Metabolic Engineering Of Bacillus Licheniformis To Enhance Poly-γ-glutamic Acid Production

With the vigorous development of the bioenergy industry,more and more inexpensive by-products of crude glycerol will be produced during the production of biodiesel.The use of these glycerol as raw materials to produce high value-added products,not only can reduce the production costs of bio-based products,but also to promote the development of bioenergy industry.As a new type of biopolymer materials,poly-γ-glutamic acid(γ-PGA)has wide application value in the food,medicine,environmental protection and agriculture and other fields.Utilize glycerol to biosynthesize γ-PGA,not only can make the rational use of resources,but also can reduce the fermentation cost of γ-PGA.However,the use of glycerol fermentation of γ-PGA in Bacillus licheniformis has the disadvantage of low utilization of glycerol and low synthesis rate of γ-PGA,which limits its industrial application.In this study,B.licheniformis WX-02 was used as the original strain to transform the glycerol metabolic pathway and the central carbon metabolic pathway through rational metabolic engineering to improve the utilization efficiency of glycerol and the availability of NADPH to highly efficient crude glycerol utilization for γ-PGA production.In this study,the glycerol respiratory metabolic pathway was metabolically engineered that the glp FK operon leader was deleted and the glycerol kinase glp K was integrated on the B.licheniformis WX-02 chromosome.It was found that deletion of glp FK operon precursors resulted in a decrease in γ-PGA yield and bacterial biomass,which did not achieve the goal of lifting tglp FK-dependent CCR to improve glycerol utilization.The γ-PGA yield and glycerol consumption of glp K intrgrative strain were increased by 32.34% and 23.21% in the medium with glycerol as the carbon source and no exogenous glutamate.It was also found that the addition of a glp K copy on the WX-02 chromosome not only improved the level of glp K transcription but also raised the transcription level of key genes in several pathways.At the same time,we focus on the carbon flux changes in DHAP(dihydroxyacetone phosphate)point of glycerol metabolism,the metabolic engineering of gluconeogenesis was studied.pyc A,pck A,pgi and glp X were used as key genes.The effects of these four genes on glycerol metabolism and γ-PGA synthesis were studied by free enhanced expression.pyc A,pck A and pgi had no significant effect on the yield of γ-PGA,but the yield of γ-PGA of fructose 1,6-diphosphatase glp X over-expression was 43.59% higher than that of the control strain.While enhancing the expression of glp X by replacing the original promoter of the glp X gene or increasing the chromosome copy of the gene glp X.It was found that the using the constituent promoter P43 that derived from B.subtilis 168,the yield and glycerol utilization of γ-PGA were increased by 26.87% and 27.86%,respectively.Meanwhile,after adding a glp X copy on B.licheniformis chromosome,the yield of γ-PGA and glycerol consumption was increased by 27.64% and 26.82%,respectively,and the transcription level was increased.Moreover,the PPP pathway was enhanced by enhancing the glucose-6-phosphate dehydrogenase zwf,transketolase tkt1 and tkt2 expression.The results showed that the addition of a zwf gene to the WX-02 chromosome resulted in 17.11% and 14.19%enhancement in the yield of γ-PGA and glycerol utilization,respectively.The integrated expression gene tkt1 increased the consumption of glycerol by 24.31%,and γ-PGA The yield was 14.39% higher than that of the original strain.To strengthen glycerol oxidation pathway and direct more carbon into gluconeogenesis and the PP pathway,three engineering strains has been constructed.Theγ-PGA production,biomass and glycerol consumption of each engineering strain were significantly increased compared with wild-type strain.And through the change of the transcription level of the key genes revealed the possible expression of the key genes to increase the utilization of glycerol and the possible causes of γ-PGA production.At the same time,we found that the combination of glp K,glp X,zwf and tkt1 on B.licheniformis WX-02 chromosome resulted in an increase of 30.45% and NADPH / NADH ratio of62.70%,respectively,compared with wild-type WX-02 The synthesis of γ-PGA provides more glutamate precursors and NADPH.