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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.

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 k cat prediction using the deep learning approach. Results According to empirical design and prediction of “Feitian” software, six single-point mutants with high k cat 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.

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.

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 ( Sm PepD) from Serratia marcescens . β-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 CH 3 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 β-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.

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 .

Alanine is the most abundant chiral amino acid that exists into the D -alanine or L-alanine forms with diverse applications in the biomedical, pharmaceutical, plastics, and food industries. D/L-alanine production can be carried out through chemical, microbial fermentation, and biocatalytic methods and not much effective due to complicated processes or purification issues and is still challenging to achieve a higher yield. In the present study, biobrick method was utilized for efficient production of D/L-alanine in the recombinant Escherichia coli BL21(DE3) with tandem three-gene co-expression plasmid. Firstly, the co-expression plasmid pET-22bNS-DadX-Ald-Gdh containing three genes, alanine dehydrogenase ( ald) , alanine racemase ( dadX) , and glucose dehydrogenase ( gdh) from Bacillus pseudofirmus OF4 were successfully constructed and introduced into the E. coli BL21(DE3) strain. Then, under optimized conditions in the whole-cell biocatalytic reaction [20 mM Na 2 CO 3 -NaHCO 3 (pH 10.1), 200 mM D-glucose, 200 mM sodium pyruvate, and 200 mM ammonium chloride], the concentration of D-alanine and L-alanine reached the maximum value (6.48 g/L and 7.05 g/L) after 3.0 h reaction time at 37°C under 180 rpm rotation. Meanwhile, promoter replacement experiments and Western blot analysis revealed that the expression level of protein OF4Ald had a significant effect on the production of D/L-alanine, indicating that alanine dehydrogenase might be the rate-limiting enzyme for D/L-alanine synthesis. This study provides a simple, feasible, and efficient biosynthesis process of D/L-alanine, which could explore emerging applications for large-scale production of industrial bioproducts.

Ectoine is a compatible solute naturally produced in some halophilic bacteria as a protective agent for survival in salty environments. It has gained special interest as a therapeutic agent in the pharmaceutical and healthcare sectors for the treatment of different diseases. Ectoine mainly produced by bacterial milking, chemical, and fed-batch fermentation methods under a high-salt medium. Unfortunately, the ectoine yield through these methods is still too low to meet high industrial demand, causing salinity issues. The biobrick method was potentially utilized for efficient ectoine biosynthesis under a low-salt medium with different conditions in E. coli BL21(DE3) harboring the pET-22bNS-EctA-EctB-EctC plasmid. Firstly, three genes, L-2,4-diamino-butyric acid acetyltransferase (ectA), L-2,4-diaminobutyric acid transaminase (ectB), and ectoine synthase (ectC) from Bacillus pseudofirmus OF4, were precisely assembled and expressed into E. coli BL21(DE3). After optimizing the reaction conditions in a whole-cell catalytic reaction [50 mM of the sodium phosphate buffer (pH~7.5) containing 300 mM L-aspartic acid, 100 mM glycerol, 1/20 g/mL cell pellets], the amount of ectoine in the plasmid pET-22bNS-ALacBTacCTac reached the maximum level of 167.2 mg/mL/d (6.97 mg/mL/h). Moreover, Western blot analysis revealed that high expression levels of EctA and EctC had a significant effect on ectoine biosynthesis, indicating that both proteins might be the key enzymes in ectoine production. We conclude that a high amount of ectoine achieved through the biobrick method and efficiently used for different industrial applications.

Background Combining experimental and computational screening methods has been of keen interest in drug discovery. In the present study, we developed an efficient screening method that has been used to screen 2100 small-molecule compounds for alanine racemase Alr-2 inhibitors. Results We identified ten novel non-substrate Alr-2 inhibitors, of which patulin, homogentisic acid, and hydroquinone were active against Aeromonas hydrophila . The compounds were found to be capable of inhibiting Alr-2 to different extents with 50% inhibitory concentrations (IC 50 ) ranging from 6.6 to 17.7 μM. These compounds inhibited the growth of A. hydrophila with minimal inhibitory concentrations (MICs) ranging from 20 to 120 μg/ml. These compounds have no activity on horseradish peroxidase and d -amino acid oxidase at a concentration of 50 μM. The MTT assay revealed that homogentisic acid and hydroquinone have minimal cytotoxicity against mammalian cells. The kinetic studies indicated a competitive inhibition of homogentisic acid against Alr-2 with an inhibition constant ( K i ) of 51.7 μM, while hydroquinone was a noncompetitive inhibitor with a K i of 212 μM. Molecular docking studies suggested that homogentisic acid binds to the active site of racemase, while hydroquinone lies near the active center of alanine racemase. Conclusions Our findings suggested that combining experimental and computational methods could be used for an efficient, large-scale screening of alanine racemase inhibitors against A. hydrophila that could be applied in the development of new antibiotics against A. hydrophila .

Pyridoxal 5'-phosphate (PLP) dependent alanine racemase catalyzes racemization of L-Ala to D-Ala, a key component of the peptidoglycan network in bacterial cell wall. It has been extensively studied as an important antimicrobial drug target due to its restriction in eukaryotes. However, many marketed alanine racemase inhibitors also act on eukaryotic PLP-dependent enzymes and cause side effects. A thermostable alanine racemase (AlrTt) from Thermoanaerobacter tengcongensis MB4 contains an evolutionarily non-conserved residue Gln360 in inner layer of the substrate entryway, which is supposed to be a key determinant in substrate specificity. Here we determined the crystal structure of AlrTt in complex with L-Ala at 2.7 Å resolution, and investigated the role of Gln360 by saturation mutagenesis and kinetic analysis. Compared to typical bacterial alanine racemase, presence of Gln360 and conformational changes of active site residues disrupted the hydrogen bonding interactions necessary for proper PLP immobilization, and decreased both the substrate affinity and turnover number of AlrTt. However, it could be complemented by introduction of hydrophobic amino acids at Gln360, through steric blocking and interactions with a hydrophobic patch near active site pocket. These observations explained the low racemase activity of AlrTt, revealed the essential role of Gln360 in substrate selection, and its preference for hydrophobic amino acids especially Tyr in bacterial alanine racemization. Our work will contribute new insights into the alanine racemization mechanism for antimicrobial drug development.