Study On The Cooperative Expression Mechanism Of Glms And Gna1 Gene

Glucosamine(GlcN)and acetylglucosamine(GlcNAc)exist in a wide range and are indispensable functional sugars for a variety of animals,plants and microorganisms,as well as important components of chitosan,chitin and cell wall.Both of them are mainly used in health products and cosmetics in people's daily life and their market value and potential are huge.At present,there are many defects in industrial production methods,so the production of GlcN and GlcNAc by microbial fermentation has become the mainstream production methods.In this paper,we mainly explored the optimal co-catalytic ratio of glucosamine synthase(Glm S)and acetylglucosamine synthase(Gna1),and realized the coordinated expression of Glm S and Gna1 by using promoter engineering technology.It is mainly reflected in the following aspects:(1)A fusion expression plasmid pET28a-egfp-gna1 was constructed to express green fluorescent protein and Gna1.The main feature of the plasmid was that the fluorescence intensity of green fluorescent protein was used as an indicator unit to analyze the optimal expression conditions of Gna1.Then,two engineering plasmids p BAD33-glms and pET28a-gna1 with different promoter strength were constructed.After the two plasmids were co-transformed into the same strain,the optimal expression conditions of Gna1 were optimized and fixed and the expression level of Glm S protein was regulated by the concentration of inducer.Combined with the yield of acetylglucosamine and the concentration of polyacrylamide gel electrophoresis bands,the optimal ratio of Glm S to Gna1 was 2:5.(2)E.coli pET28a-egfp-glms was constructed to express green fluorescent protein and Glm S fusion.The fluorescence intensity of green fluorescent protein was used to indicate the expression of Glm S.According to the optimal ratio of Glm S and Gna1,in order to reduce the expression level of Glm S,three bases of T^-10G^-11A^-12 in the-10 region of the T7 promoter were selected to construct the random mutation library of the promoter through the technology of site-specific saturation mutation.The mutant strains were screened by flow cytometry.A total of 74 strains with different fluorescence intensity were selected for fermentation culture.The T7 promoter mutant strains with reduced fluorescence intensity by 60%,50%and 40%were sequenced.(3)The co-expression plasmid pRSFDuet-1-glms-gna1 was constructed by Glm S and Gna1 double promoter.E.coli BL21(DE3)was transformed into an engineered strain E.coli BL21 pRSFDuet-1-glms-gna1.In order to achieve the coordinated expression of Glm S and Gna1 proteins,combining the coordinated proportional relationship between the two proteins and the base sequence relationship corresponding to the T7 promoter strength,the T7 promoter sequence T^-10G^-11A^-12 controlling Glm S expression in the engineering plasmid pRSFDuet-1-glms-gna1 was replaced with base sequence T^-10G^-11G^-12 and the plasmid pRSFDuet-1-glms-gna1-mutant₂ was constructed finally.After transforming into E.coli BL21(DE3)competent state,E.coli BL21 P pRSFDuet-1-glms-gna1-mutant₂ with double enzyme co-expression was constructed and the results showed that under the optimal fermentation conditions,the yield of E.coli BL21 pRSFDuet-1-glms-gna1-mutant₂ reached 1.53 g/L,which was 1.77 and 36.4 times of that of the unoptimized strain and the original strain.