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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.

Bacillus licheniformis WX-02 is a well-studied strain to produce poly-γ-glutamic acid (γ-PGA) with numerous applications. This study is to improve WX-02 strain's capability of assimilating glycerol, a major byproduct of biofuels industries, through metabolic manipulation. Through gene knockout, the GlpK pathway was identified as the sole functional glycerol catabolism pathway, while the DhaK pathway was inactive for this strain under either aerobic or anaerobic conditions. The enhancement of glycerol utilization was attempted by substituting the native glpFK promoter with the constitutive promoter (P43), ytzE promoter (PytzE), and bacABC operon promoter (PbacA), respectively. The glycerol consumptions of the corresponding mutant strains WX02-P43glpFK, WX02-PytzEglpFK, and WX02-PbacAglpFK were 30.9, 26.42, and 18.8% higher than that of the WX-02 strain, respectively. The γ-PGA concentrations produced by the three mutant strains were 33.71, 23.39, and 30.05% higher than that of WX-02 strain, respectively. When biodiesel-derived crude glycerol was used as the carbon source, the mutant WX02-P43glpFK produced 16.63 g L -1 of γ-PGA, with a productivity of 0.35 g L -1  h -1 . Collectively, this study demonstrated that glycerol can be used as an effective substrate for producing γ-PGA by metabolic engineering B. licheniformis strains.