Rewiring glycerol metabolism for enhanced production of poly-γ-glutamic acid in Bacillus licheniformis

Abstract

Abstract Background Poly-γ-glutamic acid (γ-PGA) is a natural polymer with great potential applications in areas of agriculture, industry, and pharmaceutical. The biodiesel-derived glycerol can be used as an attractive feedstock for γ-PGA production due to its availability and low price; however, insufficient production of γ-PGA from glycerol is limitation. Results The metabolic pathway of Bacillus licheniformis WX-02 was rewired to improve the efficiency of glycerol assimilation and the supply of NADPH for γ-PGA synthesis. GlpK, GlpX, Zwf, and Tkt1 were found to be the key enzymes for γ-PGA synthesis using glycerol as a feedstock. Through combinational expression of these key enzymes, the γ-PGA titer increased to 19.20 ± 1.57 g/L, which was 1.50-fold of that of the wild-type strain. Then, we studied the flux distributions, gene expression, and intracellular metabolites in WX-02 and the recombinant strain BC4 (over-expression of the above quadruple enzymes). Our results indicated that over-expression of the quadruple enzymes redistributed metabolic flux to γ-PGA synthesis. Furthermore, using crude glycerol as carbon source, the BC4 strain showed a high productivity of 0.38 g/L/h, and produced 18.41 g/L γ-PGA, with a high yield of 0.46 g γ-PGA/g glycerol. Conclusions The approach to rewiring of metabolic pathways enables B. licheniformis to efficiently synthesize γ-PGA from glycerol. The γ-PGA productivity reported in this work is the highest obtained in glutamate-free medium. The present study demonstrates that the recombinant B. licheniformis strain shows significant potential to produce valuable compounds from crude glycerol.

Keywords

• Bacillus licheniformis
• Poly-γ-glutamic acid
• Glycerol metabolism
• Gluconeogenesis pathway
• NADPH
• Combinatorial optimization