Effect Of Modification Of Escherichia Coli Metabolic Pathway On Accumulation Of Glucosamine

Glucose(GlcN)is an indispensable substance for the human body,and its market demand is also growing,especially in the areas of pharmaceutical products,cosmetics and health care products.Faced with this growing market demand,traditional glucosamine production methods have many drawbacks,and urgently need to find new ways to improve production.Therefore,more and more people are committed to reconstructing the metabolic pathways of microorganisms at the molecular level and using engineering bacteria to ferment and produce glucosamine.This experiment is mainly to study the effect of the transformation of E.coli metabolic pathway on the accumulation of GlcN.First,the accumulation of glucosamine and acetylglucosamine was increased by enhancing the expression of GlmS(glucosamine synthase)and GnaI(glucosamine acetylase)enzymes.GlmS(Bacillussubtilis),gnaI(Saccharomyces cerevisiae)and the common E.coli expression vector pET28 a were selected to construct the expression plasmid pET28a-glmS-gnaI,which was then transformed into E.coli BL21 to construct E.coli BL21-pET28a-glmS-gnaI strain.The optimum composition medium was selected,the optimum concentration of glucose was 10%,the optimal concentration of peptone was 10g/L,the optimal concentration of yeast powder was 24g/L,and the optimum concentration of manganese chloride was 15mg/L.Then,three enzymes were selected of the E.coli glucosamine metabolism: glucosamine-1-phosphateacetyltransferase(GlmU),N-acetylmuramic acid-6-phosphate(MunQ),N-Acetaminomannose-6-phosphate epimerase(NanE)three enzymes.Using homologous arm single-exchange method to knock them out(GlmU knock out the second domain),three gene-defective bacteria were obtained: E.coli BL21?glmU,E.coli BL21?murQ,E.coli BL21?nanE.In addition,shake flask fermentation was performed on these three kinds of bacteria,and the accumulation of urinary sugar increased significantly.By shaking flask fermentation,the highest value of total amount of two kinds of ammonia sugar was 0.41g/L for E.coli BL21,1.37g/L for E.coli BL21 ?glmU,and 1.04g/L for E.coli BL21?murQ.The highest value of ammonia sugar in E.coli BL21?nanE is 1.16g/L.This proves that the knockout of glmU,murQ,nanE can accumulate glutamine.The previously constructed expressionplasmid pET28a-glmS-gnaI was transformed into E.coli BL21?glmU,E.coli BL21?murQ,E.coli BL21?nanE and E.coli BL21 competent cells,and 3 strains of E.coli BL21 were constructed: E.coli BL21?glmUpET28a-glmS-gnaI,E.coli BL21?murQ-pET28a-glmS-gnaI,E.coli BL21?nanE-pET28a-glmS-gnaI.Fermentation analysis can then be performed to detect the production of glucosamine and acetylglucosamine.To verify the total ammonia sugar yield of glucosamine and acetylglucosamine by fermentation of fermentation tanks: E.coli BL21-pET28a-glmS-gnaI was 10.79g/L,E.coli BL21?glmU-pET28a-glmS-gnaI was 13.97g/L,and E.coli BL21?murQpET28a-glmS-gnaI was 9.50g/L,E.coli BL21?nanE-pET28a-glmS-gnaI was 10.09g/L,E.coli BL21 was 1.94g/L.Among them,E.coli BL21?glmU-pET28a-glmS-gnaI was selected as the highest glutamine producing engineered strain,its yield is 7.20 times that of the control bacteria.After a series of metabolic pathways in this study,the total glucosamine production had a significant increase over the control bacteria.This experiment has opened up new research ideas for the transformation of carbon metabolism pathways for the production of GlcN and complemented the studies on key genes on the glucosamine metabolic pathway.