Abstract
BackgroundPoly-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.ResultsThe 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.ConclusionsThe 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.
Highlights
Poly-γ-glutamic acid (γ-PGA) is a natural polymer with great potential applications in areas of agriculture, industry, and pharmaceutical
As a terminator-dependent carbon catabolite repression (CCR) mechanism is operative for the glpFK operon by the terminator tglpFK, whose formation is prevented by the Gly3P-activated antiterminator GlpP [18], the terminator tglpFK was knocked out by homologous recombination, and the glpK and glpP were overexpressed, respectively, by introducing an additional copy of each gene under P43 promoter
The glycerol consumption (37.59 Glycerol γ-PGA titer (g/L)) and poly-γ-glutamic acid (γ-PGA) titer (16.98 g/L) of WX02-glpK were increased by 23.20% and 32.35%, respectively, compared to the original strain WX-02, and the growth rate of WX02-glpK strain increased obviously (Fig. 2a)
Summary
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 is an ideal substrate for γ-PGA production due to its abundance and low price. About 10 kg of crude glycerol will be generated from every 100 kg of biodiesel production [6]. There are some chemical applications utilizing pure glycerol as feedstock, but it is not economical to refine the crude glycerol into pure glycerol [8]. Crude glycerol is biologically converted into the value-added products such as acetoin [9], succinate [10], n-butanol [11], 3-hydroxypropionic acid [12], 1,3-propanediol [13], and poly-3-hydroxybutyrate [14]. Glycerol can generate more reducing agents than other carbon sources, and is able to produce high amounts of biochemical products [6]
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