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Poly-γ-glutamic acid (γ-PGA) is an anionic polymer with various applications. Teichoic acid (TA) is a special component of cell wall in gram-positive bacteria, and its D-alanylation modification can change the net negative charge of cell surface, autolysin activity and cationic binding efficiency, and might further affect metabolic production. In this research, four genes (dltA, dltB, dltC , and dltD) of dlt operon were, respectively, deleted and overexpressed in the γ-PGA producing strain Bacillus licheniformis WX-02. Our results implied that overexpression of these genes could all significantly increase γ-PGA synthetic capabilities, among these strains, the dltB overexpression strain WX-02/pHY- dltB owned the highest γ-PGA yield (2.54 g/L), which was 93.42% higher than that of the control strain WX-02/pHY300 (1.31 g/L). While, the gene deletion strains produced lower γ-PGA titers. Furthermore, 13C-Metabolic flux analysis was conducted to investigate the influence of dltB overexpression on metabolic flux redistribution during γ-PGA synthesis. The simulation data demonstrated that fluxes of pentose phosphate pathway and tricarboxylic acid cycle in WX-02/pHY- dltB were 36.41 and 19.18 mmol/g DCW/h, increased by 7.82 and 38.38% compared to WX-02/pHY300 (33.77 and 13.86 mmol/g DCW/h), respectively. The synthetic capabilities of ATP and NADPH were also increased slightly. Meanwhile, the fluxes of glycolytic and by-product synthetic pathways were all reduced in WX-02/pHY- dltB. All these above phenomenons were beneficial for γ-PGA synthesis. Collectively, this study clarified that overexpression of dltB strengthened the fluxes of PPP pathway, TCA cycle and energy metabolism for γ-PGA synthesis, and provided an effective strategy for enhanced production of γ-PGA. [ABSTRACT FROM AUTHOR]

Poly‐γ‐glutamic acid (γ‐PGA) is an important multifunctional biopolymer with various applications, for which adenosine triphosphate (ATP) supply plays a vital role in biosynthesis. In this study, the enhancement of γ‐PGA production was attempted through various approaches of improving ATP supply in the engineered strains of Bacillus licheniformis. The first approach is to engineer respiration chain branches of B. licheniformis, elimination of cytochrome bd oxidase branch reduced the maintenance coefficient, leading to a 19.27% increase of γ‐PGA yield. The second approach is to introduce Vitreoscilla hemoglobin (VHB) into recombinant B. licheniformis, led to a 13.32% increase of γ‐PGA yield. In the third approach, the genes purB and adK in ATP‐biosynthetic pathway were respectively overexpressed, with the AdK overexpressed strain increased γ‐PGA yield by 14.69%. Our study also confirmed that the respiratory nitrate reductase, NarGHIJ, is responsible for the conversion of nitrate to nitrite, and assimilatory nitrate reductase NasBC is for conversion of nitrite to ammonia. Both NarGHIJ and NasBC were positively regulated by the two‐component system ResD–ResE, and overexpression of NarG, NasC, and ResD also improved the ATP supply and the consequent γ‐PGA yield. Based on the above individual methods, a method of combining the deletion of cydBC gene and overexpression of genes vgB, adK, and resD were used to enhance ATP content of the cells to 3.53 μmol/g of DCW, the mutant WX‐BCVAR with this enhancement produced 43.81 g/L of γ‐PGA, a 38.64% improvement compared to wild‐type strain WX‐02. Collectively, our results demonstrate that improving ATP content in B. licheniformis is an efficient strategy to improve γ‐PGA production. The respiratory chain is optimized to reduce metabolism coefficient, and the VHB and AdK are strengthened to improve ATP supply for enhanced γ‐PGA production. The roles of NarGHIJ, NasBC and ResD∼E are identified in the BFCNA medium during γ‐PGA production, and Strengthening of NarG, NasC and ResD improve the nitrate metabolism, ATP supply and γ‐PGA yield. The γ‐PGA yield produced by WX‐BCVAR is increased by 38.64% via improvement of ATP supply [ABSTRACT FROM AUTHOR]

Poly-γ-glutamic acid (γ-PGA) is a multifunctional biopolymer mainly produced by Bacillus. The cofactor specificity of enzymes plays a critical role in regulating metabolic process and metabolite production. Here, we present a novel approach for switching cofactor specificity of glutamate dehydrogenase RocG from nicotinamide adenine dinucleotide phosphate (NADPH) to nicotinamide adenine dinucleotide (NADH) to improve γ-PGA production. Firstly, 3D structural modeling and molecular docking were performed to predict the binding modes of NADH and NADPH. Several site-specific mutants based on the conventional and Random Accelerated Molecular Dynamics simulations were obtained to alter cofactor specificity. Then, the effects of RocG variants overexpressions on γ-PGA production were evaluated. Compared to the wild-type, the mutant RocGD276E showed highest increase in γ-PGA yield, increased by 40.50%. Meanwhile, yields of main by-products acetoin and 2,3-butandieol were decreased by 21.70% and 16.53%, respectively. Finally, the results of enzymatic properties confirmed that glutamate dehydrogenase mutant RocGD276E exhibited the higher affinity for NADH, caused a shift in coenzyme preference from NADPH to NADH, with a catalytic efficiency comparable with NADPH-dependent RocG. Taken together, this research demonstrated that switching the cofactor preference of glutamate dehydrogenase via rational design was an effective strategy for high-level production of γ-PGA in Bacillus licheniformis. [Display omitted] • Three-dimension structure of B. licheniformis RocG was attained by homology modeling. • Potential mutation sites of RocG were scoped by cMD and RAMD simulations. • The specificity of RocG was altered to NADH-dependent by rational enzyme design. • Cofactor engineering was proven as an effective strategy for increasing γ-PGA production. [ABSTRACT FROM AUTHOR]

Poly-γ-glutamic acid (γ-PGA) is a natural and multifunctional biopolymer. Glycerol, especially crude glycerol, was used as fermentation raw material for production of a variety of chemical products. Here, glycerol was used with nitrate addition for γ-PGA production by Bacillus licheniformis WX-02 at a 5 L fermenter scale. However, nitrite accumulation was discovered in batch fermentation, which resulted in low γ-PGA yield (19.58 g/L). Furthermore, based on transcription analysis and intracellular NADH measurement, nitrite accumulation was probably due to the low content of intracellular NADH in glycerol-based medium, which might be resulted from low efficiency of NADH generation rate and quick NADH consumption by nitrate reduction. Accordingly, different nitrate feeding strategies were designed and optimized in γ-PGA fermentation with glycerol, then applied in fermentation with crude glycerol. Finally, 35.63 g/L of γ-PGA was achieved with the constant-rate nitrate feeding of 0.38 g/(L h) by using crude glycerol, while 36.83 g/L of γ-PGA was achieved with pH-feedback controlling. Therefore, this study provided a novel strategy for cost-effective production of γ-PGA from crude glycerol. • Nitrite accumulation observed in glycerol medium resulted in low γ-PGA yield. • Intracellular NADH content was proven as the key factor for nitrite conversion. • 35 g/L γ-PGA was achieved by using optimized constant-rate nitrate feeding. • pH-feedback control reduced nitrate addition by half with 34 g/L γ-PGA production. • The nitrate feeding strategy was applied for γ-PGA production with crude glycerol. [ABSTRACT FROM AUTHOR]