AUTHOR=He Penghui , Wan Ni , Cai Dongbo , Hu Shiying , Chen Yaozhong , Li Shunyi , Chen Shouwen TITLE=13C-Metabolic Flux Analysis Reveals the Metabolic Flux Redistribution for Enhanced Production of Poly-γ-Glutamic Acid in dlt Over-Expressed Bacillus licheniformis JOURNAL=Frontiers in Microbiology VOLUME=10 YEAR=2019 URL=https://www.frontiersin.org/journals/microbiology/articles/10.3389/fmicb.2019.00105 DOI=10.3389/fmicb.2019.00105 ISSN=1664-302X 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.