Glutamate Racemase Research Articles

Screening and Identification of Natural Compounds as Potential Inhibitors of Glutamate Racemase and an Emerging Drug Target of Food Pathogen E. coli O157:H7: An In silico Approach to Combat Increasing Drug Resistance.

Shiga Toxin-Producing Escherichia coli (E. coli O157:H7), capable of causing serious food-borne illnesses, is extensively studied and is known to be transmitted through animal reservoirs or person-to-person contact, leading to severe disease outbreaks. The emergence of antibiotic resistance in these strains, coupled with increased adverse effects of existing therapeutics, underscores the urgent need for alternative therapeutic strategies. This study aims to evaluate Glutamate Racemase (MurI protein) of the food-path-ogenic E. coli O157:H7 (EC MurI) as a novel drug target. Furthermore, the study seeks to identify new compounds with potential inhibitory effects against this protein. Using computational tools, the study identified inhibitor binding sites on EC MurI and identified relevant inhibitors capable of binding to these sites. Molecular docking tech-niques were employed to assess potential hits, and selected compounds were further analyzed for their structural activity and binding affinity to the protein. The results of the study revealed that Frigocyclinone and Deslanoside, exhibited the best binding affinity with EC-MurI. Subsequent molecular dynamic (MD) simulations of the selected complexes indicated that both compounds were stable. This suggests that Frigocy-clinone and Deslanoside have the potential to serve as potent inhibitors of EC-MurI. In summary, this study highlights the urgent need for alternative therapies against food-pathogenic E. coli, focusing on E. coli O157:H7. Evaluation of Glutamate Race-mase as a drug target identified Frigocyclinone and Deslanoside as promising inhibitors. MD simulations indicated their stability, suggesting their potential as lead molecules for further research and treatment development.

Dual transcriptional inhibition of glutamate and alanine racemase is synergistic in Mycobacterium tuberculosis.

Synergistic interactions between chemical inhibitors, whilst informative, can be difficult to interpret, as chemical inhibitors can often have multiple targets, many of which can be unknown. Here, using multiplexed transcriptional repression, we have validated that the simultaneous repression of glutamate racemase and alanine racemase has a synergistic interaction in Mycobacterium tuberculosis. This confirms prior observations from chemical interaction studies and highlights the potential of targeting multiple enzymes involved in mycobacterial cell wall synthesis.

Genomic characterization and related functional genes of γ- poly glutamic acid producing Bacillus subtilis.

γ- poly glutamic acid (γ-PGA), a high molecular weight polymer, is synthesized by microorganisms and secreted into the extracellular space. Due to its excellent performance, γ-PGA has been widely used in various fields, including food, biomedical and environmental fields. In this study, we screened natto samples for two strains of Bacillus subtilis N3378-2at and N3378-3At that produce γ-PGA. We then identified the γ-PGA synthetase gene cluster (PgsB, PgsC, PgsA, YwtC and PgdS), glutamate racemase RacE, phage-derived γ-PGA hydrolase (PghB and PghC) and exo-γ-glutamyl peptidase (GGT) from the genome of these strains. Based on these γ-PGA-related protein sequences from isolated Bacillus subtilis and 181 B. subtilis obtained from GenBank, we carried out genotyping analysis and classified them into types 1-5. Since we found B. amyloliquefaciens LL3 can produce γ-PGA, we obtained the B. velezensis and B. amyloliquefaciens strains from GenBank and classified them into types 6 and 7 based on LL3. Finally, we constructed evolutionary trees for these protein sequences. This study analyzed the distribution of γ-PGA-related protein sequences in the genomes of B. subtilis, B. velezensis and B. amyloliquefaciens strains, then the evolutionary diversity of these protein sequences was analyzed, which provided novel information for the development and utilization of γ-PGA-producing strains.

Assaying D-Amino Acid in Japanese Sake Using L-Amino Acid Derivatizing Agent.

The D-amino acids of D-alanine, D-glutamic acid, and D-aspartic acid increase tasting evaluation scores of Sake, a Japanese traditional alcohol beverage. Sake is brewed using seed mash for growth of brewing yeast without growth of contaminating microorganisms. Kimoto is brewed using lactic acid bacteria growth to decrease pH. Sake brewed using the Kimoto method also has a rich taste and a higher tasting evaluation score than Sake brewed using the Sokujyo Syubo (Moto) method, which adds lactic acid instead of using lactic acid bacteria growth. D-alanine, D-glutamic acid, and D-aspartic acid in Sake have the function of increasing tasting evaluation scores. They are converted by enzymes in lactic acid bacteria respectively as alanine racemase (EC 5.1.1.1), glutamate racemase (EC 5.1.1.3), and aspartate racemase (EC 5.1.1.13) in Kimoto Mash. Herein, simultaneous assay methods for D-alanine, D-glutamic acid, and D-aspartic acid are explained. Sample solutions adjusted to alkalinity are derivatized by an L-FDLA solution only for L-amino acid. Results demonstrate that, for D-alanine, D-glutamic acid, and D-aspartic acid, this method can assay them easily using no expensive or specialized equipment.

Glutamate racemase is a compelling target for antibiotic design for the treatment of infections caused by Pseudomonas aeruginosa

Glutamate racemase is a compelling target for antibiotic design for the treatment of infections caused by <i>Pseudomonas aeruginosa</i>

Targeting multi-drug-resistant Acinetobacter baumannii: a structure-based approach to identify the promising lead candidates against glutamate racemase.

Acinetobacter baumannii, one of the critical ESKAPE pathogens, is a highly resilient, multi-drug-resistant, Gramnegative, rod-shaped, highly pathogenic bacteria. It is responsible for almost 1-2% of all hospital-borne infections in immunocompromised patients and causes community outbreaks. Because of its resilience and MDR characteristics, looking for new strategies to check the infections related to this pathogen becomes paramount. The enzymes involved in the peptidoglycan biosynthetic pathway are attractive and the most promising drug targets. They contribute to the formation of the bacterial envelope and help to maintain the rigidity and integrity of the cell. The MurI (glutamate racemase) is one of the crucial enzymes that aid in the formation of the pentapeptide responsible for the interlinkage of peptidoglycan chains. It converts L-glutamate to D-glutamate, which is required to synthesise the pentapeptide chain. In this study, the MurI protein of A. baumannii (strain AYE) was modelled and subjected to high-throughput virtual screening against the enamine-HTSC library, taking UDP-MurNAc-Ala binding site as the targeted site. Four ligandmolecules, Z1156941329 (N-(1-methyl-2-oxo-3,4-dihydroquinolin-6-yl)-1-phenyl-3,4-dihydro-1H-isoquinoline-2-carboxamide), Z1726360919 (1-[2-[3-(benzimidazol-1-ylmethyl)piperidin-1-yl]-2-oxo-1-phenylethyl]piperidin-2-one), Z1920314754 (N-[[3-(3-methylphenyl)phenyl]methyl]-8-oxo-2,7-diazaspiro[4.4]nonane-2-carboxamide) and Z3240755352 (4R)-4-(2,5-difluorophenyl)-1-(4-fluorophenyl)-1,3a,4,5,7,7a-hexahydro-6H-pyrazolo[3,4-b]pyridin-6-one), were identified to be the lead candidates based on Lipinski's rule of five, toxicity, ADME properties, estimated binding affinity and intermolecular interactions. The complexes of these ligands with the protein molecule were then subjected to MD simulations to scrutinise their dynamic behaviour, structural stability and effects on protein dynamics. The molecular mechanics/Poisson-Boltzmann surface area-based binding free energy analysis was also performed to compute the binding free energy of protein-ligand complexes, which offered the following values -23.32 ± 3.04 kcal/mol, -20.67 ± 2.91kcal/mol, -8.93 ± 2.90 kcal/mol and -26.73 ± 2.95 kcal/mol for MurI-Z1726360919, MurI-Z1156941329, MurI-Z3240755352 and MurI-Z3240755354 complexes respectively. Together, the results from various computational analyses utilised in this study proposed that Z1726360919, Z1920314754 and Z3240755352 could act as potential lead molecules to suppress the function of MurI protein from Acinetobacter baumannii.

Analysis of D-amino acid in Japanese post-fermented tea, Ishizuchi-kurocha.

The D-amino acid content of Ishizuchi-kurocha, a post-fermented tea produced in Ehime, Japan, was measured. Ishizuchi-kurocha mainly contains D-glutamic acid and D-alanine, but it also contains a small amount of D-aspartic acid. Two types of lactic acid bacteria, Lactiplantibacillus plantarum and Levilactobacillus brevis, are the main species involved in lactic acid fermentation during the tea fermentation process. Therefore, the D-amino acid-producing abilities of strains of these two species isolated from Ishizuchi-kurocha were examined. Specifically, the production of D-aspartic acid, D-alanine, and D-glutamic acid by L. brevis and L. plantarum strains was observed. The amount of D-aspartic acid produced by L. plantarum was low. D-glutamine was detected in culture supernatant but not in bacterial cells. D-arginine was detected in bacterial cells of the L. plantarum strains but not in the culture supernatant. Both the L. brevis and L. plantarum strains possessed at least three kinds of putative racemase genes: alanine racemase, glutamate racemase, and aspartate racemase. However, their expression and enzyme activity remain unknown. L. plantarum and L. brevis could play an important role in the production of D-amino acids in Ishizuchi-kurocha. In fact, Ishizuchi-kurocha is expected to possess the effective physiological activities of D-amino acids.

Design and evaluation of substrate-product analoginhibitors for racemases and epimerases utilizing a 1,1-proton transfer mechanism.

Racemases and epimerases catalyze the inversion of stereochemistry at asymmetric carbon atoms to generate stereoisomers that often play important roles in normal and pathological physiology. Consequently, there is interest in developing inhibitors of these enzymes for drug discovery. A strategy for the rational design of substrate-product analog (SPA) inhibitors of racemases and epimerases utilizing a direct 1,1-proton transfer mechanism is elaborated. This strategy assumes that two groups on the asymmetric carbon atom remain fixed at active-site binding determinants, while the hydrogen and third, motile group move during catalysis, with the latter potentially traveling between an R- and S-pocket at the active site. SPAs incorporate structural features of the substrate and product, often with geminal disubstitution on the asymmetric carbon atom to simultaneously present the motile group to both the R- and S-pockets. For racemases operating on substrates bearing three polar groups (glutamate, aspartate, and serine racemases) or with compact, hydrophobic binding pockets (proline racemase), substituent motion is limited and the design strategy furnishes inhibitors with poor or modest binding affinities. The approach is most successful when substrates have a large, motile hydrophobic group that binds at a plastic and/or capacious hydrophobic site. Potent inhibitors were developed for mandelate racemase, isoleucine epimerase, and α-methylacyl-CoA racemase using the SPA inhibitor design strategy, exhibiting binding affinities ranging from substrate-like to exceeding that of the substrate by 100-fold. This rational approach for designing inhibitors of racemases and epimerases having the appropriate active-site architectures is a useful strategy for furnishing compounds for drug development.

Silver Nanoparticles Synthesis using Banana Peels Extract and Its in Silico Evaluation for Antibacterial Activity against Escherichia coli

Metal nanoparticles (NPs) is widely applied in biomedical science. Silver nanoparticles (AgNPs) is one of the potential metal nanoparticles famous for its potent antibacterial properties. However, its detrimental consequences on human health have been a significant concern. In this study, AgNPs was synthesized using banana peels by environmentally friendly approach, and their antibacterial activity against Escherichia coli as well as mechanisms were studied in silico. Total phenolic content (TPC), total flavonoid content (TFC), and total tannin acid content (TTC) of the BPE were initially determined by extraction using aqueous and methanolic solutions. A disc diffusion test (DDT) against E. coli was used to confirm the antibacterial activity of the BPE and BPE-AgNPs. An in silico approach was used to conduct molecular docking research to assess the potential antibacterial mechanism of BPE phytochemicals with the proteins of E. coli. The phytochemical content analysis shows that flavonoid has the highest content in the aqueous and the methanol extract, with respective concentrations of 439.60±57.84 mg QE/g DW and 222.42±14.56 mg QE/g DW. According to the results of DDT, BPE-AgNPs demonstrated positive antibacterial activity, while BPE demonstrated negative result. Several flavonoid chemical compounds, including rutin, quercetin, myricetin, naringenin, apigenin, luteolin, morin, galangin, catechin, and chrysin, were examined for their ability to bind to E. coli proteins such penicillin-binding protein, dihydrofolate reductase, and glutamate racemase. Rutin with penicillin-binding protein demonstrated the highest binding affinity, with the lowest free binding energy of -9.96 kcal/mol and the inhibition constant of 0.05 µM. The in silico and molecular docking analysis demonstrated the ability of active components of banana peels to inhibit the activity of the E.coli proteins. These elements not only function as reducing and stabilising agents for AgNP synthesis but also prevent E. coli from growing, which is potential in future antibacterial uses.

MOLECULAR DOCKING AND COMPUTATIONAL PHARMACOKINETIC STUDY OF SOME NOVEL COUMARIN–BENZOTHIAZOLE SCHIFF’S BASE FOR ANTIMICROBIAL ACTIVITY

Objective: The present study discusses molecular docking of some novel coumarin–benzothiazole Schiff bases and the prediction of pharmacokinetic properties of potent molecules by the computational method. Methods: Five protein targets were selected for the study and their structures were taken from RCSB Protein Data Bank in PDB format. Preparation of proteins was done using Discovery Studio 2021 Client. A total of twenty derivatives were drawn using ChemDraw 20.0 and saved in Mol format. Molecular docking was performed using PyRx software. Docking results were visualized by Discovery Studio 2021 Client. The pharmacokinetic properties of the best compounds were determined using the pkCSM tool. Results: All twenty derivatives were docked against the five proteins, namely DNA Ligase (PDB ID: 3PN1), Topoisomerase (PDB ID: 3TTZ), Sterol demethylase (PDB ID: 5FSA), Enoyl-acyl-carrier protein (PDB ID: 1BVR) and Glutamate racemase (PDB ID: 5HJ7). The compound JJB18 has shown the best binding score against DNA ligase (-10.7 kcal/mol), Glutamate racemase (-8.4 kcal/mol), and Enoyl-acyl-carrier protein (-10.8 kcal/mol). Further, compound JJB19 has shown the best score for fungal sterol demethylase (-10.6 kcal/mol) and compound JJB20 towards topoisomerase (-9.4 kcal/mol) than the standard drugs. The physicochemical properties of potent derivatives were also reported. Conclusion: Molecular Docking study indicates that coumarin–benzothiazole Schiff bases may be effective inhibitors for the different microbial proteins. Additionally, in silico ADMET studies predicts drug-like features. Hence, these compounds may be considered lead molecules and further investigation of their analogues may help in the development of novel drugs for the treatment of microbial diseases.

Identification of a novel d‐amino acid aminotransferase involved in d‐glutamate biosynthetic pathways in the hyperthermophile Thermotoga maritima

The hyperthermophilic bacterium Thermotoga maritima has an atypical peptidoglycan that contains d-lysine alongside the usual d-alanine and d-glutamate. We previously identified a lysine racemase involved in d-lysine biosynthesis, and this enzyme also possesses alanine racemase activity. However, T. maritima has neither alanine racemase nor glutamate racemase enzymes; hence, the precise biosynthetic pathways of d-alanine and d-glutamate remain unclear in T. maritima. In the present study, we identified and characterized a novel d-amino acid aminotransferase (TM0831) in T. maritima. TM0831 exhibited aminotransferase activity towards 23 d-amino acids, but did not display activity towards l-amino acids. It displayed high specific activities towards d-homoserine and d-glutamine as amino donors. The most preferred acceptor was 2-oxoglutarate, followed by glyoxylate. Additionally, TM0831 displayed racemase activity towards four amino acids including aspartate and glutamate. Catalytic efficiency (kcat /Km ) for aminotransferase activity was higher than for racemase activity, and pH profiles were distinct between these two activities. To evaluate the functions of TM0831, we constructed a TTHA1643 (encoding glutamate racemase)-deficient Thermus thermophilus strain (∆TTHA1643) and integrated the TM0831 gene into the genome of ∆TTHA1643. The growth of this TM0831-integrated strain was promoted compared with ∆TTHA1643 and was restored to almost the same level as that of the wild-type strain. These results suggest that TM0831 is involved in d-glutamate production. TM0831 is a novel d-amino acid aminotransferase with racemase activity that is involved in the production of d-amino acids in T. maritima.

Decrypting a cryptic allosteric pocket in H. pylori glutamate racemase

One of our greatest challenges in drug design is targeting cryptic allosteric pockets in enzyme targets. Drug leads that do bind to these cryptic pockets are often discovered during HTS campaigns, and the mechanisms of action are rarely understood. Nevertheless, it is often the case that the allosteric pocket provides the best option for drug development against a given target. In the current studies we present a successful way forward in rationally exploiting the cryptic allosteric pocket of H. pylori glutamate racemase, an essential enzyme in this pathogen’s life cycle. A wide range of computational and experimental methods are employed in a workflow leading to the discovery of a series of natural product allosteric inhibitors which occupy the allosteric pocket of this essential racemase. The confluence of these studies reveals a fascinating source of the allosteric inhibition, which centers on the abolition of essential monomer-monomer coupled motion networks.

PGN-005 - In-silico identification of novel inhibitors for glutamate racemase (MurI) to decipher the subtle of antimicrobial resistance in Neisseria gonorrhoeae: A virtual screening and molecular dynamics approach

PGN-005 - In-silico identification of novel inhibitors for glutamate racemase (MurI) to decipher the subtle of antimicrobial resistance in Neisseria gonorrhoeae: A virtual screening and molecular dynamics approach

Identification of an l-serine/l-threonine dehydratase with glutamate racemase activity in mammals

Recent investigations have shown that multiple d-amino acids are present in mammals and these compounds have distinctive physiological functions. Free d-glutamate is present in various mammalian tissues and cells and in particular, it is presumably correlated with cardiac function, and much interest is growing in its unique metabolic pathways. Recently, we first identified d-glutamate cyclase as its degradative enzyme in mammals, whereas its biosynthetic pathway in mammals is unclear. Glutamate racemase is a most probable candidate, which catalyzes interconversion between d-glutamate and l-glutamate. Here, we identified the cDNA encoding l-serine dehydratase-like (SDHL) as the first mammalian clone with glutamate racemase activity. This rat SDHL had been deposited in mammalian databases as a protein of unknown function and its amino acid sequence shares ∼60% identity with that of l-serine dehydratase. Rat SDHL was expressed in Escherichia coli, and the enzymatic properties of the recombinant were characterized. The results indicated that rat SDHL is a multifunctional enzyme with glutamate racemase activity in addition to l-serine/l-threonine dehydratase activity. This clone is hence abbreviated as STDHgr. Further experiments using cultured mammalian cells confirmed that d-glutamate was synthesized and l-serine and l-threonine were decomposed. It was also found that SDHL (STDHgr) contributes to the homeostasis of several other amino acids.

Competing Substrates for the Bifunctional Diaminopimelic Acid Epimerase/Glutamate Racemase Modulate Peptidoglycan Synthesis in Chlamydia trachomatis.

The Chlamydia trachomatis genome encodes multiple bifunctional enzymes, such as DapF, which is capable of both diaminopimelic acid (DAP) epimerase and glutamate racemase activity. Our previous work demonstrated the bifunctional activity of chlamydial DapF in vitro and in a heterologous system (Escherichia coli). In the present study, we employed a substrate competition strategy to demonstrate DapF Ct function in vivo in C. trachomatis We reasoned that, because DapF Ct utilizes a shared substrate-binding site for both racemase and epimerase activities, only one activity can occur at a time. Therefore, an excess of one substrate relative to another must determine which activity is favored. We show that the addition of excess l-glutamate or meso-DAP (mDAP) to C. trachomatis resulted in 90% reduction in bacterial titers, compared to untreated controls. Excess l-glutamate reduced in vivo synthesis of mDAP by C. trachomatis to undetectable levels, thus confirming that excess racemase substrate led to inhibition of DapF Ct DAP epimerase activity. We previously showed that expression of dapFCt in a murI (racemase) ΔdapF (epimerase) double mutant of E. coli rescues the d-glutamate auxotrophic defect. Addition of excess mDAP inhibited growth of this strain, but overexpression of dapFCt allowed the mutant to overcome growth inhibition. These results confirm that DapF Ct is the primary target of these mDAP and l-glutamate treatments. Our findings demonstrate that suppression of either the glutamate racemase or epimerase activity of DapF compromises the growth of C. trachomatis Thus, a substrate competition strategy can be a useful tool for in vivo validation of an essential bifunctional enzyme.

Contribution of the murI Gene Encoding Glutamate Racemase in the Motility and Virulence of Ralstonia solanacearum.

Bacterial traits for virulence of Ralstonia solanacearum causing lethal wilt in plants were extensively studied but are not yet fully understood. Other than the known virulence factors of Ralstonia solanacearum, this study aimed to identify the novel gene(s) contributing to bacterial virulence of R. solanacearum. Among the transposon-inserted mutants that were previously generated, we selected mutant SL341F12 strain produced exopolysaccharide equivalent to wild type strain but showed reduced virulence compared to wild type. In this mutant, a transposon was found to disrupt the murI gene encoding glutamate racemase which converts L-glutamate to D-glutamate. SL341F12 lost its motility, and its virulence in the tomato plant was markedly diminished compared to that of the wild type. The altered phenotypes of SL341F12 were restored by introducing a full-length murI gene. The expression of genes required for flagella assembly was significantly reduced in SL341F12 compared to that of the wild type or complemented strain, indicating that the loss of bacterial motility in the mutant was due to reduced flagella assembly. A dramatic reduction of the mutant population compared to its wild type was apparent in planta (i.e., root) than its wild type but not in soil and rhizosphere. This may contribute to the impaired virulence in the mutant strain. Accordingly, we concluded that murI in R. solanacearum may be involved in controlling flagella assembly and consequently, the mutation affects bacterial motility and virulence.

From pan-genome to protein dynamics: A computational hierarchical quest to identify drug target in multi-drug resistant Burkholderia cepacia

Burkholderia cepacia has emerged as a significant infectious agent that exhibits resistance to major classes of antibiotics and novel therapeutic approaches are necessitated to address this pathogen. Glutamate racemase (GR) has been revealed as highly potent and safe drug target by pan-genome analysis. It plays role in the interconversion of L-Glutamate to D-Glutamate and remains an essential element of the peptidoglycan layer. Docking guided virtual screening is carried out predicting a lead molecule: “(R)-1-((5-((1R, 6R)-6-carboxycyclohexa-2, 4-dien-1-yl) thiazol-2-yl) methyl)-3-oxopiperazin-1-ium” possess the best fitting conformation to the target. It is found that heterocyclic ring of the molecule has a key role in the binding affinity of the target protein. During simulation, the inhibitor interacts with the allosteric site, allowing the heterocyclic ring to be positioned at the adjacent substrate binding site. Local secondary structures interconversion is seen regularly in both chains of the protein. The radial distribution function investigated Leu24 as closely placed at 1.9 Å with an average g (r) value 0.8 to the binding site residues during the course of simulation. The density of the interactive space shows that Leu24 and Asn85 are the most active residues with average binding energies of −2.65 kcal/mol and −2.36 kcal/mol, respectively and affirms strong hydrogen bonding. Total binding energies in both MMPB/GBSA results in −21.13 kcal/mol and −36.41 kcal/mol are contributing heavily towards the stability. Interestingly, the Waterswap approach exhibits accurate binding energies of −20.16 kcal/mol. This chemical entity is adjacent to medicinally active molecules and in line with the previous research which showed thiazol subunits and its derivatives bearing many biological functions. In order to validate compound antibacterial activity, it is necessary to investigate the biological potency of the compound using biological assays and could be promising for experimentalists in the future to address this multi-drug resistant pathogen.

Enzymatic properties and physiological function of glutamate racemase from Thermus thermophilus

d-Amino acids are physiologically important components of peptidoglycan in the bacterial cell wall, maintaining cell structure and aiding adaptation to environmental changes through peptidoglycan remodelling. Therefore, the biosynthesis of d-amino acids is essential for bacteria to adapt to different environmental conditions. The peptidoglycan of the extremely thermophilic bacterium Thermus thermophilus contains d-alanine (d-Ala) and d-glutamate (d-Glu), but its d-amino acid metabolism remains poorly understood. Here, we investigated the enzyme activity and function of the product of the TTHA1643 gene, which is annotated to be a Glu racemase in the T. thermophilus HB8 genome. Among 21 amino acids tested, TTHA1643 showed highly specific activity toward Glu as the substrate. The catalytic efficiency (kcat/Km) of TTHA1643 toward d- and l-Glu was comparable; however, the kcat value was 18-fold higher for l-Glu than for d-Glu. Temperature and pH profiles showed that the racemase activity of TTHA1643 is high under physiological conditions for T. thermophilus growth. To assess physiological relevance, we constructed a TTHA1643-deficient strain (∆TTHA1643) by replacing the TTHA1643 gene with the thermostable hygromycin resistance gene. Growth of the ∆TTHA1643 strain in synthetic medium without d-Glu was clearly diminished relative to wild type, although the TTHA1643 deletion was not lethal, suggesting that alternative d-Glu biosynthetic pathways may exist. The deterioration in growth was restored by adding d-Glu to the culture medium, showing that d-Glu is required for normal growth of T. thermophilus. Collectively, our findings show that TTHA1643 is a Glu racemase and has the physiological function of d-Glu production in T. thermophilus.

Lactic acid biosynthesis pathways and important genes of Lactobacillus panis L7 isolated from the Chinese liquor brewing microbiome

Lactic acid is a major organic acid and precursor of ethyl lactate, which confers the important flavor of Chinese Maotai-flavor liquor. Moreover, lactic acid helps modulate the microbiome pH, which coordinates the microbial community. Lactobacillus panis L7 is the major lactic acid producer, however, the whole genome sequence is not available and the important genes for lactic acid biosynthesis have not been unidentified, which limits understanding of lactic acid biosynthesis. Whole genome sequencing of L. panis L7 was undertaken, obtaining 127 genome sequence scaffolds with an N50 of 33,743 bp. The genome total length was 2,026,099 bp encoding 1932 genes with a GC content of 47.0%. Heterolactic acid fermentation pathways and the involved genes were identified, including multiple copies of lactate dehydrogenase encoding genes. To identify important genes for lactic acid production, L. panis L7 was genetically perturbed using atmospheric and room temperature plasma followed by high-throughput screening of mutants with improved or reduced lactic acid production. Comparative genomic analysis of L. panis L7 and its mutants identified genes encoding for glutamate racemase, methylated-DNA-protein-cysteine methyltransferase, fumarate reductase flavoprotein subunit, and amidophosphoribosyltransferase as potentially important factors to improve lactic acid biosynthesis; genes encoding for ribokinase, fructokinase, argininosuccinate lyase, and large subunit ribosomal protein L30 as potentially important factors for reduced lactic acid biosynthesis. The results improved the understanding of the mechanism of lactic acid metabolism by L. panis for the benefit of the Chinese liquor brewing industry, and will, hopefully, help develop effective measures to modulate lactic acid content during brewing.

Efficient Biosynthesis of Low-Molecular-Weight Poly-γ-glutamic Acid Based on Stereochemistry Regulation in Bacillus amyloliquefaciens.

Low-molecular-weight poly-γ-glutamic acid (LMW-γ-PGA) has attracted much attention because of its many potential applications in food, agriculture, medicine, and cosmetics. Enzymatic degradation is an efficient way for the synthesis of LMW-γ-PGA. However, the stereochemistry of γ-PGA limits the degradation of γ-PGA. This study identifies the role of γ-PGA synthase (pgsA) and glutamate racemase (racE) in the regulation of γ-PGA stereochemistry and demonstrates their combinational use for LMW-γ-PGA synthesis. First, the expression of pgsA and racE was enhanced, leading to improvements both in the molecular weight (Mw) and the d-glutamate proportion of γ-PGA. Then, an optimal combination of pgsA, racE, and γ-PGA hydrolase pgdS was constructed by exchanging the gene origins for the synthesis of LMW-γ-PGA. Finally, the Mw of γ-PGA was decreased to 6-8 kDa, which was much lower compared with the case without stereochemistry switching (20-30 kDa). This study provides a novel strategy to control the Mw of γ-PGA based on stereochemistry regulation and lays a solid foundation for synthesis of LMW-γ-PGA.