Metabolic Engineering Of Escherichia Coli For Acetylglucosamine Production

The aim of this study was to construct an efficient synthesis pathway of N-acetylglucosamine(Glc NAc)in Escherichia coli.Glucosamine synthase genes Bs Glm S and Ec Glm S from Bacillus subtilis and Escherichia coli were co-expressed with GNA1 from yeast.Recombinant plasmid p T-Bs Glm S-GNA1 and p T-Ec Glm S-GNAl with medium intensity promoter and medium copy number plasmid p Trc99A3,and the recombinant engineered strains MG:Bs Glm S-GNA1 and MG:Ec Glm S-GNA1 were constructed using E.coli MG1655 as host bacteria.The yield of Glc NAc was increased by optimizing the carbon source and inducing conditions in 2YT medium.The results showed that Glc NAc was the main product,and glycerol was the better carbon source for product synthesis than glucose,and 0.1 mm IPTG was the best induction condition at 37~oC:the Glc NAc and Glc N production of MG:Ec Glm S-GNA1 and MG:Bs Glm S-GNA1 were 4.36 g/l and 4.67 g/l,respectively,and the latter was 6.4%higher than the former.The results show that Bsglms is more suitable for the production of Glc N and Glc NAc than Ecglms.In this study,end A1 gene in E.coli was knocked out to improve plasmid stability,ato DA,yjg B,ack A-pta,nag K,nag E and man X genes were also knocked out to study their effects on the yield of Glc N and Glc NAc.The results showed that the total yield of end A1 defective engineered strain MGΔend A1:Bs Glm S-GNAl was 6.16 g/l,which was 31.9%higher than that of the wild type.The total yields of ack A-pta,nag K and man X deficient strains were 6.51 g/l,6.90 g/l and7.93 g/l,which were 5.6%,12%and 28.7%higher than that of end A1 deficient strains,respectively.In addition,the overexpression of glutamine synthase gene(gln A)in the mutant MGΔend A1Δnag K and MGΔend A1Δman X resulted in the total production of Glc NAc and Glc N reaching 10.78 g/l and 14.38 g/l,respectively.In this study,the total production of Glc NAc and Glc N was gradually increased from 4.36g/l to 14.38 g/l by overexpression of key enzyme genes in the metabolic pathway,optimizing the gene source of key enzymes,improving the stability of plasmid DNA,blocking the transport channel of products into the cell and supplementing the substrates of key catalytic steps,the increase rate reached 229.8%.Therefore,the results of this study have certain theoretical research and practical guiding significance for the final realization of the industrial efficient production of Glc N and Glc NAc.