Improving the enzymatic activity and stability of N-carbamoyl hydrolase using deep learning approach.

BACKGROUND: Optically active D-amino acids are widely used as intermediates in the synthesis of antibiotics, insecticides, and peptide hormones. Currently, the two-enzyme cascade reaction is the most efficient way to produce D-amino acids using enzymes DHdt and DCase, but DCase is susceptible to heat inactivation. Here, to enhance the enzymatic activity and thermal stability of DCase, a rational design software "Feitian" was developed based on kcat prediction using the deep learning approach. RESULTS: According to empirical design and prediction of "Feitian" software, six single-point mutants with high kcat value were selected and successfully constructed by site-directed mutagenesis. Out of six, three mutants (Q4C, T212S, and A302C) showed higher enzymatic activity than the wild-type. Furthermore, the combined triple-point mutant DCase-M3 (Q4C/T212S/A302C) exhibited a 4.25-fold increase in activity (29.77 ± 4.52 U) and a 2.25-fold increase in thermal stability as compared to the wild-type, respectively. Through the whole-cell reaction, the high titer of D-HPG (2.57 ± 0.43 mM) was produced by the mutant Q4C/T212S/A302C, which was about 2.04-fold of the wild-type. Molecular dynamics simulation results showed that DCase-M3 significantly enhances the rigidity of the catalytic site and thus increases the activity of DCase-M3. CONCLUSIONS: In this study, an efficient rational design software "Feitian" was successfully developed with a prediction accuracy of about 50% in enzymatic activity. A triple-point mutant DCase-M3 (Q4C/T212S/A302C) with enhanced enzymatic activity and thermostability was successfully obtained, which could be applied to the development of a fully enzymatic process for the industrial production of D-HPG.

[Using transporter to enhance the acid tolerance of Bacillus coagulans DSM1].

As the precursor of polylactic acid (PLA), optically pure l-lactic acid production is attracting increasing attention. The accumulation of lactic acid during fermentation inhibits strain growth. Therefore, it is necessary to improve the acid tolerance of lactic acid producers. In this study, comparative transcriptomic analysis was performed to investigate the effects of transporters on lactic acid tolerance of Bacillus coagulans DSM1, which is an l-lactic acid producer. The genes with more than two-fold up-regulation in transcriptional profile were further verified using real-time PCR. The transcriptional levels of RS06895, RS10595, RS10595, RS00500, RS00500, RS10635 and RS10635 were enhanced during lactic acid fermentation. Strain overexpressing RS10595 exhibited a retarded cell growth and low lactic acid production at pH 6.0, but an improved lactic acid production at pH 4.6. This study may facilitate the investigation of the acid tolerance mechanism in B. coagulans DSM1, as well as the construction of efficient lactic acid producers.

Enhancement in the catalytic efficiency of D-amino acid oxidase from Glutamicibacter protophormiae by multiple amino acid substitutions.

D-Amino acid oxidase (DAAO) is an imperative oxidoreductase that oxidizes D-amino acids to corresponding keto acids, producing ammonia and hydrogen peroxide. Previously, based on the sequence alignment of DAAO from Glutamicibacter protophormiae (GpDAAO-1) and (GpDAAO-2), 4 residues (E115, N119, T256, T286) at the surface regions of GpDAAO-2, were subjected to site-directed mutagenesis and achieved 4 single-point mutants with enhanced catalytic efficiency (kcat/Km) compared to parental GpDAAO-2. In the present study, to further enhance the catalytic efficiency of GpDAAO-2, a total of 11 (6 double, 4 triple, and 1 quadruple-point) mutants were prepared by the different combinations of 4 single-point mutants. All mutants and wild types were overexpressed, purified and enzymatically characterized. A triple-point mutant E115A/N119D/T286A exhibited the most significant improvement in catalytic efficiency as compared to wild-type GpDAAO-1 and GpDAAO-2. Structural modeling analysis elucidated that residue Y213 in loop region C209-Y219 might act as the active-site lid for controlling substrate access, the residue K256 substituted by threonine (K256T) might change the hydrogen bonding interaction between residue Y213 and the surrounding residues, and switch the conformation of the active-site lid from the closed state to the open state, resulting in the enhancement in substrate accessibility and catalytic efficiency.

Design of a genetically encoded biosensor to establish a high-throughput screening platform for L-cysteine overproduction.

Metabolic engineering seeks to rewire the metabolic network of cells for the efficient production of value-added compounds from renewable substrates. However, it remains challenging to evaluate and identify strains with the desired phenotype from the vast rational or random mutagenesis library. One effective approach to resolve this bottleneck is to design an efficient high-throughput screening (HTS) method to rapidly detect and analyze target candidates. L-cysteine is an important sulfur-containing amino acid and has been widely used in agriculture, pharmaceuticals, cosmetics, and food additive industries. However, HTS methods that enable monitoring of L-cysteine levels and screening of the enzyme variants and strains to confer superior L-cysteine biosynthesis remain unavailable, greatly limiting the development of efficient microbial cell factories for L-cysteine production at the industrial scale. Here, we took advantage of the L-cysteine-responsive transcriptional regulator CcdR to develop a genetically encoded biosensor for engineering and screening the L-cysteine overproducer. The in vivo L-cysteine-responsive assays and in vitro electrophoretic mobility shift assay (EMSA) and DNase I footprint analysis indicated that CcdR is a transcriptional activator that specifically interacts with L-cysteine and binds to its regulatory region to induce the expression of target genes. To improve the response performance of the L-cysteine biosensor, multilevel optimization strategies were performed, including regulator engineering by semi-rational design and systematic optimization of the genetic elements by modulating the promoter and RBS combination. As a result, the dynamic range and sensitivity of the biosensor were significantly improved. Using this the excellent L-cysteine biosensor, a HTS platform was established by coupling with fluorescence-activated cell sorting (FACS) and was successfully applied to achieve direct evolution of the key enzyme in the L-cysteine biosynthetic pathway to increase its catalytic performance and to screen the high L-cysteine-producing strains from the random mutagenesis library. These results presented a paradigm of design and optimization of biosensors to dynamically detect metabolite concentrations and provided a promising tool enabling HTS and metabolic regulation to construct L-cysteine hyperproducing strains to satisfy industrial demand.

Efficient molasses utilization for low-molecular-weight poly-γ-glutamic acid production using a novel Bacillus subtilis stain.

BACKGROUND: Poly-γ-glutamic acid (γ-PGA) is a biopolymer and has various applications based on its biocompatibility, non-toxicity, and edibility. Low-molecular-weight (Mw)-γ-PGA has promising applications in agriculture and pharmaceuticals. It is traditionally produced by enzymatic hydrolysis. Cost-effective bioproduction of low-Mw-γ-PGA is essential for commercial application of γ-PGA. RESULTS: Bacillus subtilis 242 is a newly isolated low-Mw-γ-PGA-producing strain. To develop cost-effective production of γ-PGA using this newly isolated strain, cane molasses and corn steep liquor were used to produce γ-PGA. The concentration of cane molasses was optimized and 100 g/L cane molasses resulted in high γ-PGA production. The effects of yeast extract and corn steep liquor on γ-PGA yield were investigated. High concentration of γ-PGA was obtained in the medium with corn steep liquor. A concentration of 32.14 g/L γ-PGA was achieved in fed-batch fermentation, with a productivity of 0.67 g/L/h and a percentage yield (gγ-PGA/gglutamate) of 106.39%. The Mw of γ-PGA was 27.99 kDa. CONCLUSION: This study demonstrated the potential application of B. subtilis 242 for cost-effective production of low-Mw-γ-PGA from cane molasses.

Characterization of a new L-carnosine synthase mined from deep-sea sediment metagenome.

L-Carnosine is a natural biologically active dipeptide with critical physiological functions, such as antioxidant, antiglycation, and cytoplasmic buffering properties. Direct enzymatic synthesis is a promising way for L-carnosine production. In this study, a new aminopeptidase (gene_236976) with synthetic activity toward L-carnosine was identified by a metagenome mining approach from deep-sea sediment and functionally expressed in Escherichia coli. The enzyme shared a low identity of 14.3% with reported L-carnosine dipeptidase (SmPepD) from Serratia marcescens. ß-Alanine methyl ester was proven to be the best substrate for the synthesis, and no ATP was needed for the enzymatic reaction. The enzyme activity was increased by structure-guided rational design. Only the mutant of G310 site gave positive results, and G310A mutant showed the best performance among the site-direct saturation mutagenesis, indicating that the additional CH3 group of mutant G310A was the main factor affecting the enzymatic activity. The engineered enzyme produced about 10 mM L-carnosine was produced from substrates of 50 mM ß-alanine methyl ester and 50 mM L-histidine, under a tentatively optimized condition. This study enriched the enzyme resources for developing the microbial synthesis process of L-carnosine production.

Significance of broad-spectrum racemases for the viability and pathogenicity of Aeromonas hydrophila.

Aim: To investigate the function of broad-spectrum racemases in Aeromonas hydrophila (BsrA). Results: The A. hydrophila gene encoding BsrA (bsr) mutants (AHΔbsr) exhibited a significant decrease in growth, motility, extracellular protease production and biofilm formation compared with the wild-type. Furthermore, bsr gene knockout instigated cell wall damage compared with the wild-type strains. The survival rate and replication capability in the blood and organs of the AHΔbsr-infected mice were significantly decreased. The degree of tissue injury in the AHΔbsr-infected group was lower than that of the wild-type-infected group. Moreover, there was a significant decrease in the expression of 12 AHΔbsr virulence genes. Conclusion: The bsr gene is essential for the viability and virulence of A. hydrophila.

Engineering of Corynebacterium glutamicum for high-level γ-aminobutyric acid production from glycerol by dynamic metabolic control.

Synthetic biology seeks to reprogram microbial cells for efficient production of value-added compounds from low-cost renewable substrates. A great challenge of chemicals biosynthesis is the competition between cell metabolism and target product synthesis for limited cellular resource. Dynamic regulation provides an effective strategy for fine-tuning metabolic flux to maximize chemicals production. In this work, we created a tunable growth phase-dependent autonomous bifunctional genetic switch (GABS) by coupling growth phase responsive promoters and degrons to dynamically redirect the carbon flux for metabolic state switching from cell growth mode to production mode, and achieved high-level GABA production from low-value glycerol in Corynebacterium glutamicum. A ribosome binding sites (RBS)-library-based pathway optimization strategy was firstly developed to reconstruct and optimize the glycerol utilization pathway in C. glutamicum, and the resulting strain CgGly2 displayed excellent glycerol utilization ability. Then, the initial GABA-producing strain was constructed by deleting the GABA degradation pathway and introducing an exogenous GABA synthetic pathway, which led to 5.26 g/L of GABA production from glycerol. In order to resolve the conflicts of carbon flux between cell growth and GABA production, we used the GABS to reconstruct the GABA synthetic metabolic network, in which the competitive modules of GABA biosynthesis, including the tricarboxylic acid (TCA) cycle module and the arginine biosynthesis module, were dynamically down-regulated while the synthetic modules were dynamically up-regulated after sufficient biomass accumulation. Finally, the resulting strain G7-1 accumulated 45.6 g/L of GABA with a yield of 0.4 g/g glycerol, which was the highest titer of GABA ever reported from low-value glycerol. Therefore, these results provide a promising technology to dynamically balance the metabolic flux for the efficient production of other high value-added chemicals from a low-value substrate in C. glutamicum.

Structure of an Alkaline Pectate Lyase and Rational Engineering with Improved Thermo-Alkaline Stability for Efficient Ramie Degumming.

Alkaline pectate lyases have biotechnological applications in plant fiber processing, such as ramie degumming. Previously, we characterized an alkaline pectate lyase from Bacillus clausii S10, named BacPelA, which showed potential for enzymatic ramie degumming because of its high cleavage activity toward methylated pectins in alkaline conditions. However, BacPelA displayed poor thermo-alkaline stability. Here, we report the 1.78 Å resolution crystal structure of BacPelA in apo form. The enzyme has the characteristic right-handed ß-helix fold of members of the polysaccharide lyase 1 family and shows overall structural similarity to them, but it displays some differences in the details of the secondary structure and Ca2+-binding site. On the basis of the structure, 10 sites located in flexible regions and showing high B-factor and positive ΔTm values were selected for mutation, aiming to improve the thermo-alkaline stability of the enzyme. Following site-directed saturation mutagenesis and screening, mutants A238C, R150G, and R216H showed an increase in the T5015 value at pH 10.0 of 3.0 °C, 6.5 °C, and 7.0 °C, respectively, compared with the wild-type enzyme, interestingly accompanied by a 24.5%, 46.6%, and 61.9% increase in activity. The combined mutant R150G/R216H/A238C showed an 8.5 °C increase in the T5015 value at pH 10.0, and an 86.1% increase in the specific activity at 60 °C, with approximately doubled catalytic efficiency, compared with the wild-type enzyme. Moreover, this mutant retained 86.2% activity after incubation in ramie degumming conditions (4 h, 60 °C, pH 10.0), compared with only 3.4% for wild-type BacPelA. The combined mutant increased the weight loss of ramie fibers in degumming by 30.2% compared with wild-type BacPelA. This work provides a thermo-alkaline stable, highly active pectate lyase with great potential for application in the textile industry, and also illustrates an effective strategy for rational design and improvement of pectate lyases.

Selection and validation of experimental condition-specific reference genes for qRT-PCR in Metopolophium dirhodum (Walker) (Hemiptera: Aphididae).

Metopolophium dirhodum (Walker) (Hemiptera: Aphididae) is one of the most common aphid pests of winter cereals. To facilitate accurate gene expression analyses with qRT-PCR assays, the expression stability of candidate reference genes under specific experimental conditions must be verified before they can be used to normalize target gene expression levels. In this study, 10 candidate reference genes in M. dirhodum were analyzed by qRT-PCR under various experimental conditions. Their expression stability was evaluated with delta Ct, BestKeeper, geNorm, and NormFinder methods, and the final stability ranking was determined with RefFinder. The results indicate that the most appropriate sets of internal controls were SDHB and RPL8 across geographic population; RPL8, Actin, and GAPDH across developmental stage; SDHB and NADH across body part; RPL8 and Actin across wing dimorphism and temperature; RPL4 and EF1A across starvation stress; AK and RPL4 across insecticide treatments; RPL8 and NADH across antibiotic treatments; RPL8, RPL4, Actin, and NADH across all samples. The results of this study provide useful insights for establishing a standardized qRT-PCR procedure for M. dirhodum and may be relevant for identifying appropriate reference genes for molecular analyses of related insects.

Extracellular production of active-form Streptomyces mobaraensis transglutaminase in Bacillus subtilis.

Transglutaminase (TG) from Streptomyces mobaraensis has been widely used in the food industry. It is secreted naturally as an inactive zymogen, which is then activated by the removal of the N-terminal pro-peptide. In this study, the mtg gene from S. mobaraensis was expressed in a food-grade strain of bacterium, Bacillus subtilis. When its native signal peptide was replaced by signal peptide SacB (SPsacB) and the pro-peptide was replaced by that derived from S. hygroscopicus, an extracellular activity of 16.1 U/mg was observed. A modified Saccharomyces cerevisiae vacuolar ATPase subunit (VMA) intein was introduced into the zymogen to simplify its activation process by controlling temperature. When the cleavage site in the C-terminal of VMA was placed between the pro-peptide and core domain, the activation process was carried out at 18 °C. Promoter replacement further increased the enzymatic activity. Finally, the extracellular enzymatic activity reached 2.6 U/mg under the control of the constitutive promoter PyvyD. This is the first report on the extracellular production of active-form Streptomyces TG in B. subtilis without splicing with the cleavage enzyme.

Purification, Characterization and Inhibition of Alanine Racemase from a Pathogenic Strain of Streptococcus iniae.

Streptococcus iniae is a pathogenic and zoonotic bacteria that impacted high mortality to many fish species as well as capable of causing serious disease to humans. Alanine racemase (Alr, EC 5.1.1.1) is a pyridoxal-5'-phosphate (PLP)-containing homodimeric enzyme that catalyzes the racemization of L-alanine and D-alanine. In this study, we purified alanine racemase from S. iniae that was isolated from an infected Chinese sturgeon (Acipenser sinensis), as well as determined its biochemical characteristics and inhibitors. The alr gene has an open reading frame (ORF) of 1107 bp, encoding a protein of 369 amino acids, which has a molecular mass of 40 kDa. The enzyme has optimal activity at a temperature of 35°C and a pH of 9.5. It belongs to the PLP-dependent enzymes family and is highly specific to L-alanine. S. iniae Alr (SiAlr) could be inhibited by some metal ions, hydroxylamine and dithiothreitol (DTT). The kinetic parameters K m and V max of the enzyme were 33.11 mM, 2426 units/mg for L-alanine, and 14.36 mM, 963.6 units/mg for D-alanine. Finally, the 50% inhibitory concentrations (IC50) values and antibiotic activity of two alanine racemase inhibitors (homogentisic acid and hydroquinone), were determined and found to be effective against both Gram-positive and Gram-negative bacteria employed in this study.Streptococcus iniae is a pathogenic and zoonotic bacteria that impacted high mortality to many fish species as well as capable of causing serious disease to humans. Alanine racemase (Alr, EC 5.1.1.1) is a pyridoxal-5'-phosphate (PLP)-containing homodimeric enzyme that catalyzes the racemization of L-alanine and D-alanine. In this study, we purified alanine racemase from S. iniae that was isolated from an infected Chinese sturgeon (Acipenser sinensis), as well as determined its biochemical characteristics and inhibitors. The alr gene has an open reading frame (ORF) of 1107 bp, encoding a protein of 369 amino acids, which has a molecular mass of 40 kDa. The enzyme has optimal activity at a temperature of 35°C and a pH of 9.5. It belongs to the PLP-dependent enzymes family and is highly specific to L-alanine. S. iniae Alr (SiAlr) could be inhibited by some metal ions, hydroxylamine and dithiothreitol (DTT). The kinetic parameters K m and V max of the enzyme were 33.11 mM, 2426 units/mg for L-alanine, and 14.36 mM, 963.6 units/mg for D-alanine. Finally, the 50% inhibitory concentrations (IC50) values and antibiotic activity of two alanine racemase inhibitors (homogentisic acid and hydroquinone), were determined and found to be effective against both Gram-positive and Gram-negative bacteria employed in this study.

Elucidating the Role and Regulation of a Lactate Permease as Lactate Transporter in Bacillus coagulans DSM1.

A key feature of Bacillus coagulans is its ability to produce l-lactate via homofermentative metabolism. A putative lactate permease-encoding gene (lutP) and the gene encoding its regulator (lutR) were identified in one operon in B. coagulans strains. LutP orthologs are highly conserved and located adjacent to the gene cluster related to lactate utilization in most lactate-utilizing microorganisms. However, no lactate utilization genes were found adjacent to lutP in all sequenced B. coagulans strains. The stand-alone presence of lutP in l-lactate producers indicates that it may have functions in lactate production. In this study, B. coagulans DSM1 was used as a representative strain, and the critical roles of LutP and its regulation were described. Transport property assays showed that LutP was essential for lactate uptake. Its regulator LutR directly interacted with the lutP-lutR intergenic region, and lutP transcription was activated by l-lactate via regulation by LutR. A biolayer interferometry assay further confirmed that LutR bound to an 11-bp inverted repeat in the intergenic region, and lutP transcription began when the binding of LutR to the lutP upstream sequence was inhibited. We conclusively showed that lutP encodes a functional lactate permease in B. coagulansIMPORTANCE Lactate-utilizing strains require lactate permease (LutP) to transport lactate into cells. Bacillus coagulans LutP is a previously uncharacterized lactate permease with no lactate utilization genes situated either adjacent to or remotely from it. In this study, an active lactate permease in an l-lactate producer, B. coagulans DSM1, was identified. Lactate supplementation regulated the expression of lactate permease. This study presents physiological evidence of the presence of a lactate transporter in B. coagulans Our findings indicate a potential target for the engineering of strains in order to improve their fermentation characteristics.

Knockout of alanine racemase gene attenuates the pathogenicity of Aeromonas hydrophila.

BACKGROUND: Aeromonas hydrophila is an opportunistic pathogen of poikilothermic and homoeothermic animals, including humans. In the present study, we described the role of Alanine racemase (alr-2) in the virulence of A. hydrophila using an alr-2 knockout mutant (A.H.Δalr). RESULTS: In mouse and common carp models, the survival of animals challenged with A.H.Δalr was significantly increased compared with the wild-type (WT), and the mutant was also impaired in its ability to replicate in the organs and blood of infected mice and fish. The A.H.Δalr significantly increased phagocytosis by macrophages of the mice and fish. These attenuation effects of alr-2 could be complemented by the addition of D-alanine to the A.H.Δalr strain. The histopathology results indicated that the extent of tissue injury in the WT-infected animals was more severe than in the A.H.Δalr-infected groups. The expression of 9 virulence genes was significantly down-regulated, and 3 outer membrane genes were significantly up-regulated in A.H.Δalr. CONCLUSIONS: Our data suggest that alr-2 is essential for the virulence of A. hydrophila. Our findings suggested alanine racemase could be applied in the development of new antibiotics against A. hydrophila.

Biochemical characterization and mutational analysis of alanine racemase from Clostridium perfringens.

Clostridium perfringens is a gram-positive, anaerobic, pathogenic bacterium that can cause a wide range of diseases in humans, poultry and agriculturally important livestock. A pyridoxal-5-phosphate-dependent alanine racemase with a function in the racemization of d- and l-alanine is an attractive drug target for C. perfringens and other pathogens due to its absence in animals and humans. In this study alanine racemase from C. perfringens (CPAlr) was successfully expressed and purified in Escherichia coli and biochemically characterized. The purified CPAlr protein was a dimeric PLP-dependent enzyme with high substrate specificity. The optimal racemization temperature and pH were 40°C and 8.0, respectively. The kinetic parameters Km and kcat of CPAlr, determined by HPLC at 40°C were 19.1 mM and 17.2 s-1 for l-alanine, and 10.5 mM and 8.7 s-1 for d-alanine, respectively. Gel filtration chromatographic analysis showed that the molecular weight of mutant Y359A was close to monomeric form, suggesting that the inner layer residue Tyr359 might play an essential role in dimer-formation. Furthermore, the mutation at residues Asp171 and Tyr359 resulted in a dramatic increase in Km value and/or decreased in kcat value, indicating that the middle and inner layer residues Asp171 and Tyr359 of CPAlr might have the key role in substrate binding, catalytic activity or oligomerization state through the hydrogen-bonding interaction with the pentagonal ring waters and/or PLP cofactor.

Impaired oxidative stress and sulfur assimilation contribute to acid tolerance of Corynebacterium glutamicum.

The industrial organism Corynebacterium glutamicum is often subjected to acid stress during large-scale fermentation for the production of bio-based chemicals. The capacity of the cells to thrive in acidic environments is a prerequisite for achieving high product yields. In this study, we obtained an acid-adapted strain using an adaptive laboratory evolution strategy. Physiological characterizations revealed that the adapted strain achieved improved cell viability after acid-stress challenge, with a higher cytoplasmic pHin level, a lower intracellular reactive oxygen species (ROS), and an enhanced morphological integrity of the cells, when compared to those of the original control strain. Transcriptome analysis indicated that several important cellular processes were altered in the adapted strain, including sulfur metabolism, iron transport, and central metabolic pathways. Further research displayed that KatA and Dps cooperatively mediated intracellular ROS scavenging, which was required for resistance to low-pH stress in C. glutamicum. Furthermore, the repression of sulfur assimilation by the McbR regulator also contributed to the improvement of acid-stress tolerance. Moreover, two copper chaperone genes cg1328 and cg3292 were found to be involved in promoting cell survival under acid-stress conditions. Finally, a new recombinant C. glutamicum strain with enhanced acid tolerance was generated by the combined overexpression of katA, dps, mcbR, and cg1328, showing 18.4 ± 2.5% higher biomass yields than the wild-type strain under acid-stress conditions. These findings will provide new insights into the understanding and genetic improvement of acid tolerance in C. glutamicum.

Metabolic engineering of Corynebacterium glutamicum for L-cysteine production.

L-cysteine, a valuable sulfur-containing amino acid, has been widely used in food, agriculture, and pharmaceutical industries. Due to the toxicity and complex regulation of L-cysteine, no efficient cell factory has yet been achieved for L-cysteine industrial production. In this study, the food-grade microorganism Corynebacterium glutamicum was engineered for L-cysteine production. Through deletion of the L-cysteine desulfhydrases (CD) and overexpression of the native serine acetyltransferase (CysE), the initial L-cysteine-producing strain CYS-2 was constructed to produce 58.2 ± 5.1 mg/L of L-cysteine. Subsequently, several metabolic engineering strategies were performed to further promote L-cysteine biosynthesis, including using strong promoter tac-M to enhance expression intensity of CysE, investigating the best candidate among several heterogeneous feedback-insensitive CysEs for L-cysteine biosynthesis, overexpressing L-cysteine synthase (CysK) to drive more metabolic flux, evaluating the efflux capacity of several heterogeneous L-cysteine transporters, engineering L-serine biosynthesis module to increase the precursor L-serine level and using thiosulfate as the sulfur source. Finally, the L-cysteine concentration of the engineered strain CYS-19 could produce 947.9 ± 46.5 mg/L with addition of 6 g/L Na2S2O3, approximately 14.1-fold higher than that of the initial strain CYS-2, which was the highest titer of L-cysteine ever reported in C. glutamicum. These results indicated that C. glutamicum was a promising platform for L-cysteine production.

Enzymatic characterization and crystal structure of biosynthetic alanine racemase from Pseudomonas aeruginosa PAO1.

Alanine racemase is a pyridoxal-5'-phosphate (PLP)-dependent enzyme that reversibly catalyzes the conversion of l-alanine to d-alanine. d-alanine is an essential constituent in many prokaryotic cell structures. Inhibition of alanine racemase is lethal to prokaryotes, creating an attractive target for designing antibacterial drugs. Here we report the crystal structure of biosynthetic alanine racemase (Alr) from a pathogenic bacteria Pseudomonas aeruginosa PAO1. Structural studies showed that P. aeruginosa Alr (PaAlr) adopts a conserved homodimer structure. A guest substrate d-lysine was observed in the active site and refined to dual-conformation. Two buffer ions, malonate and acetate, were bound in the proximity to d-lysine. Biochemical characterization revealed the optimal reaction conditions for PaAlr.

Structural features and kinetic characterization of alanine racemase from Bacillus pseudofirmus OF4.

Alanine racemase (Alr) is a pyridoxal-5'-phosphate-dependent (PLP) enzyme that catalyzes a reversible racemization between the enantiomers of alanine. d-Alanine is an indispensable constituent in the biosynthesis of bacterial cell-wall peptidoglycan, and its inhibition is lethal to prokaryotes, which makes it an attractive target for designing antibacterial drugs. In this study, the molecular structure of alanine racemase from Bacillus pseudofirmus OF4 (DadXOF4) was determined by X-ray crystallography to a resolution of 1.8 Å. The comparison of DadXOF4 with alanine racemases from other bacteria demonstrated a conserved overall fold. Enzyme kinetics analysis showed that the conserved residues at the substrate entryway and the salt bridge at the dimer interface are critical for enzyme activity. These structural and biochemical findings provide a template for future structure-based drug-development efforts targeting alanine racemases.