Abstract
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.
Highlights
Poly-γ-glutamic acid (γ-PGA) is an important anionic polypeptide consisting of D-glutamic acid and/or L-glutamic acid residues, which linked together via amide bonds between α-amino and γ-carboxyl (Feng et al, 2015; Sirisansaneeyakul et al, 2017)
B. licheniformis WX-02 was acted as the original strain for constructing recombinants, and E. coli DH5α was served as the host strain for plasmid construction
Three volumes of absolute ethanol were added into the supernatant after adjusting pH to 7.0, and the precipitate was dried at 80◦C to a constant weight for measuring γ-PGA yield
Summary
Poly-γ-glutamic acid (γ-PGA) is an important anionic polypeptide consisting of D-glutamic acid and/or L-glutamic acid residues, which linked together via amide bonds between α-amino and γ-carboxyl (Feng et al, 2015; Sirisansaneeyakul et al, 2017). Bacillus species have been proven as the efficient γ-PGA producers, and a number of metabolic engineering strategies have been developed to improve γ-PGA yield. Knocking out glutamate dehydrogenase genes rocG and gudB improved glutamic acid accumulation, which led to a 38% increase of γ-PGA yield in Bacillus amyloliquefaciens (Zhang et al, 2015). Cell surface engineering was proven to be an effective strategy for enhancement production of metabolics. Overexpression of phosphatidylserine synthase gene pssA could enhance the cell membrane integrity and hydrophilicity, and further improved the cell tolerance and biorenewable yields (short-chain fatty acids, organic alcohols, organic acids and other aromatic compounds, etc) in E. coli (Tan et al, 2017). Elevation of membrane cardiolipin levels via overexpressing cardiolipin synthase gene clsA significantly increased hyaluronic acid titer by 204% in Bacillus subtilis (Westbrook et al, 2018). No research has been focused on the relationship between cell surface engineering and γ-PGA synthesis
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