Enhanced Production of Poly-gamma-glutamic acid by Overexpression of the Global Anaerobic Regulator Fnr in Bacillus licheniformis WX-02

Poly-gamma-glutamic acid is a multi-functional biopolymer with various applications. ATP supply plays an important role in poly-gamma-glutamic acid (gamma-PGA) synthesis. Global anaerobic regulator Fnr plays a key role in anaerobic adaptation and nitrate respiration, which might affect ATP generation during gamma-PGA synthesis. In this study, we have improved gamma-PGA production by overexpression of Fnr in Bacillus licheniformis WX-02. First, the gene fnr was knocked out in WX-02, and the gamma-PGA yields have no significant differences between WX-02 and the fnr-deficient strain WX Delta fnr in the medium without nitrate (BFC medium). However, the gamma-PGA yield of 8.95 g/L, which was produced by WX Delta fnr in the medium with nitrate addition (BFCN medium), decreased by 74% compared to WX-02 (34.53 g/L). Then, the fnr complementation strain WX Delta fnr/pHY-fnr restored the gamma-PGA synthesis capability, and gamma-PGA yield was increased by 13% in the Fnr overexpression strain WX/pHY-fnr (39.96 g/L) in BFCN medium, compared to WX/pHY300 (35.41 g/L). Furthermore, the transcriptional levels of narK, narG, and hmp were increased by 5.41-, 4.93-, and 3.93-fold in WX/pHY-fnr, respectively, which led to the increases of nitrate consumption rate and ATP supply for gamma-PGA synthesis. Collectively, Fnr affects gamma-PGA synthesis mainly through manipulating the expression level of nitrate metabolism, and this study provides a novel strategy to improve gamma-PGA production by overexpression of Fnr.