A novel approach to improve poly-γ-glutamic acid production by NADPH Regeneration in Bacillus licheniformis WX-02

Abstract

Poly-γ-glutamic acid (γ-PGA) is an important biochemical product with a variety of applications. This work reports a novel approach to improve γ-PGA through over expression of key enzymes in cofactor NADPH generating process for NADPH pool. Six genes encoding the key enzymes in NADPH generation were over-expressed in the γ-PGA producing strain B. licheniformis WX-02. Among various recombinants, the strain over-expressing zwf gene (coding for glucose-6-phosphate dehydrogenase), WX-zwf, produced the highest γ-PGA concentration (9.13 g/L), 35% improvement compared to the control strain WX-pHY300. However, the growth rates and glucose uptake rates of the mutant WX-zwf were decreased. The transcriptional levels of the genes pgsB and pgsC responsible for γ-PGA biosynthesis were increased by 8.21- and 5.26-fold, respectively. The Zwf activity of the zwf over expression strain increased by 9.28-fold, which led to the improvement of the NADPH generation, and decrease of accumulation of by-products acetoin and 2,3-butanediol. Collectively, these results demonstrated that NADPH generation via over-expression of Zwf is as an effective strategy to improve the γ-PGA production in B. licheniformis.

Figure 1. The metabolic pathways of NADPH generation and γ-PGA production in B. licheniformis WX-02.

Figure 2. Effects of the over-expression of Zwf, Gdh, Gnd, Ppnk, pntAB and UdhA on the γ-PGA production.

Figure 3. Fermentation process curve of B. licheniformis WX-pHY300 and WX-zwf.

(A) γ-PGA yield; (B) Biomass; (C) Residue glucose; (D) Acetoin and 2,3-butanediol concentrations.

Figure 4. Effects of over-expression of Zwf on the relative transcriptional levels of the genes in the glucose metabolic and γ-PGA biosynthesis.

Figure 5. Assay of the activity of Zwf.