Genetic Engineering Of Corynebacterium Glutamicum To Produce Nitrogen Acetylglucosamine

N-acetylglucosamine(Glc NAc)is a kind of glucose monosaccharide derivatives.It is a favorable nutrient for the survival of many microorganisms.It is widely distributed and has been widely used in various fields such as medicine,cosmetics,agriculture,food and so on.The main functions are: preventing rheumatoid arthritis and other diseases,as a healthy food sweetener,improving skin lines and promoting seed germination.At present,the methods of producing Glc NAc are mainly chemical methods,but because of the disadvantages of non environmental protection,they are gradually replaced by microbial fermentation with low cost,no pollution and mild process conditions.Since its discovery,Corynebacterium glutamicum has been used as a host strain for genetic engineering because it neither produces endotoxin nor is easy to be eroded and lysed by phages.It is classified as a "recognized safe"(GRAS)organism,and is easy to grow and genetic treatment.In this paper,Corynebacterium glutamicum atcc13032 was used as the host strain of genetic engineering.Firstly,the main growth pathway competing for carbon flux with the target product Glc NAc synthesis pathway was weakened,and the reverse transport pathway of Glc NAc was blocked and screened to enhance the carbon flux to Glc NAc synthesis pathway and improve the utilization rate of raw materials;Then,the expression of key enzyme genes in Glc NAc synthesis pathway was optimized,and mannose pathway was blocked to reduce the consumption of precursors,so as to increase the yield of Glc NAc;Finally,the basic fermentation conditions were further optimized to increase the yield of Glc NAc again.In this paper,the yield and yield of Glc NAc produced by Corynebacterium glutamicum were increased by means of genetic modification and fermentation optimization.The specific research is as follows:(1)Weaken the main growth pathway competing for carbon flux with the target product Glc NAc synthesis pathway,and block and screen the reverse transport pathway of Glc NAc.Blocking the growth competition pathway pentose phosphate pathway in Corynebacterium glutamicum and knocking out the key gene zwf(encoding 6-p-glucose dehydrogenase)of this pathway increased the yield of Glc NAc by 30.7%.At 48 h,the fermentation concentration reached 19.3 g/L and the conversion rate was0.32 g/g.The two acetylaminosaccharide specific transporter genes Cgl2642 and nag E in Corynebacterium glutamicum metabolism were screened by single knockout and double knockout,and the optimal blocking scheme c.g4((△nag A/B-△ldh-△Cgl2642-△zwf))was selected as the chassis strain,Asrna weakening technology was used to weaken the glycolysis pathway,and the weakening strain with the best fermentation result was selected.The final yield could reach 23.3 g/L and the conversion rate was 0.38 g/g in 48 h.(2)Optimize the expression of key enzyme genes in Glc NAc synthesis pathway and block mannose pathway.The codons of three foreign genes Glms,Gna1 and yqa B in the intermediate synthesis pathway of Glc NAc were optimized for the glutamate rod citrus expression system.Compared with the control strain,the yield of Glc NAc increased by 1.1g/L,reached 20.2 g/L and the yield was 0.33 g/g,After 48 h fermentation,the synthesis concentration of Glc NAc reached the final yield and 11.4%higher than the former,reaching 21.2 g/L and the yield was 0.35 g/g;The key gene nane of mannose pathway was knocked out.After 48 hours of fermentation,the synthesis concentration of Glc NAc 8% higher than that the former,having 22.4 g/L and the yield was 0.37 g/g.(3)Finally,the fermentation conditions were optimized to further improve the concentration of Glc NAc synthesis.The optimal medium conditions for the production of Glc NAc by the modified Corynebacterium glutamate were optimized,and the concentration of 100 g/L glucose and20 g/L corn pulp were optimized;The time of IPTG was 3 h(OD600 ≈ 3),the optimized concentration of Glc NAc was 36.3 g/L,and the yield of glucose was 0.36 g/g.Finally,the fermentation was conducted in the 5 L tank for 120 h,and the Glc NAc concentration reached 112 g/L,and the conversion rate reached 0.348 g/g.