Fermentation Route Research Articles

New Insights into the Formation of Viable but Nonculturable Escherichia coli O157:H7 Induced by High-Pressure CO2.

ABSTRACTThe formation of viable but nonculturable (VBNC) Escherichia coli O157:H7 induced by high-pressure CO2 (HPCD) was investigated using RNA sequencing (RNA-Seq) transcriptomics and isobaric tag for relative and absolute quantitation (iTRAQ) proteomic methods. The analyses revealed that 97 genes and 56 proteins were significantly changed upon VBNC state entry. Genes and proteins related to membrane transport, central metabolisms, DNA replication, and cell division were mainly downregulated in the VBNC cells. This caused low metabolic activity concurrently with a division arrest in cells, which may be related to VBNC state formation. Cell division repression and outer membrane overexpression were confirmed to be involved in VBNC state formation by homologous expression of z2046 coding for transcriptional repressor and ompF encoding outer membrane protein F. Upon VBNC state entry, pyruvate catabolism in the cells shifted from the tricarboxylic acid (TCA) cycle toward the fermentative route; this led to a low level of ATP. Combating the low energy supply, ATP production in the VBNC cells was compensated by the degradation of l-serine and l-threonine, the increased AMP generation, and the enhanced electron transfer. Furthermore, tolerance of the cells with respect to HPCD-induced acid, oxidation, and high CO2 stresses was enhanced by promoting the production of ammonia and NADPH and by reducing CO2 production during VBNC state formation. Most genes and proteins related to pathogenicity were downregulated in the VBNC cells. This would decrease the cell pathogenicity, which was confirmed by adhesion assays. In conclusion, the decreased metabolic activity, repressed cell division, and enhanced survival ability in E. coli O157:H7 might cause HPCD-induced VBNC state formation.

Outsourcing flourishes for synthetic biology firms

Ginkgo Bioworks and Amyris develop modified organisms that produce valuable chemicals. Now, both are choosing to outsource one step of the synthetic biology to-do list: manufacturing new DNA sequences. For that, Ginkgo and Amyris have inked new deals with Twist Bioscience and Gen9, firms that specialize in automating the production of millions of base pairs. Ginkgo develops modified microbes on a contract basis for companies in the flavor, fragrance, and cosmetics industries. For example, it is working on a fermentation route to rose oil. It will tap Twist to source 300 million DNA base pairs—up from its first order of 100 million—and Gen9 for another 300 million. Last week, Ginkgo also announced it has raised $100 million in venture funding. The money will go not just to Twist and Gen9, but also to building Ginkgo’s new organism engineering facility. “We’re the world’s largest consumer of synthetic genes,” Jason Kelly, Ginkgo’s

Lignin Degradation in the Production of Bioethanol – A Review

AbstractLignin is the principal impediment in the production of biofuel from different lignocellulosic sources through hydrolysis, saccharification and fermentation route. The two main types of treatment for lignin separation from the other main constituent plant materials, hemicellulose and cellulose, are biodegradation and the chemical degradation including base‐catalyzed, acid‐catalyzed, ionic liquids‐assisted lignin depolymerization. Severe reaction conditions, such as high pressure, high temperature and extreme pH, are being in practice for chemical degradation and result in the requirement of specially designed reactors. This leads to high costs of facility and handling along with the inadvertent generation of toxins. Biological degradation seems to be widespread and cost effective if enzymes degrading lignin could be produced by isolation of fungi. Bacterial strains could also be prepared that produce the lignin‐splitting enzyme laccase. The present article reviews the available processes for lignin degradation during lignocellulosic conversion of ethanol and discusses the research gaps in these fields.

Direct fermentation route for the production of acrylic acid

There have been growing concerns regarding the limited fossil resources and global climate changes resulting from modern civilization. Currently, finding renewable alternatives to conventional petrochemical processes has become one of the major focus areas of the global chemical industry sector. Since over 4.2 million tons of acrylic acid (AA) is annually employed for the manufacture of various products via petrochemical processes, this chemical has been the target of efforts to replace the petrochemical route by ecofriendly processes. However, there has been limited success in developing an approach combining the biological production of 3-hydroxypropionic acid (3-HP) and its chemical conversion to AA. Here, we report the first direct fermentative route for producing 0.12g/L of AA from glucose via 3-HP, 3-HP-CoA, and Acryloyl-CoA, leading to a strain of Escherichia coli capable of directly producing acrylic acid. This route was developed through extensive screening of key enzymes and designing a novel metabolic pathway for AA.

Generation of bioethanol and VFA through anaerobic acidogenic fermentation route with press mud obtained from sugar mill as a feedstock

Acidogenic anaerobic fermentation route was explored for the production of bioethanol and volatile fatty acids (VFA) from the press mud (PM) obtained from sugar mill. Slurry was prepared from PM having 10% of total solids and the same was hydrolyzed under acidic thermal conditions. Both press mud slurry (PMS) and pre-treated press mud slurry (PTPMS) was used as feedstock with mixed microbial consortia (MMC) and enriched mixed microbial consortia (EMMC). Mix of bioethanol and VFA were obtained in all the four cases (PMS-MMC, PMS-EMMC, PTPMS-EMC and PTPMS-EMMC), but, bioethanol and VFA yield of 0.04g/g and 0.27g/g, respectively obtained from PTPMS with EMMC was found to be comparatively higher. Control experiments carried out with glucose yielded bioethanol and VFA of 0.042g/g and 0.28g/g, respectively demonstrating that the organism was using reducible sugars in the feedstock for the generation of bioethanol by simultaneously producing the VFA from COD.

Life cycle assessment of biomass-based ethylene production in Sweden — is gasification or fermentation the environmentally preferable route?

To reduce its environmental impact, the chemical industry is investigating the biomass-based production of chemicals such as ethylene, including fermentation and gasification conversion processes. However, a comprehensive study that compares the environmental impact of these biomass routes is missing. This study assesses and compares a wood gasification with a wood fermentation route to ethylene in Sweden, as well as compares it with the commercialized sugarcane and fossil oil alternatives. The study followed the methodology of life cycle assessment. A cradle-to-gate perspective for the production of 50,000 t ethylene/year was applied, and the following impact categories were investigated: global warming (GWP), acidification (ACP), photochemical ozone creation (POCP), and eutrophication (EP). Biomass acquisition including transport to the gasification plant was the major cause of the gasification route’s GWP and POCP, suggesting improvements with regard to fuel source and machine efficiency. NOx emissions from the gasification process had the main share on the ACP and EP. The comparison of the gasification with a wood and a sugarcane fermentation route showed a lower impact for the gasification route. Among other things, this is caused by high emissions from transport and cultivation for the sugarcane route and high emissions from enzyme and ethanol production for the wood fermentation route. The results were less distinct for a comparison of the gasification with a fossil-based route. Fossil-based ethylene production was found to be preferable for the ACP and the EP, but less preferable for the GWP and POCP. However, it needs to be considered that the fossil route has been optimized for decades and is still ahead of the gasification and other biomass routes. The study shows that a gasification-based production of ethylene could outperform a fermentation-based one; however, further investigations are recommended, given the state of development of the investigated biomass routes. Moreover, based on the limited availability of biomass, further investigations into economical and ecological restrictions are of importance.

Model-driven <em>in Silico glpC</em> Gene Knockout Predicts Increased Succinate Production from Glycerol in <em>Escherichia Coli</em>

Metabolic engineered targeting for increased succinate production in Escherichia coli using glycerol as a low cost carbon source has attracted global attention in recent years. Succinate production in engineered E. coli has progressed significantly using an experimental trial and error approach. The use of a model-guided, targeted metabolic gene knockout prediction for increased succinate production from glycerol under anaerobic conditions in E. coli still remains largely underexplored. In this study, we applied a model-driven, targeted glpC/b2243 in silico metabolic gene knockout using E. coli genome scale model iJO1366 under the OptFlux software platform with the aim of predicting high succinate flux. The results indicated that the mutant model lacking the glpC/b2243 gene will demonstrate increased succinate flux that is 30% higher than its wild-type control model. We can hypothesize that an additional NADH molecule was generated following the deletion of the gene and/or the alternatively preferred GldA-DhaKLM fermentative route for glycerol metabolism in E. coli may have been activated. Although the exact metabolic mechanism involved in increasing the succinate flux still remains obscure; the current study informs other studies that a model-driven, metabolic glpC/b2243 gene knockout could be applicable in filling our knowledge gap using a comprehensive experimental inquiry in the future; leading to a better understanding of the underlying metabolic function of this gene in relation to succinate production in E. coli from glycerol.

Fermentative production of intracellular β-galactosidase by Bacillus safensis (JUCHE 1) growing on lactose and glucose—Modeling and experimental

Abstract Bacillus safensis (JUCHE 1) is an isolated bacterial strain which is capable to produce lactose hydrolyzing enzyme, β-galactosidase through fermentative route. Therefore, to enhance the production of β-galactosidase, systematic and programmed investigations are prerequisite. Unfortunately no attempt has yet been made to develop the mathematical model of B. safensis (JUCHE 1) growing on dual carbohydrate sources. In the present investigation a non-segregated structured model for B. safensis (JUCHE 1), growing in a medium containing multiple carbohydrate sources, namely, lactose and glucose has been developed. In the microbial growth medium lactose to glucose ratio has been varied from 3:1 to 39:1, and the total concentration of carbohydrate has been kept at 20 g L−1. The deterministic structured model, so developed, has been utilized to predict the concentration time histories of biomass generation, glucose, and lactose in the abiotic phase as well as the intracellular concentrations of lactose, lactose-6p, glucose-6p, β-galactosidase, and phospho-β-galactosidase through a combination of experimental, and computational studies. The model parameters were either evaluated through non-linear regression analysis of data obtained from programmed batch experiments in Erlenmeyer flasks with high correlation coefficient, or in some cases obtained from the literature. The model has been validated by comparison with some of the experimental data obtained from a large scale (5 L) bioreactor. It is expected that the developed model could be effectively used to predict the behavior of bioreactors, optimal operating conditions for enzyme production using B. safensis (JUCHE 1).

Stepwise metabolic engineering of Gluconobacter oxydans WSH-003 for the direct production of 2-keto-l-gulonic acid from d-sorbitol

2-Keto-l-gulonic acid (2-KLG), the direct precursor of vitamin C, is currently produced by a two-step fermentation route from d-sorbitol. However, this route involves three bacteria, making the mix-culture system complicated and redundant. Thus, replacement of the conventional two-step fermentation process with a one-step process could be revolutionary in vitamin C industry. In this study, different combinations of five l-sorbose dehydrogenases (SDH) and two l-sorbosone dehydrogenases (SNDH) from Ketogulonicigenium vulgare WSH-001 were introduced into Gluconobacter oxydans WSH-003, an industrial strain used for the conversion of d-sorbitol to l-sorbose. The optimum combination produced 4.9g/L of 2-KLG. In addition, 10 different linker peptides were used for the fusion expression of SDH and SNDH in G. oxydans. The best recombinant strain (G. oxydans/pGUC-k0203-GS-k0095) produced 32.4g/L of 2-KLG after 168h. Furthermore, biosynthesis of pyrroloquinoline quinine (PQQ), a cofactor of those dehydrogenases, was enhanced to improve 2-KLG production. With the stepwise metabolic engineering of G. oxydans, the final 2-KLG production was improved to 39.2g/L, which was 8.0-fold higher than that obtained using independent expression of the dehydrogenases. These results bring us closer to the final one-step industrial-scale production of vitamin C.

Production, purification, characterization, immobilization, and application of β‐galactosidase: a review

ABSTRACTBiopharmaceuticals are new categorizing biomolecules, which are the results of incredible proliferation in the field of biotechnology. One of the challenging biomolecule β‐galactosidase (β‐galactosidase galacto hydrolysase, trivially lactase) catalyzes hydrolysis of lactose to produce glucose and galactose, and in some cases, it takes part in transgalactosylation reaction that produces functional food galato‐oligosaccharide. A wide variety of strategies had been already attempted for production of β‐galactosidase through fermentative route. Beside the upstream process, downstream technology towards purification and immobilization of target enzyme also create great attentions. Subsequently, its wide applications in the field of food, biopharmaceuticals, dairy, diagnosis, and waste treatment boost up biotechnological economy as well as zero‐effluent discharge. In dairy industry, β‐galactosidase has been used to degrade lactose, prevent crystallization of lactose, improve sweetness, and increase the solubility of milk product, otherwise which would be an environmental pollutant. In food and pharmaceutical industries, β‐galactosidase has been used to prepare low lactose‐containing food products for low lactose‐tolerant people. Therefore, it is obvious to elucidate different technological aspects of β‐galactosidase, which may provide a great knowledge in educational and industrial field. Taking the enzyme into account, a ready review has been made about its production, purification, characterization, and immobilization technology. The review also addresses wide applications of β‐galactosidase in different fields. © 2014 Curtin University of Technology and John Wiley & Sons, Ltd.

Catalytic Conversion of Empty Fruit Bunch of Palm Oil for Producing Lactic Acid

Catalytic conversion of lignocellulosic biomass such as Empty Fruit Bunch (EFB) of Palm Oil into valuable chemicals such as lactic acid (LA) is an alternative route to currently fermentation route and it is a sustainable process for substituting petro-based chemical with biomass-based chemical. Conversion of lignocellulosic biomass (EFB) was conducted in a 100-ml batch reactor in aqueous solution using homogeneous AlCl3 catalyst at mild temperature in the range of 140-240°C, under hydrothermal condition. Some chemicals are identified in the product especialy LA and levulinic acid which are valuable chemicals produced by hydrolysis of lignocellulosic biomass and yield of LA compared with cellulosic biomass for comparison of feedstock. The sugar units in the lignocellulosic biomass (EFB) was around 48.75%, dominated by glucose (28%), xylose (16.5%), and followed with lower composistion of other sugar units such as galactose (1%), arabinose (2.5%) and mannose (0.75%). The yield of EFB conversion to lactic acid was found in the range 6-8.9% at temperature 140-240°C while for cellulosic material 19%, with reaction time 3hours and range of fixed AlCl3 concentration 50-100mM. Experimental results show that higher operating temperature and higher concentration of catalyst will produce higher percentage of LA. However, pretreatment of lignocelluloses with NaOH produced lower yield of LA 3.18 to 5.69% and probably the pretreatment leading to degradation of sugar units in the biomass.

Enhanced photo-fermentative H2 production using Rhodobacter sphaeroides by ethanol addition and analysis of soluble microbial products.

BackgroundBiological fermentation routes can provide an environmentally friendly way of producing H2 since they use renewable biomass as feedstock and proceed under ambient temperature and pressure. In particular, photo-fermentation has superior properties in terms of achieving high H2 yield through complete degradation of substrates. However, long-term H2 production data with stable performance is limited, and this data is essential for practical applications. In the present work, continuous photo-fermentative H2 production from lactate was attempted using the purple non-sulfur bacterium, Rhodobacter sphaeroides KD131. As a gradual drop in H2 production was observed, we attempted to add ethanol (0.2% v/v) to the medium.ResultsAs continuous operation went on, H2 production was not sustained and showed a negligible H2 yield (< 0.5 mol H2/mol lactateadded) within two weeks. Electron balance analysis showed that the reason for the gradual drop in H2 production was ascribed to the increase in production of soluble microbial products (SMPs). To see the possible effect of ethanol addition, a batch test was first conducted. The presence of ethanol significantly increased the H2 yield from 1.15 to 2.20 mol H2/mol lactateadded, by suppressing the production of SMPs. The analysis of SMPs by size exclusion chromatography showed that, in the later period of fermentation, more than half of the low molecular weight SMPs (< 1 kDa) were consumed and used for H2 production when ethanol had been added, while the concentration of SMPs continuously increased in the absence of ethanol. It was found that the addition of ethanol facilitated the utilization of reducing power, resulting in an increase in the cellular levels of NAD+ and NADP+. In continuous operation, ethanol addition was effective, such that stable H2 production was attained with an H2 yield of 2.5 mol H2/mol lactateadded. Less than 15% of substrate electrons were used for SMP production, whereas 35% were used in the control.ConclusionsWe have found that SMPs are the key factor in photo-fermentative H2 production, and their production can be suppressed by ethanol addition. However, since external addition of ethanol to the medium represents an extra economic burden, ethanol should be prepared in a cost-effective way.

Algal biomass conversion to bioethanol – a step‐by‐step assessment

The continuous growth in global population and the ongoing development of countries such as China and India have contributed to a rapid increase in worldwide energy demand. Fossil fuels such as oil and gas are finite resources, and their current rate of consumption cannot be sustained. This, coupled with fossil fuels' role as pollutants and their contribution to global warming, has led to increased interest in alternative sources of energy production. Bioethanol, presently produced from energy crops, is one such promising alternative future energy source and much research is underway in optimizing its production. The economic and temporal constraints that crop feedstocks pose are the main downfalls in terms of the commercial viability of bioethanol production. As an alternative to crop feedstocks, significant research efforts have been put into utilizing algal biomass as a feedstock for bioethanol production. Whilst the overall process can vary, the conversion of biomass to bioethanol usually contains the following steps: (i) pretreatment of feedstock; (ii) hydrolysis; and (iii) fermentation of bioethanol. This paper reviews different technologies utilized in the pretreatment and fermentation steps, and critically assesses their applicability to bioethanol production from algal biomass. Two different established fermentation routes, single-stage fermentation and two-stage gasification/fermentation processes, are discussed. The viability of algal biomass as an alternative feedstock has been assessed adequately, and further research optimisation must be guided toward the development of cost-effective scalable methods to produce high bioethanol yield under optimum economy.

Plastics production from biomass: assessing feedstock requirement

Biomass, as an alternative and renewable feedstock, has recently received increasing attention in the process industry due to its potential in producing sustainable energy, chemicals and materials. The competing uses of biomass make it important to understand the feedstock requirement for each purpose in order to quantify the true potential for replacing fossil-based feedstocks. Focusing on bio-based plastics, this work attempts to estimate the percentage of lignocellulosic agricultural residues and woody biomass residues resulting from logging and wood processing that is required for producing five main plastics (polyethylene, polypropylene, polyvinylchloride, polystyrene, polyethylenetherepthalate) world-wide and in Europe. The theoretical yields of three different production routes, namely direct fermentation, syngas fermentation, and chemical synthesis, are calculated, and the gap with the realistic yields is considered. The analysis shows that the chemical synthesis route and the syngas fermentation route for converting lignocellulosics to plastics are more productive than the direct fermentation route and have the potential to produce ethylene and propylene required by these plastics by consuming 28–47 and 48–80 % of the considered feedstock available world-wide and in Europe, respectively, for meeting the corresponding demands. It also reveals the challenges in feedstock sufficiency for the production of benzene and terephthalic acid (as plastics components) from lignin.

Succinic acid production derived from carbohydrates: An energy and greenhouse gas assessment of a platform chemical toward a bio‐based economy

AbstractBio‐based succinic acid has the potential to become a platform chemical, i.e. a key building block for deriving both commodity and high‐value chemicals, which makes it an attractive compound in a bio‐based economy. A few companies and industrial consortia have begun to develop its industrial production on a large scale. A life cycle assessment of different bio‐based succinic acid production processes, based on dextrose from corn, was performed to investigate their non‐renewable energy use (NREU) and greenhouse gas (GHG) emissions, from cradle‐to‐factory gate in Europe. Three processes were studied, i.e. (i) low pH yeast fermentation with downstream processing (DSP) by direct crystallization, (ii) anaerobic fermentation to succinate salt at neutral pH (pH7) and subsequent DSP by electrodialysis, and (iii) a similar process producing ammonium sulfate as co‐product in DSP. These processes are compared to the production of petrochemical maleic anhydride, succinic acid, and adipic acid. Low pH yeast fermentation to succinic acid with direct crystallization was found to have significantly lower GHG emissions and NREU, compared to other fermentation routes and three petrochemical routes. However, the disparity in GHG emissions between this process and the electrodialysis process becomes less prominent if one considers a cleaner electricity mix than the current European production mix. Moreover, this study highlights that the allocation approach in corn wet milling and the succinic acid plant location strongly influence the results. Overall, the results suggest that low pH yeast fermentation with direct crystallization is the most beneficial process to bio‐based succinic acid from an environmental perspective. © 2013 Society of Chemical Industry and John Wiley &amp; Sons, Ltd

Investigations on the kinetics and thermodynamics of dilute acid hydrolysis of Parthenium hysterophorus L. substrate

Parthenium hysterophorus L. commonly known as parthenium weed, is one of the seven obnoxious weed in the world. Being a lignocellulosic biomass, with a composition (w/w) of cellulose: 27.8%, hemicellulose: 21.01% and lignin: 13.9%; as analyzed in the present work, it may be considered as the potential source of fuel ethanol production through pretreatment, saccharification and fermentation route. Batch hydrolysis of P. Hysterophorus L. with 1–5% sulfuric acid at a temperature range of 150–210°C for 4–30min treatment time and 2–6h soaking period has been conducted to study the xylose yield. It is realized through the kinetic plots and analysis of various parameters that the process undergoes through first order kinetics and consequently follows Arrhenius law. It has also been understood that the reaction rate, the activation energy and the frequency factor of Arrhenius equation as well as all other related thermodynamic parameters depend largely on the treatment temperature during hydrolysis, the concentration of acid medium used and the soaking period of parthenium substrate before the actual hydrolysis reaction. The present investigation demonstrated that maximum xylose yield was 46.1% of original hemicelluloses present in the biomass under the process parameters considered, where higher treatment temperature, higher concentration of acid medium and higher soaking time favors the formation of stable depolymerised products.

A Natural Isolate Producing Shikimic Acid: Isolation, Identification, and Culture Condition Optimization

Shikimic acid has wide use in pharmaceuticals due to its application in the synthesis of drug Tamiflu used in the treatment of Swine flu. The high cost and limited availability of shikimic acid isolated from plants has impeded the use of this valuable building block of the drug. In this context, fermentation route to produce shikimic acid from renewable resources has become increasingly attractive. The present study was embarked upon isolation of wild-type microorganisms able to produce shikimic acid. Out of the 42 isolates obtained from the soil, isolate GR-21 was selected as the best with initial production of 0.54 g/L shikimic acid and later identified as Citrobacter sp. The process optimization resulted in 14-fold increase in the shikimic acid production, thereby claiming this process to be a sustainable alternative for the production of this important biomolecule. The process was further scaled up to 14 L bioreactor to validate the production of shikimic acid. Further, the product formed is shikimic acid was confirmed by FTIR analysis. The current studies suggest that the selected isolate could be used as a promising agent to fulfill the worldwide demand of shikimic acid.

Lactic acid as a platform chemical in the biobased economy: the role of chemocatalysis

Upcoming bio-refineries will be at the heart of the manufacture of future transportation fuels, chemicals and materials. A narrow number of platform molecules are envisioned to bridge nature's abundant polysaccharide feedstock to the production of added-value chemicals and intermediate building blocks. Such platform molecules are well-chosen to lie at the base of a large product assortment, while their formation should be straightforward from the refined biomass, practical and energy efficient, without unnecessary loss of carbon atoms. Lactic acid has been identified as one such high potential platform. Despite its established fermentation route, sustainability issues – like gypsum waste and cost factors due to multi-step purification and separation requirements – will arise as soon as the necessary orders of magnitude larger volumes are needed. Innovative production routes to lactic acid and its esters are therefore under development, converting sugars and glycerol in the presence of chemocatalysts. Moreover, catalysis is one of the fundamental routes to convert lactic acid into a range of useful chemicals in a platform approach. This contribution attempts a critical overview of all advances in the field of homogeneous and heterogeneous catalysis and recognises a great potential of some of these chemocatalytic approaches to produce and transform lactic acid as well as some other promising α-hydroxy acids.

Carbon isotope fractionation of 11 acetogenic strains grown on H2 and CO2.

Acetogenic bacteria are able to grow autotrophically on hydrogen and carbon dioxide by using the acetyl coenzyme A (acetyl-CoA) pathway. Acetate is the end product of this reaction. In contrast to the fermentative route of acetate production, which shows almost no fractionation of carbon isotopes, the acetyl-CoA pathway has been reported to exhibit a preference for light carbon. In Acetobacterium woodii the isotope fractionation factor (ε) for (13)C and (12)C has previously been reported to be ε = -58.6‰. To investigate whether such a strong fractionation is a general feature of acetogenic bacteria, we measured the stable carbon isotope fractionation factor of 10 acetogenic strains grown on H(2) and CO(2). The average fractionation factor was ε(TIC) = -57.2‰ for utilization of total inorganic carbon and ε(acetate) = -54.6‰ for the production of acetate. The strongest fractionation was found for Sporomusa sphaeroides (ε(TIC) = -68.3‰), the lowest fractionation for Morella thermoacetica (ε(TIC) = -38.2‰). To investigate the reproducibility of our measurements, we determined the fractionation factor of 21 biological replicates of Thermoanaerobacter kivui. In general, our study confirmed the strong fractionation of stable carbon during chemolithotrophic acetate formation in acetogenic bacteria. However, the specific characteristics of the bacterial strain, as well as the cultural conditions, may have a moderate influence on the overall fractionation.

Structural Determinants of the β-Selectivity of a Bacterial Aminotransferase

Chiral β-amino acids occur as constituents of various natural and synthetic compounds with potentially useful bioactivities. The pyridoxal 5'-phosphate (PLP)-dependent S-selective transaminase from Mesorhizobium sp. strain LUK (MesAT) is a fold type I aminotransferase that can be used for the preparation of enantiopure β-Phe and derivatives thereof. Using x-ray crystallography, we solved structures of MesAT in complex with (S)-β-Phe, (R)-3-amino-5-methylhexanoic acid, 2-oxoglutarate, and the inhibitor 2-aminooxyacetic acid, which allowed us to unveil the molecular basis of the amino acid specificity and enantioselectivity of this enzyme. The binding pocket of the side chain of a β-amino acid is located on the 3'-oxygen side of the PLP cofactor. The same binding pocket is utilized by MesAT to bind the α-carboxylate group of an α-amino acid. A β-amino acid thus binds in a reverse orientation in the active site of MesAT compared with an α-amino acid. Such a binding mode has not been reported before for any PLP-dependent aminotransferase and shows that the active site of MesAT has specifically evolved to accommodate both β- and α-amino acids.