encoding mitochondrial C1-tetrahydrofolate synthaseShannon, Karen W.; Rabinowitz, Jesse C.Journal of Biological Chemistry (1988), 263 (16), 7717-25CODEN: JBCHA3; ISSN:0021-9258.C1-Tetrahydrofolate synthase is a trifunctional polypeptide found in eukaryotic organisms that catalyzes 10-formyltetrahydrofolate synthetase (EC 6.3.4.3), 5,10-methenyltetrahydrofolate cyclohydrolase (EC 3.5.4.9), and 5,10-methylenetetrahydrofolate dehydrogenase (EC 1.5.1.5) activities. In S. cereivisae, C1-tetrahydrofolate synthase is found in both the cytoplasm and the mitochondria. The gene encoding yeast mitochondrial C1-tetrahydrofolate synthase was isolated using synthetic oligonucleotide probes based on the N-terminal sequence of the purified protein. Hybridization anal. shows that the gene (designated MIS1) has a single copy in the yeast genome. The predicted amino acid sequence of mitochondrial C1-tetrahydrofolate synthase and shares 39% identity with clostridial 10-formyltetrahydrofolate synthetase. Chromosomal deletions of the mitochondrial C1-tetrahydrofolate synthase gene were generated using the cloned MIS1 gene. Mutant strains which lack a functional MIS1 gene are viable and can grow in medium contg. a nonfermentable carbon source. In fact, deletion of the MIS1 locus has no detectable effect on cell growth.
- 21Hochrein, L., Machens, F., Gremmels, J., Schulz, K., Messerschmidt, K., and Mueller-Roeber, B. (2017) AssemblX: a user-friendly toolkit for rapid and reliable multi-gene assemblies. Nucleic Acids Res. gkx034, DOI: 10.1093/nar/gkx034Google ScholarThere is no corresponding record for this reference.
- 22Klein, S. M. and Sagers, R. D. (1966) Glycine metabolism. II. Kinetic and optical studies on the glycine decarboxylase system from Peptococcus glycinophilus. J. Biol. Chem. 241, 206– 209Google Scholar22https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaF2MXltVWhtrg%253D&md5=046a9abbb589332545a46ef7542b8d7cGlycine metabolism. II. Kinetic and optical studies on the glycine decarboxylase system from Peptococcus glycinophilusKlein, Sigrid M.; Sagers, Richard D.Journal of Biological Chemistry (1966), 241 (1), 206-9CODEN: JBCHA3; ISSN:0021-9258.Kinetic and optical properties of the pyridoxal phosphate-contg. enzyme and the heat-stable, low mol. wt. protein required for the exchange of bicarbonate with the glycine carboxyl group are described. Satn. curves were detd. for glycine, bicarbonate, pyridoxal phosphate, and the heat-stable protein P2; and Michaelis consts., resp., of 0.032M, 0.931M, 4.6μM, and 1.3 mg./ml. were obtained. Treatment of the enzyme with cysteine removed the coenzyme and resulted in greatly decreased activity, but the activity was fully restored by incubation of the apoenzyme with pyridoxal phosphate. The ultraviolet absorption spectrum of the holoenzyme showed a max. at 430 mμ which was reduced 85% by treatment with cysteine, but which was restored by incubation with pyridoxal phosphate. Fluorescence max. at 390 and-500 mμ were-observed when the holoenzyme was subjected to activating light at 330 and 430 mμ, resp. The 500-mμ peak was assocd. with the azomethine linkage between the coenzyme and the enzyme since it was decreased by removal of pyridoxal phosphate, but the 390-mμ peak appeared to be assocd. only with the protein since it was not significantly altered by the above treatment. Cf. preceding abstr.
- 23Hiraga, K. and Kikuchi, G. (1980) The mitochondrial glycine cleavage system. Functional association of glycine decarboxylase and aminomethyl carrier protein. J. Biol. Chem. 255, 11671– 11676Google Scholar23https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaL3cXmtlekt7Y%253D&md5=98b0d17460c4c9406d6c14ec5eace94dThe mitochondrial glycine cleavage system. Functional association of glycine decarboxylase and aminomethyl carrier proteinHiraga, Koichi; Kikuchi, GoroJournal of Biological Chemistry (1980), 255 (24), 11671-6CODEN: JBCHA3; ISSN:0021-9258.The activity of the purified glycine decarboxylase, tentatively called P-protein and considered a constituent of the glycine cleavage system (glycine synthase, EC 2.1.2.10), which catalyzed the exchange of the glycine carboxyl C with CO2 was increased >100,000-fold by the addn. of the aminomethyl carrier protein (tentatively called H-protein), and the Km for glycine was reduced to ∼1/4 of that in the absence of H-protein. Decarboxylation of glycine was also greatly stimulated by the addn. of H-protein and the decarboxylation yielded the H-protein-bound aminomethyl moiety and CO2 at a stoichiometric ratio of unity. P-protein and H-protein formed a fairly stable complex which could be demonstrated by gel filtration and by sucrose d. gradient centrifugation. H-protein caused a significant change in the absorption spectrum of P-protein, and a titrn. expt. indicated that 2 mols. of H-protein bind to 1 mol. of P-protein, or 1 mol. of H-protein to each subunit of P-protein. H-protein also acted to reduce the dissocn. const. for methylamine, as estd. by the degree of spectral change of P-protein caused by the addn. of methylamine, from 63 mM to 27 mM, and the Kd value of 27 mM was practically equal to the Ki value (28 mM) for methylamine obtained in the glycine-CO2 exchange reaction in the presence of H-protein. H-protein seems to bring about a conformational change of P-protein which may be relevant to the expression of the decarboxylase activity of P-protein. In other words, although P-protein is, by nature, glycine decarboxylase, the functional glycine decarboxylase may be an enzyme complex composed of both P-protein and H-protein. H-protein seems to play a dual role in the glycine decarboxylation; the one as a regulatory protein of P-protein, and the other as an electron-pulling agent and concomitantly as a carrier of the aminomethyl moiety derived from glycine by decarboxylation.
- 24Warnecke, T. and Gill, R. T. (2005) Organic acid toxicity, tolerance, and production in Escherichia coli biorefining applications. Microb. Cell Fact. 4, 25, DOI: 10.1186/1475-2859-4-25Google Scholar24https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A280%3ADC%252BD2Mvpt1CgsA%253D%253D&md5=629ae62fd55f8487c1fde673ab1eac1aOrganic acid toxicity, tolerance, and production in Escherichia coli biorefining applicationsWarnecke Tanya; Gill Ryan TMicrobial cell factories (2005), 4 (), 25 ISSN:.Organic acids are valuable platform chemicals for future biorefining applications. Such applications involve the conversion of low-cost renewable resources to platform sugars, which are then converted to platform chemicals by fermentation and further derivatized to large-volume chemicals through conventional catalytic routes. Organic acids are toxic to many of the microorganisms, such as Escherichia coli, proposed to serve as biorefining platform hosts at concentrations well below what is required for economical production. The toxicity is two-fold including not only pH based growth inhibition but also anion-specific effects on metabolism that also affect growth. E. coli maintain viability at very low pH through several different tolerance mechanisms including but not limited to the use of decarboxylation reactions that consume protons, ion transporters that remove protons, increased expression of known stress genes, and changing membrane composition. The focus of this mini-review is on organic acid toxicity and associated tolerance mechanisms as well as several examples of successful organic acid production processes for E. coli.
- 25Nicholls, P. (1975) Formate as an inhibitor of cytochrome c oxidase. Biochem. Biophys. Res. Commun. 67, 610– 616, DOI: 10.1016/0006-291X(75)90856-6Google Scholar25https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaE28XhsVSitw%253D%253D&md5=5273ecd954058e9353dee6685ac0b726Formate as an inhibitor of cytochrome c oxidaseNicholls, PeterBiochemical and Biophysical Research Communications (1975), 67 (2), 610-16CODEN: BBRCA9; ISSN:0006-291X.Formate inhibited cytochrome c oxidase with a Ki of 5-30mM at pH 7.4 (depending on assay conditions). The formate binding site is accessible in the fully oxidized (a3+ a33+) and partially reduced (a2+ a33+) states, but not in the fully reduced (a2+ a32+ state). Azide competed with formate for the binding site. Formate induced a blue shift in the Soret peak of fully oxidized enzyme. The rate of formate binding, and the apparent affinity (1/Ki), increased as the pH was diminished, suggesting that HCOOH is the bound species.
- 26Zaldivar, J. and Ingram, L. O. (1999) Effect of organic acids on the growth and fermentation of ethanologenic Escherichia coli LY01. Biotechnol. Bioeng. 66, 203– 210, DOI: 10.1002/(SICI)1097-0290(1999)66:4<203::AID-BIT1>3.0.CO;2-#Google Scholar26https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaK1MXotFamu7g%253D&md5=bbb857f3ceb346ff331df9f2fb22e1efEffect of organic acids on the growth and fermentation of ethanologenic Escherichia coli LY01Zaldivar, Jesus; Ingram, Lonnie O.Biotechnology and Bioengineering (1999), 66 (4), 203-210CODEN: BIBIAU; ISSN:0006-3592. (John Wiley & Sons, Inc.)Hemicellulose residues can be hydrolyzed into a sugar syrup using dil. mineral acids. Although this syrup represents a potential feedstock for biofuel prodn., toxic compds. generated during hydrolysis limit microbial metab. E. coli LY01, an ethanologenic biocatalyst engineered to ferment the mixed sugars in hemicellulose syrups, has been tested for resistance to selected org. acids that are present in hemicellulose hydrolyzates. Compds. tested include arom. acids derived from lignin (ferulic, gallic, 4-hydroxybenzoic, syringic, and vanillic acids), acetic acid from the hydrolysis of acetylxylan, and others derived from sugar destruction (furoic, formic, levulinic, and caproic acids). Toxicity was related to hydrophobicity. Combinations of acids were roughly additive as inhibitors of cell growth. When tested at concns. that inhibited growth by 80%, none appeared to strongly inhibit glycolysis and energy generation or to disrupt membrane integrity. Toxicity was not markedly affected by inoculum size or incubation temp. The toxicity of all acids except gallic acid was reduced by an increase in initial pH (from pH 6.0 to pH 7.0 to pH 8.0). Together, these results are consistent with the hypothesis that both aliph. and mononuclear org. acids inhibit growth and ethanol prodn. in LY01 by collapsing ion gradients and increasing internal anion concns.
- 27Overkamp, K. M., Kotter, P., van der Hoek, R., Schoondermark-Stolk, S., Luttik, M. A., van Dijken, J. P., and Pronk, J. T. (2002) Functional analysis of structural genes for NAD(+)-dependent formate dehydrogenase in Saccharomyces cerevisiae. Yeast 19, 509– 520, DOI: 10.1002/yea.856Google Scholar27https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD38Xjt1eis70%253D&md5=3b8989ba195bb5193eb4f0c698e248a9Functional analysis of structural genes for NAD+-dependent formate dehydrogenase in Saccharomyces cerevisiaeOverkamp, Karin M.; Kotter, Peter; Van der Hoek, Richard; Schoondermark-Stolk, Sung; Luttik, Marijke A. H.; Van Dijken, Johannes P.; Pronk, Jack T.Yeast (2002), 19 (6), 509-520CODEN: YESTE3; ISSN:0749-503X. (John Wiley & Sons Ltd.)Co-consumption of formate by aerobic, glucose-limited chemostat cultures of Saccharomyces cerevisiae CEN.PK 113-7D led to an increased biomass yield relative to cultures grown on glucose as the sole carbon and energy substrate. In this respect, this strain differed from two previously investigated S. cerevisiae strains, in which formate oxidn. did not lead to an increased biomass yield on glucose. Enzyme assays confirmed the presence of a formate-inducible, cytosolic and NAD+-dependent formate dehydrogenase. To investigate whether this enzyme activity was entirely encoded by the previously reported FDH1 gene, an fdh1Δ null mutant was constructed. This mutant strain still contained formate dehydrogenase activity and remained capable of co-consumption of formate. The formate dehydrogenase activity in the mutant was demonstrated to be encoded by a second structural gene for formate dehydrogenase (FDH2) in S. cerevisiae CEN.PK 113-7D. FDH2 was highly homologous to FDH1 and consisted of a fusion of two open reading frames (ORFs) (YPL275w and YPL276w) reported in the S. cerevisiae genome databases. Sequence anal. confirmed that, in the database genetic background, the presence of two single-nucleotide differences led to two truncated ORFs rather than the full-length FDH2 gene present in strain CEN.PK 113-7D. In the latter strain background an fdh1Δfdh2Δ double mutant lacked formate dehydrogenase activity and was unable to co-consume formate. Absence of formate dehydrogenase activity did not affect growth on glucose as sole carbon source, but led to a reduced biomass yield on glucose-formate mixts. These findings are consistent with a role of formate dehydrogenase in the detoxification of exogenous formate.
- 28Babel, W. (2009) The Auxiliary Substrate Concept: From simple considerations to heuristically valuable knowledge. Eng. Life Sci. 9, 285– 290, DOI: 10.1002/elsc.200900027Google Scholar28https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD1MXhtV2hs7fJ&md5=410fd45ff37ed049cd5db6a4a7c24141The Auxiliary Substrate Concept: From simple considerations to heuristically valuable knowledgeBabel, WolfgangEngineering in Life Sciences (2009), 9 (4), 285-290CODEN: ELSNAE; ISSN:1618-0240. (Wiley-VCH Verlag GmbH & Co. KGaA)A review. Microorganisms are used in biotechnol. They are either (i) aim and purpose of a process, e.g. with the prodn. of single cell proteins, or (ii) mean to an end insofar as they serve as a catalyst or "factory" for syntheses (e.g. of products of primary and secondary metab., of enzymes and antibiotics) or for the degrdn. and detoxification of harmful orgs. and inorgs. In all cases, the efficiency and velocity, finally the productivity, are parameters which essentially det. the economy of the processes. Therefore, search for approaches to optimize these processes is a permanent task and challenge for scientists and engineers. It is shown that the auxiliary substrate concept is suitable to increase the yield coeffs. It is based on the energetic evaluation of orgs., on the knowledge that orgs. as sources of carbon and energy for growth are deficient in ATP and/or reducing equiv., and says that it is possible to improve the carbon conversion efficiency up to the carbon metab. detd. upper limit. The latter is detd. by inevitable losses of carbon along the way of assimilation and anabolism and amts. to about 85% for so-called glycolytic substrates, e.g. glucose, methanol, and to about 75% for gluconeogenetic substrates, e.g. C2-substrates (acetic acid, hexadecane). The approach is explained and some exptl. examples are presented. By simultaneous utilization of an extra energy source (auxiliary substrate) the yield coeff. can be increased (i) in glucose from about 0.5 to 0.7 g/g (by means of formate), (ii) in acetate from 0.34-0.4 to 0.5-0.65 g/g (by means of formate and thiosulfate, resp.), and (iii) in hexadecane from about 0.94 to 1.26 g/g (by means of formate). The precalcd. yield coeffs. and mixing ratios agree well with the exptl. attained ones. The approach is easily feasible and economically valuable.
- 29Christensen, K. E. and Mackenzie, R. E. (2008) Mitochondrial methylenetetrahydrofolate dehydrogenase, methenyltetrahydrofolate cyclohydrolase, and formyltetrahydrofolate synthetases. Vitam. Horm. 79, 393– 410, DOI: 10.1016/S0083-6729(08)00414-7Google Scholar29https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD1cXhsVOnsr7M&md5=e693f775711f681580245f24dce74ab1Mitochondrial methylenetetrahydrofolate dehydrogenase, methenyltetrahydrofolate cyclohydrolase, and formyltetrahydrofolate synthetasesChristensen, Karen E.; MacKenzie, Robert E.Vitamins and Hormones (San Diego, CA, United States) (2008), 79 (Folic Acid and Folates), 393-410CODEN: VIHOAQ; ISSN:0083-6729. (Elsevier Inc.)A review. Folate-mediated metab. involves enzyme-catalyzed reactions that occur in the cytoplasmic, mitochondrial, and nuclear compartments in mammalian cells. Which of the folate-dependent enzymes are expressed in these compartments depends on the stage of development, cell type, cell cycle, and whether or not the cell is transformed. Mitochondria become formate-generating organelles in cells and tissues expressing the MTHFD2 and MTHFD1L genes. The products of these nuclear genes were derived from trifunctional precursor proteins, expressing methylenetetrahydrofolate dehydrogenase-methenyltetrahydrofolate cyclohydrolase, and formyltetrahydrofolate synthetase activities. The MTHFD2 protein is a bifunctional protein with dehydrogenase and cyclohydrolase activities that arose from a trifunctional precursor through the loss of the synthetase domain and a novel adaptation to NAD rather than NADP specificity for the dehydrogenase. The MTHFD1L protein retains the size of its trifunctional precursor, but through the mutation of crit. residues, both the dehydrogenase and cyclohydrolase activities have been silenced. MTHFD1L is thus a monofunctional formyltetrahydrofolate synthetase. This review discusses the properties and functions of these mitochondrial proteins and their role in supporting cytosolic purine synthesis during embryonic development and in cells undergoing rapid growth.
- 30Figueroa, I. A., Barnum, T. P., Somasekhar, P. Y., Carlstrom, C. I., Engelbrektson, A. L., and Coates, J. D. (2018) Metagenomics-guided analysis of microbial chemolithoautotrophic phosphite oxidation yields evidence of a seventh natural CO2 fixation pathway. Proc. Natl. Acad. Sci. U. S. A. 115, E92– E101, DOI: 10.1073/pnas.1715549114Google Scholar30https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2sXhvVOrtrfI&md5=c396ad05612addfe07c21b2937a03049Metagenomics-guided analysis of microbial chemolithoautotrophic phosphite oxidation yields evidence of a seventh natural CO2 fixation pathwayFigueroa, Israel A.; Barnum, Tyler P.; Somasekhar, Pranav Y.; Carlstrom, Charlotte I.; Engelbrektson, Anna L.; Coates, John D.Proceedings of the National Academy of Sciences of the United States of America (2018), 115 (1), E92-E101CODEN: PNASA6; ISSN:0027-8424. (National Academy of Sciences)Dissimilatory phosphite oxidn. (DPO), a microbial metab. by which phosphite (HPO32-) is oxidized to phosphate (PO43-), is the most energetically favorable chemotrophic electron-donating process known. Only one DPO organism has been described to date, and little is known about the environmental relevance of this metab. In this study, we used 16S rRNA gene community anal. and genome-resolved metagenomics to characterize anaerobic wastewater treatment sludge enrichments performing DPO coupled to CO2 redn. We identified an uncultivated DPO bacterium, Candidatus Phosphitivorax (Ca. P.) anaerolimi strain Phox-21, that belongs to candidate order GW-28 within the Deltaproteobacteria, which has no known cultured isolates. Genes for phosphite oxidn. and for CO2 redn. to formate were found in the genome of Ca. P. anaerolimi, but it appears to lack any of the known natural carbon fixation pathways. These observations led us to propose a metabolic model for autotrophic growth by Ca. P. anaerolimi whereby DPO drives CO2 redn. to formate, which is then assimilated into biomass via the reductive glycine pathway.
- 31Brachmann, C. B., Davies, A., Cost, G. J., Caputo, E., Li, J., Hieter, P., and Boeke, J. D. (1998) Designer deletion strains derived from Saccharomyces cerevisiae S288C: a useful set of strains and plasmids for PCR-mediated gene disruption and other applications. Yeast 14, 115– 132, DOI: 10.1002/(SICI)1097-0061(19980130)14:2<115::AID-YEA204>3.0.CO;2-2Google Scholar31https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaK1cXptVyjsA%253D%253D&md5=33320be31df69f6abe592fb0692fd24cDesigner deletion strains derived from Saccharomyces cerevisiae S288C: a useful set of strains and plasmids for PCR-mediated gene disruption and other applicationsBrachmann, Carrie Baker; Davies, Adrian; Cost, Gregory J.; Caputo, Emerita; Li, Joachim; Hieter, Philip; Boeke, Jef D.Yeast (1998), 14 (2), 115-132CODEN: YESTE3; ISSN:0749-503X. (John Wiley & Sons Ltd.)A set of yeast strains based on Saccharomyces cerevisiae S288C in which commonly used selectable marker genes are deleted by design based on the yeast genome sequence has been constructed and analyzed. These strains minimize or eliminate the homol. to the corresponding marker genes in commonly used vectors without significantly affecting adjacent gene expression. Because the homol. between commonly used auxotrophic marker gene segments and genomic sequences has been largely or completely abolished, these strains will also reduce plasmid integration events which can interfere with a wide variety of mol. genetic applications. We also report the construction of new members of the pRS400 series of vectors, contg. the kanMX, ADE2 and MET15 genes.
- 32Looke, M., Kristjuhan, K., and Kristjuhan, A. (2011) Extraction of genomic DNA from yeasts for PCR-based applications. BioTechniques 50, 325– 328, DOI: 10.2144/000113672Google Scholar32https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3MXntVGjsbs%253D&md5=6948314df60f45a5754923cdd0c6ee70Extraction of genomic DNA from yeasts for PCR-based applicationsLooke, Marko; Kristjuhan, Kersti; Kristjuhan, ArnoldBioTechniques (2011), 50 (5), 325-328CODEN: BTNQDO; ISSN:0736-6205. (Informa Healthcare)The authors have developed a quick and low-cost genomic DNA extn. protocol from yeast cells for PCR-based applications. This method does not require any enzymes, hazardous chems., or extreme temps., and is esp. powerful for simultaneous anal. of a large no. of samples. DNA can be efficiently extd. from different yeast species (Kluyveromyces lactis, Hansenula polymorpha, Schizosaccharomyces pombe, Candida albicans, Pichia pastoris, and Saccharomyces cerevisiae). The protocol involves lysis of yeast colonies or cells from liq. culture in a lithium acetate (LiOAc)-SDS soln. and subsequent pptn. of DNA with ethanol. Approx. 100 ng of total genomic DNA can be extd. from 1 × 107 cells. DNA extd. by this method is suitable for a variety of PCR-based applications (including colony PCR, real-time qPCR, and DNA sequencing) for amplification of DNA fragments of ≤3500 bp.
- 33Gietz, R. D. and Schiestl, R. H. (2007) Quick and easy yeast transformation using the LiAc/SS carrier DNA/PEG method. Nat. Protoc. 2, 35– 37, DOI: 10.1038/nprot.2007.14Google Scholar33https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD2sXhtFGntb%252FE&md5=ca175dd794b225c28baa8e9b9b348e5eQuick and easy yeast transformation using the LiAc/SS carrier DNA/PEG methodGietz, R. Daniel; Schiestl, Robert H.Nature Protocols (2007), 2 (1), 35-37CODEN: NPARDW; ISSN:1750-2799. (Nature Publishing Group)Here, we describe a quick and easy version of the lithium acetate/single-stranded carrier DNA/PEG method of transformation for Saccharomyces cerevisiae. This method can be performed when only a few transformants are needed. The procedure can take less than an hour, depending on the duration of the heat shock. It can be used to transform yeast cells from various stages of growth and storage. Cells can be transformed from freshly grown cells as well as cells stored on a plate at room temp. or in a refrigerator.
- 34Hillson, N. J., Rosengarten, R. D., and Keasling, J. D. (2012) j5 DNA assembly design automation software. ACS Synth. Biol. 1, 14– 21, DOI: 10.1021/sb2000116Google Scholar34https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3MXhsFygt7vM&md5=368ad18ea12fb4c9995f7dbb38096ca0j5 DNA Assembly Design Automation SoftwareHillson, Nathan J.; Rosengarten, Rafael D.; Keasling, Jay D.ACS Synthetic Biology (2012), 1 (1), 14-21CODEN: ASBCD6; ISSN:2161-5063. (American Chemical Society)Recent advances in Synthetic Biol. have yielded standardized and automatable DNA assembly protocols that enable a broad range of biotechnol. research and development. Unfortunately, the exptl. design required for modern scar-less multipart DNA assembly methods is frequently laborious, time-consuming, and error-prone. Here, we report the development and deployment of a web-based software tool, j5, which automates the design of scar-less multipart DNA assembly protocols including SLIC, Gibson, CPEC, and Golden Gate. The key innovations of the j5 design process include cost optimization, leveraging DNA synthesis when cost-effective to do so, the enforcement of design specification rules, hierarchical assembly strategies to mitigate likely assembly errors, and the instruction of manual or automated construction of scar-less combinatorial DNA libraries. Using a GFP expression testbed, we demonstrate that j5 designs can be executed with the SLIC, Gibson, or CPEC assembly methods, used to build combinatorial libraries with the Golden Gate assembly method, and applied to the prepn. of linear gene deletion cassettes for E. coli. The DNA assembly design algorithms reported here are generally applicable to broad classes of DNA construction methodologies and could be implemented to supplement other DNA assembly design tools. Taken together, these innovations save researchers time and effort, reduce the frequency of user design errors and off-target assembly products, decrease research costs, and enable scar-less multipart and combinatorial DNA construction at scales unfeasible without computer-aided design.
Cited By
Click to copy section linkSection link copied!
Citation Statements
beta
Smart citations by scite.ai include citation statements extracted from the full text of the citing article. The number of the statements may be higher than the number of citations provided by ACS Publications if one paper cites another multiple times or lower if scite has not yet processed some of the citing articles.
Explore this article's citation statements on scite.ai
This article is cited by 81 publications.
- Shaafique Chowdhury, Ray Westenberg, Kimberly Wennerholm, Ryan A.L. Cardiff, Alexander S. Beliaev, Vincent Noireaux, James M. Carothers, Pamela Peralta-Yahya. Carbon Negative Synthesis of Amino Acids Using a Cell-Free-Based Biocatalyst. ACS Synthetic Biology 2024, 13
(12)
, 3961-3975. https://doi.org/10.1021/acssynbio.4c00359
- Fabian Machens, Guangyao Ran, Ciaran Ruehmkorff, Julie Meyer auf der Heyde, Bernd Mueller-Roeber, Lena Hochrein. PhiReX 2.0: A Programmable and Red Light-Regulated CRISPR-dCas9 System for the Activation of Endogenous Genes in Saccharomyces cerevisiae. ACS Synthetic Biology 2023, 12
(4)
, 1046-1057. https://doi.org/10.1021/acssynbio.2c00517
- Philip A. Kelso, Louise K. M. Chow, Alex C. Carpenter, Ian T. Paulsen, Thomas C. Williams. Toward Methanol-Based Biomanufacturing: Emerging Strategies for Engineering Synthetic Methylotrophy in Saccharomyces cerevisiae. ACS Synthetic Biology 2022, 11
(8)
, 2548-2563. https://doi.org/10.1021/acssynbio.2c00110
- Shengyuan Guo, Tristan Asset, Plamen Atanassov. Catalytic Hybrid Electrocatalytic/Biocatalytic Cascades for Carbon Dioxide Reduction and Valorization. ACS Catalysis 2021, 11
(9)
, 5172-5188. https://doi.org/10.1021/acscatal.0c04862
- Cong Fan, Jian Fan, Haofeng Chen, Shujin Lin, Danli Zhang, Jingya Song, Junyi Wang, Yan Wang, Xiao Han, Jifeng Yuan. Switching the yeast metabolism via manipulation of sugar phosphorylation. Metabolic Engineering 2025, 89 , 76-85. https://doi.org/10.1016/j.ymben.2025.02.008
- Mingming Qi, Chao Zhu, Chi Cheng, Wei Kang, Chuang Xue. Rewiring methanol assimilation and reductive glycine pathways in
Saccharomyces cerevisiae
to increase one-carbon recovery. Green Chemistry 2025, 27
(12)
, 3261-3271. https://doi.org/10.1039/D4GC05254D
- Feng Guo, Kang Liu, Yangyi Qiao, YongMin Zheng, Chenguang Liu, Yi Wu, Zhonghai Zhang, Wankui Jiang, Yujia Jiang, Fengxue Xin, Min Jiang, Wenming Zhang. Evolutionary engineering of
Saccharomyces cerevisiae
: Crafting a synthetic methylotroph via self-reprogramming. Science Advances 2024, 10
(51)
https://doi.org/10.1126/sciadv.adq3484
- Yan Shi, Kejing Zhang, Jianxin Chen, Bingtian Zhang, Xun Guan, Xin Wang, Tong Zhang, Han Song, Long Zou, Xiangfeng Duan, Haichun Gao, Zhang Lin. Long‐Term Autotrophic Growth and Solar‐to‐Chemical Conversion in
Shewanella Oneidensis
MR‐1 through Light‐Driven Electron Transfer. Angewandte Chemie 2024, 136
(51)
https://doi.org/10.1002/ange.202412072
- Yan Shi, Kejing Zhang, Jianxin Chen, Bingtian Zhang, Xun Guan, Xin Wang, Tong Zhang, Han Song, Long Zou, Xiangfeng Duan, Haichun Gao, Zhang Lin. Long‐Term Autotrophic Growth and Solar‐to‐Chemical Conversion in
Shewanella Oneidensis
MR‐1 through Light‐Driven Electron Transfer. Angewandte Chemie International Edition 2024, 63
(51)
https://doi.org/10.1002/anie.202412072
- Yuanyi Li, Wei Zhou, Ruijing Ling, Shuting Hou, Lujia Zhang, Bei Gao. Revealing the endogenous homoserine cycle for the effective methanol conversion in Pichia pastoris. Process Biochemistry 2024, 144 , 287-293. https://doi.org/10.1016/j.procbio.2024.06.002
- Yuanke Guo, Rui Zhang, Jing Wang, Ruirui Qin, Jiao Feng, Kequan Chen, Xin Wang. Engineering yeasts to Co-utilize methanol or formate coupled with CO2 fixation. Metabolic Engineering 2024, 84 , 1-12. https://doi.org/10.1016/j.ymben.2024.05.002
- Qian Chen, Yunhong Chen, Zeming Hou, Yuyue Ma, Jianfeng Huang, Zhidan Zhang, Yefu Chen, Xue Yang, Yanfei Zhang, Guoping Zhao. Unlocking the formate utilization of wild‐type
Yarrowia lipolytica
through adaptive laboratory evolution. Biotechnology Journal 2024, 19
(6)
https://doi.org/10.1002/biot.202400290
- Kai Li, Xue Zhang, Cheng Li, Yu-Cheng Liang, Xin-Qing Zhao, Chen-Guang Liu, Anthony J. Sinskey, Feng-Wu Bai. Systems metabolic engineering of Corynebacterium glutamicum to assimilate formic acid for biomass accumulation and succinic acid production. Bioresource Technology 2024, 402 , 130774. https://doi.org/10.1016/j.biortech.2024.130774
- Rishi Gupta, Archana Mishra, Yeruva Thirupathaiah, Anuj Kumar Chandel. Biochemical conversion of CO2 in fuels and chemicals: status, innovation, and industrial aspects. Biomass Conversion and Biorefinery 2024, 14
(3)
, 3007-3030. https://doi.org/10.1007/s13399-022-02552-8
- Pei-Ru Chen, Peng-Fei Xia. Carbon recycling with synthetic CO2 fixation pathways. Current Opinion in Biotechnology 2024, 85 , 103023. https://doi.org/10.1016/j.copbio.2023.103023
- Xue Yang, Yanfei Zhang, Guoping Zhao. Artificial carbon assimilation: From unnatural reactions and pathways to synthetic autotrophic systems. Biotechnology Advances 2024, 70 , 108294. https://doi.org/10.1016/j.biotechadv.2023.108294
- Qi Xia, Junzhu Yang, Liangwei Hu, Hongxin Zhao, Yuan Lu. Biotransforming CO2 into valuable chemicals. Journal of Cleaner Production 2024, 434 , 140185. https://doi.org/10.1016/j.jclepro.2023.140185
- Viswanada R. Bysani, Ayesha S. Alam, Arren Bar-Even, Fabian Machens. Engineering and evolution of the complete Reductive Glycine Pathway in Saccharomyces cerevisiae for formate and CO2 assimilation. Metabolic Engineering 2024, 81 , 167-181. https://doi.org/10.1016/j.ymben.2023.11.007
- Evelyn Vásquez Castro, Golnaz Memari, Özge Ata, Diethard Mattanovich. Carbon efficient production of chemicals with yeasts. Yeast 2023, 40
(12)
, 583-593. https://doi.org/10.1002/yea.3909
- Enrico Orsi, Pablo Ivan Nikel, Lars Keld Nielsen, Stefano Donati. Synergistic investigation of natural and synthetic C1-trophic microorganisms to foster a circular carbon economy. Nature Communications 2023, 14
(1)
https://doi.org/10.1038/s41467-023-42166-w
- Jinzhong Tian, Wangshuying Deng, Ziwen Zhang, Jiaqi Xu, Guiling Yang, Guoping Zhao, Sheng Yang, Weihong Jiang, Yang Gu. Discovery and remodeling of Vibrio natriegens as a microbial platform for efficient formic acid biorefinery. Nature Communications 2023, 14
(1)
https://doi.org/10.1038/s41467-023-43631-2
- Shijie Xu, Weibo Qiao, Zuanwen Wang, Xiaoying Fu, Zihe Liu, Shuobo Shi. Exploiting a heterologous construction of the 3-hydroxypropionic acid carbon fixation pathway with mesaconate as an indicator in Saccharomyces cerevisiae. Bioresources and Bioprocessing 2023, 10
(1)
https://doi.org/10.1186/s40643-023-00652-5
- Elif Kurt, Jiansong Qin, Alexandria Williams, Youbo Zhao, Dongming Xie. Perspectives for Using CO2 as a Feedstock for Biomanufacturing of Fuels and Chemicals. Bioengineering 2023, 10
(12)
, 1357. https://doi.org/10.3390/bioengineering10121357
- Du-Kyeong Kang, Seung-Hwa Kim, Jung-Hoon Sohn, Bong Hyun Sung. Insights into Enzyme Reactions with Redox Cofactors in Biological Conversion of CO
2. Journal of Microbiology and Biotechnology 2023, 33
(11)
, 1403-1411. https://doi.org/10.4014/jmb.2306.06005
- Wang Ge-Ge, Zhang Yuan, Wang Xiao-Yan, Zhang Gen-Lin. Microbial Conversion and Utilization of CO2. Annals of Civil and Environmental Engineering 2023, 7
(1)
, 045-060. https://doi.org/10.29328/journal.acee.1001055
- Qing Wang, Jianfeng Zhang, Qiulan Dai, Meijie Cui, Hao Yang, Peijian Cao, Lei Zhao. When green carbon plants meet synthetic biology. Modern Agriculture 2023, 1
(2)
, 98-111. https://doi.org/10.1002/moda.17
- Yamei Gan, Xin Meng, Cong Gao, Wei Song, Liming Liu, Xiulai Chen. Metabolic engineering strategies for microbial utilization of methanol. Engineering Microbiology 2023, 3
(3)
, 100081. https://doi.org/10.1016/j.engmic.2023.100081
- Kai Wang, Yining Liu, Zhuoheng Wu, Yilu Wu, Haoran Bi, Yanhui Liu, Meng Wang, Biqiang Chen, Jens Nielsen, Zihe Liu, Tianwei Tan. Investigating formate tolerance mechanisms in Saccharomyces cerevisiae and its application. Green Carbon 2023, 1
(1)
, 65-74. https://doi.org/10.1016/j.greenca.2023.08.003
- Lukas R. Dahlin, Alex W. Meyers, Skylar W. Stefani, Ellsbeth G. Webb, Benton Wachter, Venkataramanan Subramanian, Michael T. Guarnieri. Heterologous expression of formate dehydrogenase enables photoformatotrophy in the emerging model microalga, Picochlorum renovo. Frontiers in Bioengineering and Biotechnology 2023, 11 https://doi.org/10.3389/fbioe.2023.1162745
- Feng Guo, Yangyi Qiao, Fengxue Xin, Wenming Zhang, Min Jiang. Bioconversion of C1 feedstocks for chemical production using Pichia pastoris. Trends in Biotechnology 2023, 41
(8)
, 1066-1079. https://doi.org/10.1016/j.tibtech.2023.03.006
- Viswanada R Bysani, Ayesha S M Alam, Arren Bar-Even, Fabian Machens. Engineering and Evolution of the Complete Reductive Glycine Pathway in
Saccharomyces cerevisiae
for Formate and CO
2
Assimilation. 2023https://doi.org/10.1101/2023.07.10.548313
- Shuobo Shi, Yubo Wang, Weibo Qiao, Longhao Wu, Zihe Liu, Tianwei Tan. Challenges and opportunities in the third-generation biorefinery. Chinese Science Bulletin 2023, 68
(19)
, 2489-2503. https://doi.org/10.1360/TB-2022-1210
- Yongfei Liu, Jianming Liu, Jinglei Nie, Anping Zeng. Advances and perspectives of biosynthesis of chemicals based on CO2 and other one-carbon feedstocks. Chinese Science Bulletin 2023, 68
(19)
, 2470-2488. https://doi.org/10.1360/TB-2022-1300
- Ran Zhao, Wenyue Dong, Chen Yang, Weihong Jiang, Jinzhong Tian, Yang Gu. Formate as a supplementary substrate facilitates sugar metabolism and solvent production by Clostridium beijerinckii NCIMB 8052. Synthetic and Systems Biotechnology 2023, 8
(2)
, 196-205. https://doi.org/10.1016/j.synbio.2023.01.005
- Kai Wang, Yangyang Da, Haoran Bi, Yanhui Liu, Biqiang Chen, Meng Wang, Zihe Liu, Jens Nielsen, Tianwei Tan. A one-carbon chemicals conversion strategy to produce precursor of biofuels with Saccharomyces cerevisiae. Renewable Energy 2023, 208 , 331-340. https://doi.org/10.1016/j.renene.2023.03.058
- Ranran Wu, Fei Li, Xinyu Cui, Zehua Li, Chunling Ma, Huifeng Jiang, Lingling Zhang, Yi‐Heng P. Job Zhang, Tongxin Zhao, Yanping Zhang, Yin Li, Hui Chen, Zhiguang Zhu. Enzymatic Electrosynthesis of Glycine from CO
2
and NH
3. Angewandte Chemie 2023, 135
(14)
https://doi.org/10.1002/ange.202218387
- Ranran Wu, Fei Li, Xinyu Cui, Zehua Li, Chunling Ma, Huifeng Jiang, Lingling Zhang, Yi‐Heng P. Job Zhang, Tongxin Zhao, Yanping Zhang, Yin Li, Hui Chen, Zhiguang Zhu. Enzymatic Electrosynthesis of Glycine from CO
2
and NH
3. Angewandte Chemie International Edition 2023, 62
(14)
https://doi.org/10.1002/anie.202218387
- Yuanke Guo, Rui Zhang, Jing Wang, Ruirui Qin, Jiao Feng, Kequan Chen, Xin Wang. Engineering Yeasts to Grow Solely on Methanol or Formic acid coupled with CO2 fixation. 2023https://doi.org/10.21203/rs.3.rs-2694097/v1
- Jian Xu, Jie Wang, Chunling Ma, Zuoxi Wei, Yida Zhai, Na Tian, Zhiguang Zhu, Min Xue, Demao Li. Embracing a low-carbon future by the production and marketing of C1 gas protein. Biotechnology Advances 2023, 63 , 108096. https://doi.org/10.1016/j.biotechadv.2023.108096
- Lyon Bruinsma, Sebastian Wenk, Nico J. Claassens, Vitor A.P. Martins dos Santos. Paving the way for synthetic C1 - Metabolism in Pseudomonas putida through the reductive glycine pathway. Metabolic Engineering 2023, 76 , 215-224. https://doi.org/10.1016/j.ymben.2023.02.004
- Jian Zhang, Liang Guo, Cong Gao, Wei Song, Jing Wu, Liming Liu, Xiulai Chen. Metabolic engineering strategies for microbial utilization of C1 feedstocks. Systems Microbiology and Biomanufacturing 2023, 3
(1)
, 122-136. https://doi.org/10.1007/s43393-022-00135-2
- Hermann Bauwe. Humboldt Review: Photorespiration – Rubisco's repair crew. Journal of Plant Physiology 2023, 280 , 153899. https://doi.org/10.1016/j.jplph.2022.153899
- Wei Zhong, Hailong Li, Yajie Wang. Design and Construction of Artificial Biological Systems for One-Carbon Utilization. BioDesign Research 2023, 5 , 0021. https://doi.org/10.34133/bdr.0021
- Jinzhong Tian, Wangshuying Deng, Ziwen Zhang, Jiaqi Xu, Guoping Zhao, Sheng Yang, Weihong Jiang, Yang Gu. Discovery and remodeling of
Vibrio natriegens
as a microbial platform for efficient formic acid biorefinery. 2022https://doi.org/10.1101/2022.12.15.520533
- Bing Liu, Haijian Li, Hualan Zhou, Jianguo Zhang. Enhancing xylanase expression by Komagataella phaffii by formate as carbon source and inducer. Applied Microbiology and Biotechnology 2022, 106
(23)
, 7819-7829. https://doi.org/10.1007/s00253-022-12249-7
- Briardo Llorente, Thomas C. Williams, Hugh D. Goold, Isak S. Pretorius, Ian T. Paulsen. Harnessing bioengineered microbes as a versatile platform for space nutrition. Nature Communications 2022, 13
(1)
https://doi.org/10.1038/s41467-022-33974-7
- Vanessa Wegat, Jonathan T. Fabarius, Volker Sieber. Synthetic methylotrophic yeasts for the sustainable fuel and chemical production. Biotechnology for Biofuels and Bioproducts 2022, 15
(1)
https://doi.org/10.1186/s13068-022-02210-1
- Hawaibam Birla Singh, Min-Kyoung Kang, Moonhyuk Kwon, Seon-Won Kim. Developing methylotrophic microbial platforms for a methanol-based bioindustry. Frontiers in Bioengineering and Biotechnology 2022, 10 https://doi.org/10.3389/fbioe.2022.1050740
- Weibo Qiao, Shijie Xu, Zihe Liu, Xiaoying Fu, Huimin Zhao, Shuobo Shi. Challenges and opportunities in C1-based biomanufacturing. Bioresource Technology 2022, 364 , 128095. https://doi.org/10.1016/j.biortech.2022.128095
- Nils Guntermann, Giancarlo Franciò, Walter Leitner. Hydrogenation of CO
2
to formic acid in biphasic systems using aqueous solutions of amino acids as the product phase. Green Chemistry 2022, 24
(20)
, 8069-8075. https://doi.org/10.1039/D2GC02598A
- Bernd M. Mitic, Christina Troyer, Stephan Hann, Diethard Mattanovich. The oxygen tolerant reductive glycine pathway in eukaryotes – a native methanol, formate and CO
2
assimilation pathway in the yeast
Komagataella phaffii. 2022https://doi.org/10.1101/2022.09.01.506198
- David N. Carruthers, Taek Soon Lee. Translating advances in microbial bioproduction to sustainable biotechnology. Frontiers in Bioengineering and Biotechnology 2022, 10 https://doi.org/10.3389/fbioe.2022.968437
- Lyon Bruinsma, Sebastian Wenk, Nico J. Claassens, Vitor A.P. Martins dos Santos. Paving the way for synthetic C1- metabolism in
Pseudomonas putida
through the reductive glycine pathway. 2022https://doi.org/10.1101/2022.07.10.499465
- Congqiang Zhang, Christoph Ottenheim, Melanie Weingarten, LiangHui Ji. Microbial Utilization of Next-Generation Feedstocks for the Biomanufacturing of Value-Added Chemicals and Food Ingredients. Frontiers in Bioengineering and Biotechnology 2022, 10 https://doi.org/10.3389/fbioe.2022.874612
- Cong Du, Yimin Li, Ruijuan Xiang, Wenjie Yuan. Formate Dehydrogenase Improves the Resistance to Formic Acid and Acetic Acid Simultaneously in Saccharomyces cerevisiae. International Journal of Molecular Sciences 2022, 23
(6)
, 3406. https://doi.org/10.3390/ijms23063406
- Lena Ullmann, Lars M. Blank. Entwicklung von Ustilago als Chassis für die CO2-neutrale Itakonatproduktion. BIOspektrum 2022, 28
(1)
, 97-100. https://doi.org/10.1007/s12268-022-1689-6
- Liliana Calzadiaz-Ramirez, Anne S Meyer. Formate dehydrogenases for CO2 utilization. Current Opinion in Biotechnology 2022, 73 , 95-100. https://doi.org/10.1016/j.copbio.2021.07.011
- Yingying Xu, Jie Ren, Wei Wang, An‐Ping Zeng. Improvement of glycine biosynthesis from one‐carbon compounds and ammonia catalyzed by the glycine cleavage system in vitro. Engineering in Life Sciences 2022, 22
(1)
, 40-53. https://doi.org/10.1002/elsc.202100047
- Nico J. Claassens, Ari Satanowski, Viswanada R. Bysani, Beau Dronsella, Enrico Orsi, Vittorio Rainaldi, Suzan Yilmaz, Sebastian Wenk, Steffen N. Lindner. Engineering the Reductive Glycine Pathway: A Promising Synthetic Metabolism Approach for C1-Assimilation. 2022, 299-350. https://doi.org/10.1007/10_2021_181
- Jie Ren, Wei Wang, Jinglei Nie, Wenqiao Yuan, An-Ping Zeng. Understanding and Engineering Glycine Cleavage System and Related Metabolic Pathways for C1-Based Biosynthesis. 2022, 273-298. https://doi.org/10.1007/10_2021_186
- Yaeseong Hong, An-Ping Zeng. Biosynthesis Based on One-Carbon Mixotrophy. 2022, 351-371. https://doi.org/10.1007/10_2021_198
- Thomas A. Dixon, Thomas C. Williams, Isak S. Pretorius. Bioinformational trends in grape and wine biotechnology. Trends in Biotechnology 2022, 40
(1)
, 124-135. https://doi.org/10.1016/j.tibtech.2021.05.001
- Nils Guntermann, Hendrik G. Mengers, Giancarlo Franciò, Lars M. Blank, Walter Leitner. Bio-energy conversion with carbon capture and utilization (BECCU): integrated biomass fermentation and chemo-catalytic CO
2
hydrogenation for bioethanol and formic acid co-production. Green Chemistry 2021, 23
(24)
, 9860-9864. https://doi.org/10.1039/D1GC02915K
- Christian Simon Neuendorf, Gabriel A. Vignolle, Christian Derntl, Tamara Tomin, Katharina Novak, Robert L. Mach, Ruth Birner-Grünberger, Stefan Pflügl. A quantitative metabolic analysis reveals Acetobacterium woodii as a flexible and robust host for formate-based bioproduction. Metabolic Engineering 2021, 68 , 68-85. https://doi.org/10.1016/j.ymben.2021.09.004
- Mihris Ibnu Saleem Naduthodi, Nico J. Claassens, Sarah D’Adamo, John van der Oost, Maria J. Barbosa. Synthetic Biology Approaches To Enhance Microalgal Productivity. Trends in Biotechnology 2021, 39
(10)
, 1019-1036. https://doi.org/10.1016/j.tibtech.2020.12.010
- Wei Jiang, David Hernández Villamor, Huadong Peng, Jian Chen, Long Liu, Victoria Haritos, Rodrigo Ledesma-Amaro. Metabolic engineering strategies to enable microbial utilization of C1 feedstocks. Nature Chemical Biology 2021, 17
(8)
, 845-855. https://doi.org/10.1038/s41589-021-00836-0
- Nico J. Claassens. Reductive Glycine Pathway: A Versatile Route for One-Carbon Biotech. Trends in Biotechnology 2021, 39
(4)
, 327-329. https://doi.org/10.1016/j.tibtech.2021.02.005
- Yingying Xu, Yuchen Li, Han Zhang, Jinglei Nie, Jie Ren, Wei Wang, An-Ping Zeng. Stand-alone lipoylated H-protein of the glycine cleavage system enables glycine cleavage and the synthesis of glycine from one-carbon compounds
in vitro. 2021https://doi.org/10.1101/2021.03.28.437365
- Yaeseong Hong, Philipp Arbter, Wei Wang, Lilian N. Rojas, An‐Ping Zeng. Introduction of glycine synthase enables uptake of exogenous formate and strongly impacts the metabolism in
Clostridium pasteurianum. Biotechnology and Bioengineering 2021, 118
(3)
, 1366-1380. https://doi.org/10.1002/bit.27658
- Diep Thi Ngoc Nguyen, Ok Kyung Lee, Thu Thi Nguyen, Eun Yeol Lee. Type II methanotrophs: A promising microbial cell-factory platform for bioconversion of methane to chemicals. Biotechnology Advances 2021, 47 , 107700. https://doi.org/10.1016/j.biotechadv.2021.107700
- Yiming Zhang, Jens Nielsen, Zihe Liu. Yeast based biorefineries for oleochemical production. Current Opinion in Biotechnology 2021, 67 , 26-34. https://doi.org/10.1016/j.copbio.2020.11.009
- Qiaoyu Yang, Xiaoxian Guo, Yuwan Liu, Huifeng Jiang. Biocatalytic C-C Bond Formation for One Carbon Resource Utilization. International Journal of Molecular Sciences 2021, 22
(4)
, 1890. https://doi.org/10.3390/ijms22041890
- Lena Ullmann, An N. T. Phan, Daniel K. P. Kaplan, Lars M. Blank. Ustilaginaceae Biocatalyst for Co-Metabolism of CO2-Derived Substrates toward Carbon-Neutral Itaconate Production. Journal of Fungi 2021, 7
(2)
, 98. https://doi.org/10.3390/jof7020098
- Feng Guo, Shangjie Zhang, Yujia Jiang, Huixin Xu, Fengxue Xin, Wenming Zhang, Min Jiang. Bioconversion of Methanol by Synthetic Methylotrophy. 2021, 149-168. https://doi.org/10.1007/10_2021_176
- Irene Sánchez-Andrea, Iame Alves Guedes, Bastian Hornung, Sjef Boeren, Christopher E. Lawson, Diana Z. Sousa, Arren Bar-Even, Nico J. Claassens, Alfons J. M. Stams. The reductive glycine pathway allows autotrophic growth of Desulfovibrio desulfuricans. Nature Communications 2020, 11
(1)
https://doi.org/10.1038/s41467-020-18906-7
- Monica I. Espinosa, Ricardo A. Gonzalez-Garcia, Kaspar Valgepea, Manuel R. Plan, Colin Scott, Isak S. Pretorius, Esteban Marcellin, Ian T. Paulsen, Thomas C. Williams. Adaptive laboratory evolution of native methanol assimilation in Saccharomyces cerevisiae. Nature Communications 2020, 11
(1)
https://doi.org/10.1038/s41467-020-19390-9
- Daniel Schindler. Genetic Engineering and Synthetic Genomics in Yeast to Understand Life and Boost Biotechnology. Bioengineering 2020, 7
(4)
, 137. https://doi.org/10.3390/bioengineering7040137
- Bo Liang, Yukun Zhao, Jianming Yang. Recent Advances in Developing Artificial Autotrophic Microorganism for Reinforcing CO2 Fixation. Frontiers in Microbiology 2020, 11 https://doi.org/10.3389/fmicb.2020.592631
- Wen Mao, Qianqian Yuan, Hongge Qi, Zhiwen Wang, Hongwu Ma, Tao Chen. Recent progress in metabolic engineering of microbial formate assimilation. Applied Microbiology and Biotechnology 2020, 104
(16)
, 6905-6917. https://doi.org/10.1007/s00253-020-10725-6
- Yaeseong Hong, Jie Ren, Xinyi Zhang, Wei Wang, An-Ping Zeng. Quantitative analysis of glycine related metabolic pathways for one-carbon synthetic biology. Current Opinion in Biotechnology 2020, 64 , 70-78. https://doi.org/10.1016/j.copbio.2019.10.001
- Charles AR Cotton, Nico J Claassens, Sara Benito-Vaquerizo, Arren Bar-Even. Renewable methanol and formate as microbial feedstocks. Current Opinion in Biotechnology 2020, 62 , 168-180. https://doi.org/10.1016/j.copbio.2019.10.002
- Taicheng Zhu, Tongxin Zhao, Olufemi Emmanuel Bankefa, Yin Li. Engineering unnatural methylotrophic cell factories for methanol-based biomanufacturing: Challenges and opportunities. Biotechnology Advances 2020, 39 , 107467. https://doi.org/10.1016/j.biotechadv.2019.107467
- Zihe Liu, Kai Wang, Yun Chen, Tianwei Tan, Jens Nielsen. Third-generation biorefineries as the means to produce fuels and chemicals from CO2. Nature Catalysis 2020, 3
(3)
, 274-288. https://doi.org/10.1038/s41929-019-0421-5
- Rahul Kumar, Petri-Jaan Lahtvee. Proteome overabundance enables respiration but limitation onsets carbon overflow. 2020https://doi.org/10.1101/2020.02.20.957662
- Xiaojia Guo, Yuxue Liu, Qian Wang, Xueying Wang, Qing Li, Wujun Liu, Zongbao K. Zhao. Non‐natural Cofactor and Formate‐Driven Reductive Carboxylation of Pyruvate. Angewandte Chemie 2020, 132
(8)
, 3167-3170. https://doi.org/10.1002/ange.201915303
- Xiaojia Guo, Yuxue Liu, Qian Wang, Xueying Wang, Qing Li, Wujun Liu, Zongbao K. Zhao. Non‐natural Cofactor and Formate‐Driven Reductive Carboxylation of Pyruvate. Angewandte Chemie International Edition 2020, 59
(8)
, 3143-3146. https://doi.org/10.1002/anie.201915303
- Zhengshan Luo, Weizhu Zeng, Guocheng Du, Jian Chen, Jingwen Zhou. Enhancement of pyruvic acid production in Candida glabrata by engineering hypoxia-inducible factor 1. Bioresource Technology 2020, 295 , 122248. https://doi.org/10.1016/j.biortech.2019.122248
- Monica I. Espinosa, Thomas C. Williams, Isak S. Pretorius, Ian T. Paulsen. Benchmarking two Saccharomyces cerevisiae laboratory strains for growth and transcriptional response to methanol. Synthetic and Systems Biotechnology 2019, 4
(4)
, 180-188. https://doi.org/10.1016/j.synbio.2019.10.001
- Monica I. Espinosa, Ricardo A. Gonzalez-Garcia, Kaspar Valgepea, Manuel Plan, Colin Scott, Isak S. Pretorius, Esteban Marcellin, Ian T. Paulsen, Thomas C. Williams. Engineering and Evolution of Methanol Assimilation in
Saccharomyces cerevisiae. 2019https://doi.org/10.1101/717942
Learn about these metrics
Article Views are the COUNTER-compliant sum of full text article downloads since November 2008 (both PDF and HTML) across all institutions and individuals. These metrics are regularly updated to reflect usage leading up to the last few days.
Citations are the number of other articles citing this article, calculated by Crossref and updated daily. Find more information about Crossref citation counts.
The Altmetric Attention Score is a quantitative measure of the attention that a research article has received online. Clicking on the donut icon will load a page at altmetric.com with additional details about the score and the social media presence for the given article. Find more information on the Altmetric Attention Score and how the score is calculated.
Recommended Articles
Abstract
Figure 1
Figure 1. Reductive glycine pathway and a selection scheme for its activity in yeast. (A) The “metabolic engine” of the reductive glycine pathway: condensation of C1-moieties into the C2 compound glycine. Substructure of tetrahydrofolate (THF) is shown in brown. Lipoic acid attached to the H-protein of the glycine cleavage/synthase system (GCS) is shown in green. (B) Gene deletions (marked in red) required for the construction of a glycine auxotroph strain, which we used to select for glycine biosynthesis from the activity of the reductive glycine pathway; pathway enzymes are shown in green.
Figure 2
Figure 2. Three plasmids harboring genes encoding for different subsets of the enzymes of the reductive glycine pathway. pJGC1 harbors only the gene that encodes for MIS1, a trifunctional enzyme that converts formate to methylene-THF. pJGC2 harbors the genes encoding for the subunits of the GCS (the gene encoding for dihydrolipoamide dehydrogenase, LPD1, was not overexpressed since we reasoned its native expression would suffice as it participates in other complexes in the mitochondria, i.e., pyruvate dehydrogenase and 2-ketoglutarate dehydrogenase). pJGC3 harbors the genes encoding for MIS1 and the enzymes of the GCS. Each gene was regulated by a different strong, constitutive promoter as shown in the figure. Each plasmid was based on the pL1A-lc vector backbone as explained in the Methods section.
Figure 3
Figure 3. Formate-dependent growth. (A) Growth of the glycine auxotroph strain harboring the pJGC3 plasmid using different concentrations of formate, 2% glucose and 10% CO2. “No OE” refers to the negative control, i.e., a glycine auxotroph strain without a plasmid, while “No OE + glycine” refers to the positive control, i.e., a glycine auxotroph strain without a plasmid where glycine was added to the medium. Each curve represents the average of three replicates, which were not different by more than 10%. Growth curves were cut after reaching stationary phase. (B) Calculated growth rate as a function of formate concentration. Growth rate increases with increasing formate concentration up to 1 mM, remains rather stable up to 500 mM, and then sharply decreases with higher concentrations. .
Figure 4
Figure 4. 13C-labeling experiments confirm glycine production from formate. Fraction of labeling of different amino acids in different strains and labeled feedstocks is shown. “G” corresponds to glycine, “S” to serine, “A” to alanine, “M” to methionine, and “T” to threonine. Complete labeling of glycine in the glycine auxotroph strain harboring pJGC3 upon feeding with 13C-formate confirms that glycine biosynthesis occurs only via the reductive glycine pathway. Partial labeling of glycine with 13C-CO2 is attributed to the high production rate of unlabeled CO2 in the mitochondria. See main text for a detailed discussion on the labeling pattern of these amino acids.
References
This article references 34 other publications.
- 1Vorholt, J. A. (2012) Microbial life in the phyllosphere. Nat. Rev. Microbiol. 10, 828– 840, DOI: 10.1038/nrmicro29101https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC38Xhs1Ogs7jO&md5=04dcdc5f7e3ba05dda35863a5ea327bfMicrobial life in the phyllosphereVorholt, Julia A.Nature Reviews Microbiology (2012), 10 (12), 828-840CODEN: NRMACK; ISSN:1740-1526. (Nature Publishing Group)A review. Our knowledge of the microbiol. of the phyllosphere, or the aerial parts of plants, has historically lagged behind our knowledge of the microbiol. of the rhizosphere, or the below-ground habitat of plants, particularly with respect to fundamental questions such as which microorganisms are present and what they do there. In recent years, however, this has begun to change. Cultivation-independent studies have revealed that a few bacterial phyla predominate in the phyllosphere of different plants and that plant factors are involved in shaping these phyllosphere communities, which feature specific adaptations and exhibit multipartite relationships both with host plants and among community members. Insights into the underlying structural principles of indigenous microbial phyllosphere populations will help us to develop a deeper understanding of the phyllosphere microbiota and will have applications in the promotion of plant growth and plant protection.
- 2Abubackar, H. N., eiga, M. C., and Kennes, C. (2011) Biological conversion of carbon monoxide: rich syngas or waste gases to bioethanol. Biofuels, Bioprod. Biorefin. 5, 93– 114, DOI: 10.1002/bbb.2562https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3MXnvVaruw%253D%253D&md5=ca9c58a89bbb972d7c61e1b727ea1cbcBiological conversion of carbon monoxide: rich syngas or waste gases to bioethanolAbubackar, Haris Nalakath; Veiga, Maria C.; Kennes, ChristianBiofuels, Bioproducts & Biorefining (2011), 5 (1), 93-114CODEN: BBBICH; ISSN:1932-104X. (John Wiley & Sons Ltd.)A review. Bioconversion of syngas/waste gas components to produce ethanol appears to be a promising alternative compared to the existing chem. techniques. Recently, several lab.-scale studies have demonstrated the use of acetogens that have the ability to convert various syngas components (CO, CO2, and H2) to multicarbon compds., such as acetate, butyrate, butanol, lactate, and ethanol, in which ethanol is often produced as a minor end-product. This bioconversion process has several advantages, such as its high specificity, the fact that it does not require a highly specific H2/CO ratio, and that biocatalysts are less susceptible to metal poisoning. Furthermore, this process occurs under mild temp. and pressure and does not require any costly pre-treatment of the feed gas or costly metal catalysts, making the process superior over the conventional chem. catalytic conversion process. The main challenge faced for commercializing this technol. is the poor aq. soly. of the gaseous substrates (mainly CO and H2). In this paper, a crit. review of CO-rich gas fermn. to produce ethanol has been analyzed systematically and published results have been compared. Special emphasis has been given to understand the microbial aspects of the conversion process, by highlighting the role of different micro-organisms used, pathways, and parameters affecting the bioconversion. An anal. of the process fundamentals of various bioreactors used for the biol. conversion of CO-rich gases, mainly syngas to ethanol, has been made and reported in this paper. Various challenges faced by the syngas fermn. process for commercialization and future research requirements are also discussed.
- 3Kopljar, D., Inan, A., Vindayer, P., Wagner, N., and Klemm, E. (2014) Electrochemical reduction of CO2 to formate at high current density using gas diffusion electrodes. J. Appl. Electrochem. 44, 1107– 1116, DOI: 10.1007/s10800-014-0731-x3https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2cXhsVWkt7vF&md5=fb54c80ec5d782ad34f53b4e73d93d22Electrochemical reduction of CO2 to formate at high current density using gas diffusion electrodesKopljar, D.; Inan, A.; Vindayer, P.; Wagner, N.; Klemm, E.Journal of Applied Electrochemistry (2014), 44 (10), 1107-1116CODEN: JAELBJ; ISSN:0021-891X. (Springer)The electrochem. redn. of carbon dioxide into formate was studied using gas diffusion electrodes (GDE) with Sn as electrocatalyst in order to overcome mass transport limitations and to achieve high current densities. For this purpose, a dry pressing method was developed for GDE prepn. and optimized with respect to mech. stability and the performance in the redn. of CO2. Using this approach, GDEs can be obtained with a high reproducibility in a very simple, fast, and straightforward manner. The influence of the metal loading on c.d. and product distribution was investigated. Furthermore, the effect of changing the electrolyte pH was evaluated. Under optimized conditions, the GDE allowed current densities up to 200 mA cm-2 to be achieved with a Faradaic efficiency of around 90 % toward formate and a substantial suppression of hydrogen prodn. (<3 %) at ambient pressure. At higher current densities mass transport issues come into effect and hydrogen is increasingly produced. The corresponding cathode potential was found to be 1.57 V vs. SHE.
- 4Yang, H., Kaczur, J. J., Sajjad, S. D., and Masel, R. I. (2017) Electrochemical conversion of CO2 to formic acid utilizing Sustainion membranes. Journal of CO2 Utilization 20, 208– 217, DOI: 10.1016/j.jcou.2017.04.0114https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2sXpvFeltb4%253D&md5=2c2323d1e4095c98baabe3fe925b7e81Electrochemical conversion of CO2 to formic acid utilizing Sustainion membranesYang, Hongzhou; Kaczur, Jerry J.; Sajjad, Syed Dawar; Masel, Richard I.Journal of CO2 Utilization (2017), 20 (), 208-217CODEN: JCUOAJ; ISSN:2212-9839. (Elsevier Ltd.)Formic acid generated from CO2 has been proposed both as a key intermediate renewable chem. feedstock as well as a potential chem.-based energy storage media for hydrogen. In this paper, we describe a novel three-compartment electrochem. cell configuration with the capability of directly producing a pure formic acid product in the concn. range of 5-20 wt% at high current densities and Faradaic yields. The electrochem. cell employs a Dioxide Materials Sustainion anion exchange membrane and a nanoparticle Sn GDE cathode contg. an imidazole ionomer, allowing for improved CO2 electrochem. redn. performance. Stable electrochem. cell performance for more than 500 h was exptl. demonstrated at a c.d. of 140 mA cm-2 at a cell voltage of only 3.5 V. Future work will include cell scale-up and increasing cell Faradaic performance using selected electrocatalysts and membranes.
- 5Bennett, R. K., Steinberg, L. M., Chen, W., and Papoutsakis, E. T. (2018) Engineering the bioconversion of methane and methanol to fuels and chemicals in native and synthetic methylotrophs. Curr. Opin. Biotechnol. 50, 81– 93, DOI: 10.1016/j.copbio.2017.11.0105https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2sXhvVOrsLvL&md5=e54377475ba45693d57a2a4385b88372Engineering the bioconversion of methane and methanol to fuels and chemicals in native and synthetic methylotrophsBennett, R. Kyle; Steinberg, Lisa M.; Chen, Wilfred; Papoutsakis, Eleftherios T.Current Opinion in Biotechnology (2018), 50 (), 81-93CODEN: CUOBE3; ISSN:0958-1669. (Elsevier B.V.)Methylotrophy describes the ability of organisms to utilize reduced one-carbon compds., notably methane and methanol, as growth and energy sources. Abundant natural gas supplies, composed primarily of methane, have prompted interest in using these compds., which are more reduced than sugars, as substrates to improve product titers and yields of bioprocesses. Engineering native methylotophs or developing synthetic methylotrophs are emerging fields to convert methane and methanol into fuels and chems. under aerobic and anaerobic conditions. This review discusses recent progress made toward engineering native methanotrophs for aerobic and anaerobic methane utilization and synthetic methylotrophs for methanol utilization. Finally, strategies to overcome the limitations involved with synthetic methanol utilization, notably methanol dehydrogenase kinetics and ribulose 5-phosphate regeneration, are discussed.
- 6Bengelsdorf, F. R. and Durre, P. (2017) Gas fermentation for commodity chemicals and fuels. Microb. Biotechnol. 10, 1167– 1170, DOI: 10.1111/1751-7915.127636https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A280%3ADC%252BC1cjmsVSksg%253D%253D&md5=19d3e29d23f4194e3806cfecece8cdbaGas fermentation for commodity chemicals and fuelsBengelsdorf Frank R; Durre PeterMicrobial biotechnology (2017), 10 (5), 1167-1170 ISSN:.Gas fermentation is a microbial process that contributes to at least four of the sustainable development goals (SDGs) of the United Nations. The process converts waste and greenhouse gases into commodity chemicals and fuels. Thus, world's climate is positively affected. Briefly, we describe the background of the process, some biocatalytic strains, and legal implications.
- 7Yishai, O., Lindner, S. N., Gonzalez de la Cruz, J., Tenenboim, H., and Bar-Even, A. (2016) The formate bio-economy. Curr. Opin. Chem. Biol. 35, 1– 9, DOI: 10.1016/j.cbpa.2016.07.0057https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC28Xht1CksL%252FL&md5=f265a337019047fe6d9233688a70a52bThe formate bio-economyYishai, Oren; Lindner, Steffen N.; Gonzalez de la Cruz, Jorge; Tenenboim, Hezi; Bar-Even, ArrenCurrent Opinion in Chemical Biology (2016), 35 (), 1-9CODEN: COCBF4; ISSN:1367-5931. (Elsevier B.V.)In this review we discuss the concept of the formate bio-economy: formate can be produced efficiently from various available resources and can be consumed by microbes as the sole carbon source for the prodn. of value-added chems., directly addressing major challenges in energy storage and chem. prodn. We show that the formate assimilation pathways utilized by natural formatotrophs are either inefficient or are constrained to organisms that are difficult to cultivate and engineer. Instead, adapting model industrial organisms to formatotrophic growth using synthetic, specially tailored formate-assimilation routes could prove an advantageous strategy. Several studies have started to tackle this challenge, but a fully active synthetic pathway has yet to be established, leaving room for future undertakings.
- 8Naik, S. N., Goud, V. V., Rout, P. K., and Dalai, A. K. (2010) Production of first and second generation biofuels: a comprehensive review. Renewable Sustainable Energy Rev. 14, 578– 597, DOI: 10.1016/j.rser.2009.10.0038https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD1MXhsF2isbvF&md5=5c93f3970ae56e2ee6b7a9763c1677beProduction of first and second generation biofuels: A comprehensive reviewNaik, S. N.; Goud, Vaibhav V.; Rout, Prasant K.; Dalai, Ajay K.Renewable & Sustainable Energy Reviews (2010), 14 (2), 578-597CODEN: RSERFH; ISSN:1364-0321. (Elsevier Ltd.)A review. Sustainable economic and industrial growth requires safe, sustainable resources of energy. For the future re-arrangement of a sustainable economy to biol. raw materials, completely new approaches in research and development, prodn., and economy are necessary. The first-generation' biofuels appear unsustainable because of the potential stress that their prodn. places on food commodities. For org. chems. and materials these needs to follow a biorefinery model under environmentally sustainable conditions. Where these operate at present, their product range is largely limited to simple materials (i.e. cellulose, ethanol, and biofuels). Second generation biorefineries need to build on the need for sustainable chem. products through modern and proven green chem. technologies such as bioprocessing including pyrolysis, Fisher Tropsch, and other catalytic processes in order to make more complex mols. and materials on which a future sustainable society will be based. This review focus on cost effective technologies and the processes to convert biomass into useful liq. biofuels and bioproducts, with particular focus on some biorefinery concepts based on different feedstocks aiming at the integral utilization of these feedstocks for the prodn. of value added chems.
- 9Drake, H. L., Küsel, K., and Matthies, C. (2013) Acetogenic prokaryotes, In The Prokaryotes, pp 3– 60, Springer, Berlin, Heidelberg.There is no corresponding record for this reference.
- 10Anthony, C. (1982) The Biochemistry of Methylotrophs, Academic Press, London, New York.There is no corresponding record for this reference.
- 11Bar-Even, A. (2016) Formate Assimilation: The Metabolic Architecture of Natural and Synthetic Pathways. Biochemistry 55, 3851– 3863, DOI: 10.1021/acs.biochem.6b0049511https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC28XhtVGjtLjJ&md5=e866cc5b94b857c1b8038b370c208db3Formate Assimilation: The Metabolic Architecture of Natural and Synthetic PathwaysBar-Even, ArrenBiochemistry (2016), 55 (28), 3851-3863CODEN: BICHAW; ISSN:0006-2960. (American Chemical Society)Formate may become an ideal mediator between the physicochem. and biol. realms, as it can be produced efficiently from multiple available sources, such as electricity and biomass, and serve as one of the simplest org. compds. for providing both carbon and energy to living cells. However, limiting the realization of formate as a microbial feedstock is the low diversity of formate-fixing enzymes and thereby the small no. of naturally occurring formate-assimilation pathways. Here, the natural enzymes and pathways supporting formate assimilation are presented and discussed together with proposed synthetic routes that could permit growth on formate via existing as well as novel formate-fixing reactions. By considering such synthetic routes, the diversity of metabolic solns. for formate assimilation can be expanded dramatically, such that different host organisms, cultivation conditions, and desired products could be matched with the most suitable pathway. Astute application of old and new formate-assimilation pathways may thus become a cornerstone in the development of sustainable strategies for microbial prodn. of value-added chems.
- 12Erb, T. J., Jones, P. R., and Bar-Even, A. (2017) Synthetic metabolism: metabolic engineering meets enzyme design. Curr. Opin. Chem. Biol. 37, 56– 62, DOI: 10.1016/j.cbpa.2016.12.02312https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2sXhvFyrsLs%253D&md5=325943c541fdc461f8d7f2548ee8fe7eSynthetic metabolism: metabolic engineering meets enzyme designErb, Tobias J.; Jones, Patrik R.; Bar-Even, ArrenCurrent Opinion in Chemical Biology (2017), 37 (), 56-62CODEN: COCBF4; ISSN:1367-5931. (Elsevier B.V.)Metabolic engineering aims at modifying the endogenous metabolic network of an organism to harness it for a useful biotechnol. task, for example, prodn. of a value-added compd. Several levels of metabolic engineering can be defined and are the topic of this review. Basic 'copy, paste and fine-tuning' approaches are limited to the structure of naturally existing pathways. 'Mix and match' approaches freely recombine the repertoire of existing enzymes to create synthetic metabolic networks that are able to outcompete naturally evolved pathways or redirect flux toward non-natural products. The space of possible metabolic soln. can be further increased through approaches including 'new enzyme reactions', which are engineered on the basis of known enzyme mechanisms. Finally, by considering completely 'novel enzyme chemistries' with de novo enzyme design, the limits of nature can be breached to derive the most advanced form of synthetic pathways. We discuss the challenges and promises assocd. with these different metabolic engineering approaches and illuminate how enzyme engineering is expected to take a prime role in synthetic metabolic engineering for biotechnol., chem. industry and agriculture of the future.
- 13Bar-Even, A., Noor, E., Flamholz, A., and Milo, R. (2013) Design and analysis of metabolic pathways supporting formatotrophic growth for electricity-dependent cultivation of microbes. Biochim. Biophys. Acta, Bioenerg. 1827, 1039– 1047, DOI: 10.1016/j.bbabio.2012.10.01313https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC38Xhs1KqsrjF&md5=9ae940dc5a31b77357bc6d44ac9fdccbDesign and analysis of metabolic pathways supporting formatotrophic growth for electricity-dependent cultivation of microbesBar-Even, Arren; Noor, Elad; Flamholz, Avi; Milo, RonBiochimica et Biophysica Acta, Bioenergetics (2013), 1827 (8-9), 1039-1047CODEN: BBBEB4; ISSN:0005-2728. (Elsevier B. V.)A review, with commentary. Electrosynthesis is a promising approach that enables the biol. prodn. of commodities, like fuels and fine chems., using renewably produced electricity. Several techniques have been proposed to mediate the transfer of electrons from the cathode to living cells. Of these, the electroprodn. of formate as a mediator seems esp. promising: formate is readily sol., of low toxicity and can be produced at relatively high efficiency and at reasonable c.d. While organisms that are capable of formatotrophic growth, i.e. growth on formate, exist naturally, they are generally less suitable for bulk cultivation and industrial needs. Hence, it may be helpful to engineer a model organism of industrial relevance, such as E. coli, for growth on formate. There are numerous metabolic pathways that can potentially support formatotrophic growth. Here we analyze these diverse pathways according to various criteria including biomass yield, thermodn. favorability, chem. motive force, kinetics and the practical challenges posed by their expression. We find that the reductive glycine pathway, composed of the tetrahydrofolate system, the glycine cleavage system, serine hydroxymethyltransferase and serine deaminase, is a promising candidate to support electrosynthesis in E. coli. The approach presented here exemplifies how combining different computational approaches into a systematic anal. methodol. provides assistance in redesigning metab. This article is part of a Special Issue entitled: Metals in Bioenergetics and Biomimetics Systems.
- 14Fuchs, G. (1986) CO2 fixation in acetogenic bacteria: variations on a theme. FEMS Microbiol. Lett. 39, 181– 213, DOI: 10.1111/j.1574-6968.1986.tb01859.xThere is no corresponding record for this reference.
- 15Schneeberger, A., Frings, J., and Schink, B. (1999) Net synthesis of acetate from CO2 by Eubacterium acidaminophilum through the glycine reductase pathway. FEMS Microbiol. Lett. 177, 1, DOI: 10.1111/j.1574-6968.1999.tb13705.x15https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaK1MXls1SmsLo%253D&md5=2bf6878a461fe7c70762757287d4145bNet synthesis of acetate from CO2 by Eubacterium acidaminophilum through the glycine reductase pathwaySchneeberger, Anne; Frings, Jochen; Schink, BernhardFEMS Microbiology Letters (1999), 177 (1), 1-6CODEN: FMLED7; ISSN:0378-1097. (Elsevier Science B.V.)Eubacterium acidaminophilum combines the oxidn. of amino acids such as alanine or valine with the redn. of glycine to acetate in a two-substrate fermn. (Stickland reaction). In the absence of glycine, dense cell suspensions oxidized alanine or valine only to a small extent, with limited prodn. of hydrogen and acetate. Expts. with 14C-labeled carbonate revealed that acetate was formed under these conditions by net redn. of CO2/HCO3-; 14C-labeled formate was formed as an intermediate. E. acidaminophilum did not grow with hydrogen plus CO2; dense cell suspensions under H2/CO2 produced only very small amts. (<0.5 mM) of acetate. There was no activity of carbon monoxide dehydrogenase, indicating that the glycine pathway was used for acetate synthesis. The results are explained on the basis of biochem. and energetic considerations.
- 16Yishai, O., Bouzon, M., Doring, V., and Bar-Even, A. (2018) In Vivo Assimilation of One-Carbon via a Synthetic Reductive Glycine Pathway in Escherichia coli. ACS Synth. Biol. 7, 2023, DOI: 10.1021/acssynbio.8b0013116https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC1cXpsVChs7s%253D&md5=5f9029d1c836fcf2c889c15b830dfd04In Vivo Assimilation of One-Carbon via a Synthetic Reductive Glycine Pathway in Escherichia coliYishai, Oren; Bouzon, Madeleine; Doering, Volker; Bar-Even, ArrenACS Synthetic Biology (2018), 7 (9), 2023-2028CODEN: ASBCD6; ISSN:2161-5063. (American Chemical Society)Assimilation of one-carbon compds. presents a key biochem. challenge that limits their use as sustainable feedstocks for microbial growth and prodn. The reductive glycine pathway is a synthetic metabolic route that could provide an optimal way for the aerobic assimilation of reduced C1 compds. Here, we show that a rational integration of native and foreign enzymes enables the tetrahydrofolate and glycine cleavage/synthase systems to operate in the reductive direction, such that Escherichia coli satisfies all of its glycine and serine requirements from the assimilation of formate and CO2. Importantly, the biosynthesis of serine from formate and CO2 does not lower the growth rate, indicating high flux that is able to provide 10% of cellular carbon. Our findings assert that the reductive glycine pathway could support highly efficient aerobic assimilation of C1-feedstocks.
- 17Pasternack, L. B., Laude, D. A., Jr., and Appling, D. R. (1992) 13C NMR detection of folate-mediated serine and glycine synthesis in vivo in Saccharomyces cerevisiae. Biochemistry 31, 8713– 8719, DOI: 10.1021/bi00152a00517https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaK38XlsVKru7s%253D&md5=d0d13ec236e34bf5bc874461c21b980aCarbon-13 NMR detection of folate-mediated serine and glycine synthesis in vivo in Saccharomyces cerevisiaePasternack, Laura B.; Laude, David A., Jr.; Appling, Dean R.Biochemistry (1992), 31 (37), 8713-19CODEN: BICHAW; ISSN:0006-2960.S. cerevisiae has both cytoplasmic and mitochondrial C1-tetrahydrofolate (THF) synthases. These trifunctional isoenzymes are central to C1 metab. and are responsible for interconversion of the THF derivs. in the resp. compartments. 13C-NMR was used to study folate-mediated C1 metab. in these 2 compartments, using glycine and serine synthesis as metabolic endpoints. The availability of yeast strains carrying deletions of cytoplasmic and/or mitochondrial C1-THF synthase allows a dissection of the role each compartment plays in this metab. When yeast are incubated with [13C]formate, 13C-NMR spectra establish that prodn. of [3-13C]serine is dependent on C1-THF synthase and occurs primarily in the cytosol. However, in a strain lacking cytoplasmic C1-THF synthase but possessing the mitochondrial isoenzyme, [13C]formate can be metabolized to [2-13C]glycine and [3-13C]serine. This provides in vivo evidence for the mitochondrial assimilation of formate, activation and conversion to [13C]CH2-THF via mitochondrial C1-THF synthase, and subsequent glycine synthesis via reversal of the glycine cleavage system. Addnl. supporting evidence of reversibility of the glycine cleavage system in vivo is the prodn. of [2-13C]glycine and [2,3-13C]serine in yeast strains grown with [3-13C]serine. This metab. is independent of C1-THF synthase, since these products were obsd. in strains lacking both the cytoplasmic and mitochondrial isoenzymes. These results suggest that when formate is the C1 donor, assimilation is primarily cytoplasmic, whereas when serine serves as C1 donor, considerable metab. occurs via mitochondrial pathways.
- 18Maaheimo, H., Fiaux, J., Cakar, Z. P., Bailey, J. E., Sauer, U., and Szyperski, T. (2001) Central carbon metabolism of Saccharomyces cerevisiae explored by biosynthetic fractional (13)C labeling of common amino acids. Eur. J. Biochem. 268, 2464– 2479, DOI: 10.1046/j.1432-1327.2001.02126.x18https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD3MXjt1Wlsrw%253D&md5=122ba33dbef44434483755f2bc398f23Central carbon metabolism of Saccharomyces cerevisiae explored by biosynthetic fractional 13C labeling of common amino acidsMaaheimo, Hannu; Fiaux, Jocelyne; Cakar, Z. Petek; Bailey, James E.; Sauer, Uwe; Szyperski, ThomasEuropean Journal of Biochemistry (2001), 268 (8), 2464-2479CODEN: EJBCAI; ISSN:0014-2956. (Blackwell Science Ltd.)Aerobic and anaerobic central metab. of Saccharomyces cerevisiae cells was explored in batch cultures on a minimal medium contg. glucose as the sole carbon source, using biosynthetic fractional 13C labeling of proteinogenic amino acids. This allowed, firstly, unravelling of the network of active central pathways in cytosol and mitochondria, secondly, detn. of flux ratios characterizing glycolysis, pentose phosphate cycle, tricarboxylic acid cycle and Cl-metab., and thirdly, assessment of intercompartmental transport fluxes of pyruvate, acetyl-CoA, oxaloacetate and glycine. The data also revealed that alanine aminotransferase is located in the mitochondria, and that amino acids are synthesized according to documented pathways. In both the aerobic and the anaerobic regime: (a) the mitochondrial glycine cleavage pathway is active, and efflux of glycine into the cytosol is obsd.; (b) the pentose phosphate pathways serve for biosynthesis only, i.e. phosphoenolpyruvate is entirely generated via glycolysis; (c) the majority of the cytosolic oxaloacetate is synthesized via anaplerotic carboxylation of pyruvate; (d) the malic enzyme plays a key role for mitochondrial pyruvate metab.; (e) the transfer of oxaloacetate from the cytosol to the mitochondria is largely unidirectional, and the activity of the malate-aspartate shuttle and the succinate-fumarate carrier is low; (e) a large fraction of the mitochondrial pyruvate is imported from the cytosol; and (f) the glyoxylate cycle is inactive. In the aerobic regime, 75% of mitochondrial oxaloacetate arises from anaplerotic carboxylation of pyruvate, while in the anaerobic regime, the tricarboxylic acid cycle is operating in a branched fashion to fulfill biosynthetic demands only. The present study shows that fractional 13C labeling of amino acids represents a powerful approach to study compartmented eukaryotic systems.
- 19Schlosser, T., Gatgens, C., Weber, U., and Stahmann, K. P. (2004) Alanine: glyoxylate aminotransferase of Saccharomyces cerevisiae-encoding gene AGX1 and metabolic significance. Yeast 21, 63– 73, DOI: 10.1002/yea.105819https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A280%3ADC%252BD2c%252FksFWitw%253D%253D&md5=c8543750a284cef4e2ac7fd254c876c7Alanine : glyoxylate aminotransferase of Saccharomyces cerevisiae-encoding gene AGX1 and metabolic significanceSchlosser Thomas; Gatgens Cornelia; Weber Ulrike; Stahmann K-PeterYeast (Chichester, England) (2004), 21 (1), 63-73 ISSN:0749-503X.Alanine : glyoxylate aminotransferase is one of three different enzymes used for glycine synthesis in Saccharomyces cerevisiae. The open reading frame YFL030w (named AGX1 in the following), encoding this enzyme, was identified by comparing enzyme specific activities in knockout strains. While 100% activity was detectable in the parental strain, 2% was found in a YFL030w::kanMX4 strain. The ORF found at that locus was suspected to encode alanine : glyoxylate aminotransferase because its predicted amino acid sequence showed 23% identity to the human homologue. Since the YFL030w::kanMX4 strain showed no glycine auxtrophic phenotype, AGX1 was replaced by KanMX4 in a Delta GLY1 Delta SHM1 Delta SHM2 background. These background mutations, which cause inactivation of threonine aldolase, mitochondrial and cytosolic serine hydroxymethyltransferase, respectively, lead to a conditional glycine auxotrophy. This means that growth is not possible on glucose but on ethanol as the sole carbon source. Additional disruption of AGX1 revealed a complete glycine auxotrophy. Complementation was observed by transformation with a plasmid-encoded AGX1.
- 20Shannon, K. W. and Rabinowitz, J. C. (1988) Isolation and characterization of the Saccharomyces cerevisiae MIS1 gene encoding mitochondrial C1-tetrahydrofolate synthase. J. Biol. Chem. 263, 7717– 772520https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaL1MXjsVGksA%253D%253D&md5=b0c4885ba2870f799227fe8c39ab9836Isolation and characterization of the Saccharomyces cerevisiae MIS1 gene encoding mitochondrial C1-tetrahydrofolate synthaseShannon, Karen W.; Rabinowitz, Jesse C.Journal of Biological Chemistry (1988), 263 (16), 7717-25CODEN: JBCHA3; ISSN:0021-9258.C1-Tetrahydrofolate synthase is a trifunctional polypeptide found in eukaryotic organisms that catalyzes 10-formyltetrahydrofolate synthetase (EC 6.3.4.3), 5,10-methenyltetrahydrofolate cyclohydrolase (EC 3.5.4.9), and 5,10-methylenetetrahydrofolate dehydrogenase (EC 1.5.1.5) activities. In S. cereivisae, C1-tetrahydrofolate synthase is found in both the cytoplasm and the mitochondria. The gene encoding yeast mitochondrial C1-tetrahydrofolate synthase was isolated using synthetic oligonucleotide probes based on the N-terminal sequence of the purified protein. Hybridization anal. shows that the gene (designated MIS1) has a single copy in the yeast genome. The predicted amino acid sequence of mitochondrial C1-tetrahydrofolate synthase and shares 39% identity with clostridial 10-formyltetrahydrofolate synthetase. Chromosomal deletions of the mitochondrial C1-tetrahydrofolate synthase gene were generated using the cloned MIS1 gene. Mutant strains which lack a functional MIS1 gene are viable and can grow in medium contg. a nonfermentable carbon source. In fact, deletion of the MIS1 locus has no detectable effect on cell growth.
- 21Hochrein, L., Machens, F., Gremmels, J., Schulz, K., Messerschmidt, K., and Mueller-Roeber, B. (2017) AssemblX: a user-friendly toolkit for rapid and reliable multi-gene assemblies. Nucleic Acids Res. gkx034, DOI: 10.1093/nar/gkx034There is no corresponding record for this reference.
- 22Klein, S. M. and Sagers, R. D. (1966) Glycine metabolism. II. Kinetic and optical studies on the glycine decarboxylase system from Peptococcus glycinophilus. J. Biol. Chem. 241, 206– 20922https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaF2MXltVWhtrg%253D&md5=046a9abbb589332545a46ef7542b8d7cGlycine metabolism. II. Kinetic and optical studies on the glycine decarboxylase system from Peptococcus glycinophilusKlein, Sigrid M.; Sagers, Richard D.Journal of Biological Chemistry (1966), 241 (1), 206-9CODEN: JBCHA3; ISSN:0021-9258.Kinetic and optical properties of the pyridoxal phosphate-contg. enzyme and the heat-stable, low mol. wt. protein required for the exchange of bicarbonate with the glycine carboxyl group are described. Satn. curves were detd. for glycine, bicarbonate, pyridoxal phosphate, and the heat-stable protein P2; and Michaelis consts., resp., of 0.032M, 0.931M, 4.6μM, and 1.3 mg./ml. were obtained. Treatment of the enzyme with cysteine removed the coenzyme and resulted in greatly decreased activity, but the activity was fully restored by incubation of the apoenzyme with pyridoxal phosphate. The ultraviolet absorption spectrum of the holoenzyme showed a max. at 430 mμ which was reduced 85% by treatment with cysteine, but which was restored by incubation with pyridoxal phosphate. Fluorescence max. at 390 and-500 mμ were-observed when the holoenzyme was subjected to activating light at 330 and 430 mμ, resp. The 500-mμ peak was assocd. with the azomethine linkage between the coenzyme and the enzyme since it was decreased by removal of pyridoxal phosphate, but the 390-mμ peak appeared to be assocd. only with the protein since it was not significantly altered by the above treatment. Cf. preceding abstr.
- 23Hiraga, K. and Kikuchi, G. (1980) The mitochondrial glycine cleavage system. Functional association of glycine decarboxylase and aminomethyl carrier protein. J. Biol. Chem. 255, 11671– 1167623https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaL3cXmtlekt7Y%253D&md5=98b0d17460c4c9406d6c14ec5eace94dThe mitochondrial glycine cleavage system. Functional association of glycine decarboxylase and aminomethyl carrier proteinHiraga, Koichi; Kikuchi, GoroJournal of Biological Chemistry (1980), 255 (24), 11671-6CODEN: JBCHA3; ISSN:0021-9258.The activity of the purified glycine decarboxylase, tentatively called P-protein and considered a constituent of the glycine cleavage system (glycine synthase, EC 2.1.2.10), which catalyzed the exchange of the glycine carboxyl C with CO2 was increased >100,000-fold by the addn. of the aminomethyl carrier protein (tentatively called H-protein), and the Km for glycine was reduced to ∼1/4 of that in the absence of H-protein. Decarboxylation of glycine was also greatly stimulated by the addn. of H-protein and the decarboxylation yielded the H-protein-bound aminomethyl moiety and CO2 at a stoichiometric ratio of unity. P-protein and H-protein formed a fairly stable complex which could be demonstrated by gel filtration and by sucrose d. gradient centrifugation. H-protein caused a significant change in the absorption spectrum of P-protein, and a titrn. expt. indicated that 2 mols. of H-protein bind to 1 mol. of P-protein, or 1 mol. of H-protein to each subunit of P-protein. H-protein also acted to reduce the dissocn. const. for methylamine, as estd. by the degree of spectral change of P-protein caused by the addn. of methylamine, from 63 mM to 27 mM, and the Kd value of 27 mM was practically equal to the Ki value (28 mM) for methylamine obtained in the glycine-CO2 exchange reaction in the presence of H-protein. H-protein seems to bring about a conformational change of P-protein which may be relevant to the expression of the decarboxylase activity of P-protein. In other words, although P-protein is, by nature, glycine decarboxylase, the functional glycine decarboxylase may be an enzyme complex composed of both P-protein and H-protein. H-protein seems to play a dual role in the glycine decarboxylation; the one as a regulatory protein of P-protein, and the other as an electron-pulling agent and concomitantly as a carrier of the aminomethyl moiety derived from glycine by decarboxylation.
- 24Warnecke, T. and Gill, R. T. (2005) Organic acid toxicity, tolerance, and production in Escherichia coli biorefining applications. Microb. Cell Fact. 4, 25, DOI: 10.1186/1475-2859-4-2524https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A280%3ADC%252BD2Mvpt1CgsA%253D%253D&md5=629ae62fd55f8487c1fde673ab1eac1aOrganic acid toxicity, tolerance, and production in Escherichia coli biorefining applicationsWarnecke Tanya; Gill Ryan TMicrobial cell factories (2005), 4 (), 25 ISSN:.Organic acids are valuable platform chemicals for future biorefining applications. Such applications involve the conversion of low-cost renewable resources to platform sugars, which are then converted to platform chemicals by fermentation and further derivatized to large-volume chemicals through conventional catalytic routes. Organic acids are toxic to many of the microorganisms, such as Escherichia coli, proposed to serve as biorefining platform hosts at concentrations well below what is required for economical production. The toxicity is two-fold including not only pH based growth inhibition but also anion-specific effects on metabolism that also affect growth. E. coli maintain viability at very low pH through several different tolerance mechanisms including but not limited to the use of decarboxylation reactions that consume protons, ion transporters that remove protons, increased expression of known stress genes, and changing membrane composition. The focus of this mini-review is on organic acid toxicity and associated tolerance mechanisms as well as several examples of successful organic acid production processes for E. coli.
- 25Nicholls, P. (1975) Formate as an inhibitor of cytochrome c oxidase. Biochem. Biophys. Res. Commun. 67, 610– 616, DOI: 10.1016/0006-291X(75)90856-625https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaE28XhsVSitw%253D%253D&md5=5273ecd954058e9353dee6685ac0b726Formate as an inhibitor of cytochrome c oxidaseNicholls, PeterBiochemical and Biophysical Research Communications (1975), 67 (2), 610-16CODEN: BBRCA9; ISSN:0006-291X.Formate inhibited cytochrome c oxidase with a Ki of 5-30mM at pH 7.4 (depending on assay conditions). The formate binding site is accessible in the fully oxidized (a3+ a33+) and partially reduced (a2+ a33+) states, but not in the fully reduced (a2+ a32+ state). Azide competed with formate for the binding site. Formate induced a blue shift in the Soret peak of fully oxidized enzyme. The rate of formate binding, and the apparent affinity (1/Ki), increased as the pH was diminished, suggesting that HCOOH is the bound species.
- 26Zaldivar, J. and Ingram, L. O. (1999) Effect of organic acids on the growth and fermentation of ethanologenic Escherichia coli LY01. Biotechnol. Bioeng. 66, 203– 210, DOI: 10.1002/(SICI)1097-0290(1999)66:4<203::AID-BIT1>3.0.CO;2-#26https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaK1MXotFamu7g%253D&md5=bbb857f3ceb346ff331df9f2fb22e1efEffect of organic acids on the growth and fermentation of ethanologenic Escherichia coli LY01Zaldivar, Jesus; Ingram, Lonnie O.Biotechnology and Bioengineering (1999), 66 (4), 203-210CODEN: BIBIAU; ISSN:0006-3592. (John Wiley & Sons, Inc.)Hemicellulose residues can be hydrolyzed into a sugar syrup using dil. mineral acids. Although this syrup represents a potential feedstock for biofuel prodn., toxic compds. generated during hydrolysis limit microbial metab. E. coli LY01, an ethanologenic biocatalyst engineered to ferment the mixed sugars in hemicellulose syrups, has been tested for resistance to selected org. acids that are present in hemicellulose hydrolyzates. Compds. tested include arom. acids derived from lignin (ferulic, gallic, 4-hydroxybenzoic, syringic, and vanillic acids), acetic acid from the hydrolysis of acetylxylan, and others derived from sugar destruction (furoic, formic, levulinic, and caproic acids). Toxicity was related to hydrophobicity. Combinations of acids were roughly additive as inhibitors of cell growth. When tested at concns. that inhibited growth by 80%, none appeared to strongly inhibit glycolysis and energy generation or to disrupt membrane integrity. Toxicity was not markedly affected by inoculum size or incubation temp. The toxicity of all acids except gallic acid was reduced by an increase in initial pH (from pH 6.0 to pH 7.0 to pH 8.0). Together, these results are consistent with the hypothesis that both aliph. and mononuclear org. acids inhibit growth and ethanol prodn. in LY01 by collapsing ion gradients and increasing internal anion concns.
- 27Overkamp, K. M., Kotter, P., van der Hoek, R., Schoondermark-Stolk, S., Luttik, M. A., van Dijken, J. P., and Pronk, J. T. (2002) Functional analysis of structural genes for NAD(+)-dependent formate dehydrogenase in Saccharomyces cerevisiae. Yeast 19, 509– 520, DOI: 10.1002/yea.85627https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD38Xjt1eis70%253D&md5=3b8989ba195bb5193eb4f0c698e248a9Functional analysis of structural genes for NAD+-dependent formate dehydrogenase in Saccharomyces cerevisiaeOverkamp, Karin M.; Kotter, Peter; Van der Hoek, Richard; Schoondermark-Stolk, Sung; Luttik, Marijke A. H.; Van Dijken, Johannes P.; Pronk, Jack T.Yeast (2002), 19 (6), 509-520CODEN: YESTE3; ISSN:0749-503X. (John Wiley & Sons Ltd.)Co-consumption of formate by aerobic, glucose-limited chemostat cultures of Saccharomyces cerevisiae CEN.PK 113-7D led to an increased biomass yield relative to cultures grown on glucose as the sole carbon and energy substrate. In this respect, this strain differed from two previously investigated S. cerevisiae strains, in which formate oxidn. did not lead to an increased biomass yield on glucose. Enzyme assays confirmed the presence of a formate-inducible, cytosolic and NAD+-dependent formate dehydrogenase. To investigate whether this enzyme activity was entirely encoded by the previously reported FDH1 gene, an fdh1Δ null mutant was constructed. This mutant strain still contained formate dehydrogenase activity and remained capable of co-consumption of formate. The formate dehydrogenase activity in the mutant was demonstrated to be encoded by a second structural gene for formate dehydrogenase (FDH2) in S. cerevisiae CEN.PK 113-7D. FDH2 was highly homologous to FDH1 and consisted of a fusion of two open reading frames (ORFs) (YPL275w and YPL276w) reported in the S. cerevisiae genome databases. Sequence anal. confirmed that, in the database genetic background, the presence of two single-nucleotide differences led to two truncated ORFs rather than the full-length FDH2 gene present in strain CEN.PK 113-7D. In the latter strain background an fdh1Δfdh2Δ double mutant lacked formate dehydrogenase activity and was unable to co-consume formate. Absence of formate dehydrogenase activity did not affect growth on glucose as sole carbon source, but led to a reduced biomass yield on glucose-formate mixts. These findings are consistent with a role of formate dehydrogenase in the detoxification of exogenous formate.
- 28Babel, W. (2009) The Auxiliary Substrate Concept: From simple considerations to heuristically valuable knowledge. Eng. Life Sci. 9, 285– 290, DOI: 10.1002/elsc.20090002728https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD1MXhtV2hs7fJ&md5=410fd45ff37ed049cd5db6a4a7c24141The Auxiliary Substrate Concept: From simple considerations to heuristically valuable knowledgeBabel, WolfgangEngineering in Life Sciences (2009), 9 (4), 285-290CODEN: ELSNAE; ISSN:1618-0240. (Wiley-VCH Verlag GmbH & Co. KGaA)A review. Microorganisms are used in biotechnol. They are either (i) aim and purpose of a process, e.g. with the prodn. of single cell proteins, or (ii) mean to an end insofar as they serve as a catalyst or "factory" for syntheses (e.g. of products of primary and secondary metab., of enzymes and antibiotics) or for the degrdn. and detoxification of harmful orgs. and inorgs. In all cases, the efficiency and velocity, finally the productivity, are parameters which essentially det. the economy of the processes. Therefore, search for approaches to optimize these processes is a permanent task and challenge for scientists and engineers. It is shown that the auxiliary substrate concept is suitable to increase the yield coeffs. It is based on the energetic evaluation of orgs., on the knowledge that orgs. as sources of carbon and energy for growth are deficient in ATP and/or reducing equiv., and says that it is possible to improve the carbon conversion efficiency up to the carbon metab. detd. upper limit. The latter is detd. by inevitable losses of carbon along the way of assimilation and anabolism and amts. to about 85% for so-called glycolytic substrates, e.g. glucose, methanol, and to about 75% for gluconeogenetic substrates, e.g. C2-substrates (acetic acid, hexadecane). The approach is explained and some exptl. examples are presented. By simultaneous utilization of an extra energy source (auxiliary substrate) the yield coeff. can be increased (i) in glucose from about 0.5 to 0.7 g/g (by means of formate), (ii) in acetate from 0.34-0.4 to 0.5-0.65 g/g (by means of formate and thiosulfate, resp.), and (iii) in hexadecane from about 0.94 to 1.26 g/g (by means of formate). The precalcd. yield coeffs. and mixing ratios agree well with the exptl. attained ones. The approach is easily feasible and economically valuable.
- 29Christensen, K. E. and Mackenzie, R. E. (2008) Mitochondrial methylenetetrahydrofolate dehydrogenase, methenyltetrahydrofolate cyclohydrolase, and formyltetrahydrofolate synthetases. Vitam. Horm. 79, 393– 410, DOI: 10.1016/S0083-6729(08)00414-729https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD1cXhsVOnsr7M&md5=e693f775711f681580245f24dce74ab1Mitochondrial methylenetetrahydrofolate dehydrogenase, methenyltetrahydrofolate cyclohydrolase, and formyltetrahydrofolate synthetasesChristensen, Karen E.; MacKenzie, Robert E.Vitamins and Hormones (San Diego, CA, United States) (2008), 79 (Folic Acid and Folates), 393-410CODEN: VIHOAQ; ISSN:0083-6729. (Elsevier Inc.)A review. Folate-mediated metab. involves enzyme-catalyzed reactions that occur in the cytoplasmic, mitochondrial, and nuclear compartments in mammalian cells. Which of the folate-dependent enzymes are expressed in these compartments depends on the stage of development, cell type, cell cycle, and whether or not the cell is transformed. Mitochondria become formate-generating organelles in cells and tissues expressing the MTHFD2 and MTHFD1L genes. The products of these nuclear genes were derived from trifunctional precursor proteins, expressing methylenetetrahydrofolate dehydrogenase-methenyltetrahydrofolate cyclohydrolase, and formyltetrahydrofolate synthetase activities. The MTHFD2 protein is a bifunctional protein with dehydrogenase and cyclohydrolase activities that arose from a trifunctional precursor through the loss of the synthetase domain and a novel adaptation to NAD rather than NADP specificity for the dehydrogenase. The MTHFD1L protein retains the size of its trifunctional precursor, but through the mutation of crit. residues, both the dehydrogenase and cyclohydrolase activities have been silenced. MTHFD1L is thus a monofunctional formyltetrahydrofolate synthetase. This review discusses the properties and functions of these mitochondrial proteins and their role in supporting cytosolic purine synthesis during embryonic development and in cells undergoing rapid growth.
- 30Figueroa, I. A., Barnum, T. P., Somasekhar, P. Y., Carlstrom, C. I., Engelbrektson, A. L., and Coates, J. D. (2018) Metagenomics-guided analysis of microbial chemolithoautotrophic phosphite oxidation yields evidence of a seventh natural CO2 fixation pathway. Proc. Natl. Acad. Sci. U. S. A. 115, E92– E101, DOI: 10.1073/pnas.171554911430https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2sXhvVOrtrfI&md5=c396ad05612addfe07c21b2937a03049Metagenomics-guided analysis of microbial chemolithoautotrophic phosphite oxidation yields evidence of a seventh natural CO2 fixation pathwayFigueroa, Israel A.; Barnum, Tyler P.; Somasekhar, Pranav Y.; Carlstrom, Charlotte I.; Engelbrektson, Anna L.; Coates, John D.Proceedings of the National Academy of Sciences of the United States of America (2018), 115 (1), E92-E101CODEN: PNASA6; ISSN:0027-8424. (National Academy of Sciences)Dissimilatory phosphite oxidn. (DPO), a microbial metab. by which phosphite (HPO32-) is oxidized to phosphate (PO43-), is the most energetically favorable chemotrophic electron-donating process known. Only one DPO organism has been described to date, and little is known about the environmental relevance of this metab. In this study, we used 16S rRNA gene community anal. and genome-resolved metagenomics to characterize anaerobic wastewater treatment sludge enrichments performing DPO coupled to CO2 redn. We identified an uncultivated DPO bacterium, Candidatus Phosphitivorax (Ca. P.) anaerolimi strain Phox-21, that belongs to candidate order GW-28 within the Deltaproteobacteria, which has no known cultured isolates. Genes for phosphite oxidn. and for CO2 redn. to formate were found in the genome of Ca. P. anaerolimi, but it appears to lack any of the known natural carbon fixation pathways. These observations led us to propose a metabolic model for autotrophic growth by Ca. P. anaerolimi whereby DPO drives CO2 redn. to formate, which is then assimilated into biomass via the reductive glycine pathway.
- 31Brachmann, C. B., Davies, A., Cost, G. J., Caputo, E., Li, J., Hieter, P., and Boeke, J. D. (1998) Designer deletion strains derived from Saccharomyces cerevisiae S288C: a useful set of strains and plasmids for PCR-mediated gene disruption and other applications. Yeast 14, 115– 132, DOI: 10.1002/(SICI)1097-0061(19980130)14:2<115::AID-YEA204>3.0.CO;2-231https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaK1cXptVyjsA%253D%253D&md5=33320be31df69f6abe592fb0692fd24cDesigner deletion strains derived from Saccharomyces cerevisiae S288C: a useful set of strains and plasmids for PCR-mediated gene disruption and other applicationsBrachmann, Carrie Baker; Davies, Adrian; Cost, Gregory J.; Caputo, Emerita; Li, Joachim; Hieter, Philip; Boeke, Jef D.Yeast (1998), 14 (2), 115-132CODEN: YESTE3; ISSN:0749-503X. (John Wiley & Sons Ltd.)A set of yeast strains based on Saccharomyces cerevisiae S288C in which commonly used selectable marker genes are deleted by design based on the yeast genome sequence has been constructed and analyzed. These strains minimize or eliminate the homol. to the corresponding marker genes in commonly used vectors without significantly affecting adjacent gene expression. Because the homol. between commonly used auxotrophic marker gene segments and genomic sequences has been largely or completely abolished, these strains will also reduce plasmid integration events which can interfere with a wide variety of mol. genetic applications. We also report the construction of new members of the pRS400 series of vectors, contg. the kanMX, ADE2 and MET15 genes.
- 32Looke, M., Kristjuhan, K., and Kristjuhan, A. (2011) Extraction of genomic DNA from yeasts for PCR-based applications. BioTechniques 50, 325– 328, DOI: 10.2144/00011367232https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3MXntVGjsbs%253D&md5=6948314df60f45a5754923cdd0c6ee70Extraction of genomic DNA from yeasts for PCR-based applicationsLooke, Marko; Kristjuhan, Kersti; Kristjuhan, ArnoldBioTechniques (2011), 50 (5), 325-328CODEN: BTNQDO; ISSN:0736-6205. (Informa Healthcare)The authors have developed a quick and low-cost genomic DNA extn. protocol from yeast cells for PCR-based applications. This method does not require any enzymes, hazardous chems., or extreme temps., and is esp. powerful for simultaneous anal. of a large no. of samples. DNA can be efficiently extd. from different yeast species (Kluyveromyces lactis, Hansenula polymorpha, Schizosaccharomyces pombe, Candida albicans, Pichia pastoris, and Saccharomyces cerevisiae). The protocol involves lysis of yeast colonies or cells from liq. culture in a lithium acetate (LiOAc)-SDS soln. and subsequent pptn. of DNA with ethanol. Approx. 100 ng of total genomic DNA can be extd. from 1 × 107 cells. DNA extd. by this method is suitable for a variety of PCR-based applications (including colony PCR, real-time qPCR, and DNA sequencing) for amplification of DNA fragments of ≤3500 bp.
- 33Gietz, R. D. and Schiestl, R. H. (2007) Quick and easy yeast transformation using the LiAc/SS carrier DNA/PEG method. Nat. Protoc. 2, 35– 37, DOI: 10.1038/nprot.2007.1433https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD2sXhtFGntb%252FE&md5=ca175dd794b225c28baa8e9b9b348e5eQuick and easy yeast transformation using the LiAc/SS carrier DNA/PEG methodGietz, R. Daniel; Schiestl, Robert H.Nature Protocols (2007), 2 (1), 35-37CODEN: NPARDW; ISSN:1750-2799. (Nature Publishing Group)Here, we describe a quick and easy version of the lithium acetate/single-stranded carrier DNA/PEG method of transformation for Saccharomyces cerevisiae. This method can be performed when only a few transformants are needed. The procedure can take less than an hour, depending on the duration of the heat shock. It can be used to transform yeast cells from various stages of growth and storage. Cells can be transformed from freshly grown cells as well as cells stored on a plate at room temp. or in a refrigerator.
- 34Hillson, N. J., Rosengarten, R. D., and Keasling, J. D. (2012) j5 DNA assembly design automation software. ACS Synth. Biol. 1, 14– 21, DOI: 10.1021/sb200011634https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3MXhsFygt7vM&md5=368ad18ea12fb4c9995f7dbb38096ca0j5 DNA Assembly Design Automation SoftwareHillson, Nathan J.; Rosengarten, Rafael D.; Keasling, Jay D.ACS Synthetic Biology (2012), 1 (1), 14-21CODEN: ASBCD6; ISSN:2161-5063. (American Chemical Society)Recent advances in Synthetic Biol. have yielded standardized and automatable DNA assembly protocols that enable a broad range of biotechnol. research and development. Unfortunately, the exptl. design required for modern scar-less multipart DNA assembly methods is frequently laborious, time-consuming, and error-prone. Here, we report the development and deployment of a web-based software tool, j5, which automates the design of scar-less multipart DNA assembly protocols including SLIC, Gibson, CPEC, and Golden Gate. The key innovations of the j5 design process include cost optimization, leveraging DNA synthesis when cost-effective to do so, the enforcement of design specification rules, hierarchical assembly strategies to mitigate likely assembly errors, and the instruction of manual or automated construction of scar-less combinatorial DNA libraries. Using a GFP expression testbed, we demonstrate that j5 designs can be executed with the SLIC, Gibson, or CPEC assembly methods, used to build combinatorial libraries with the Golden Gate assembly method, and applied to the prepn. of linear gene deletion cassettes for E. coli. The DNA assembly design algorithms reported here are generally applicable to broad classes of DNA construction methodologies and could be implemented to supplement other DNA assembly design tools. Taken together, these innovations save researchers time and effort, reduce the frequency of user design errors and off-target assembly products, decrease research costs, and enable scar-less multipart and combinatorial DNA construction at scales unfeasible without computer-aided design.
Supporting Information
Supporting Information
The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acssynbio.8b00464.
Table S1: Genetic constructs used in this study; Table S2: DNA primers used in this study (PDF)
Terms & Conditions
Most electronic Supporting Information files are available without a subscription to ACS Web Editions. Such files may be downloaded by article for research use (if there is a public use license linked to the relevant article, that license may permit other uses). Permission may be obtained from ACS for other uses through requests via the RightsLink permission system: http://pubs.acs.org/page/copyright/permissions.html.