Modification Of Glucosamine Metabolic Pathways In Escherichia Coli

Glucosamine,referred to as Glc N,is widely found in animals,plants and microorganisms.As a Glc N acetylation product,Acetylglucosamine(Glc NAc)also has a good production and sales market.With the development of biology,the production of ammonia sugar gradually changed from the traditional chitin hydrolysis method to the more popular microbial fermentation method,and the latter mainly relies on the transformation of the microbial strain metabolic pathway.In this study,the double promoter vector p ETDuet-1 was first used to heterologously overexpress Glc N synthesis key proteins glucosamine synthetase(Glms)and acetylglucosamine synthase(Gna1)in E.coli BL21(DE3)strain;Secondly based on Red homologous recombination technology,new genes yoa D and yoa E that can affect the production of E.coli Glc N were discovered,and the effect of the deletion of the new gene on Glms enzyme activity and Glc N production was explored;Finally,the key gene of Glc N intracellular transport,man X,was knocked out,and the effects of carbon source,temperature and p H on the synthesis of engineering bacteria Glc N were studied,including the following three aspects:(1)Glucosamine synthase(Glms)and acetylglucosamine synthase(Gna1)are the key proteins for Glc N synthesis.In this study,the p ETDuet-1 plasmid was used for heterologous overexpression of the glms gene(from strain Bacillus subtilis168)and the gna1 gene(from strain Saccharomyces cerevisiae S288C).By constructing the plasmid p ETDuet-1-bsglms-scgna1 and successfully transferring it into the E.coli BL21(DE3)competent state,the E.coli BL21-bsglms-scgna1 engineering strain was successfully constructed.It successfully induced the expression of Glms(molecular weight 65 k Da)and Gna1(molecular weight 18 k Da)under low temperature conditions.(2)The yoa D and yoa E genes are genes encoding E.coli cell membrane proteins,and they have the functions of stimulating the degradation of the second messenger cycle digmp and regulating the transfer of certain ions.Their effects on Glc N synthesis have not been reported.In this paper,three engineering strains of E.coli BL21-?yoa D,E.coli BL21-?yoa E,and E.coli BL21-?yoa D-?yoa E were constructed in E.coli BL21(DE3)strain using Red homologous recombination technology,subsequently,the vector p ETDuet-1-bsglms-scgna1 was transferred into the above three engineering strains to construct the engineering strains E.coli BL21-?yoa D-bsglms-scgnal,E.coli BL21-?yoa E-bsglms-scgnal and E.coli BL21-?yoa D-?yoa E-bsglms-scgnal.The results show that the co-deletion of gene yoa D and gene yoa E can increase the expression activity of exogenous Glms by 2 times,and the production of Glc N reaches 1.39 g/L,which is about 3 times higher than that of original E.coli BL21.(3)In order to further increase the synthesis amount of Glc N,the gene sequence of protein Man X capable of transporting extracellular Glc N into cells was knocked out to construct the engineering strain E.coli BL21-?yoa D-?yoa E-?man X;subsequently,the engineering strain E.coli BL21-?yoa D-?yoa E-?man X-bsglms-scgnal was constructed and used as the starting strain to study the effects of carbon source,temperature and p H on the fermentation of ammonia sugar.It was found that the addition of fructose can increase the production of ammonia sugar by about 20%.Under the optimal culture conditions,the E.coli BL21-?yoa D-?yoa E-?man X-bsglms-scgnal strains produced Glc N and Glc NAc yields of 1.80 g/L and 0.80 g/L during fermentation,respectively.Compared with the original E.coli BL21 strain,it was improved about 4.0 times and 2.0 times respectively.