Efficient Synthesis Of N-acetylglucosamine By Recombinant Corynebacterium Glutamicum

N-acetylglucosamine(GlcNAc)and its deacetylated derivative glucosamine(GlcN)are important functional monosaccharides,which are widely used in the fields of medicine,food and cosmetics.The main functions include repairing cartilage tissue,improving resistance,as a dietary supplement and slow down cellular aging and more.At present,GlcNAc produced by microbial fermentation has become the most promising synthesis method due to its advantages such as low cost,mild process conditions,and environmentally friendly production methods.Corynebacterium glutamicum is easy to be cultured,does not produce spores,and is free from phage infection.In this thesis,the production strain of Corynebacterium glutamicum ATCC13032,which is a General Recognized as Safe(GRAS)strain,was used as the starting strain for the research.First,open the transport pathway of the target product GlcNAc and block the reuse of GlcNAc in the late fermentation period to promote the accumulation of the target product GlcNAc.Then,select the optimal chassis strain and strengthen the GlcNAc synthesis pathway to pull the carbon metabolism flow towards the product GlcNAc Synthetic pathway to increase GlcNAc synthesis concentration;Finally,the fermentation conditions were optimized to increase the density of C.glutamicum strains in the fermentation medium and to increase the concentration of GlcNAc synthesized by C.glutamicum.In this thesis,a series of experimental studies were conducted to promote the synthesis of GlcNAc by Corynebacterium glutamicum and increase the glucose conversion rate,which laid a certain foundation for the production of GlcNAc by recombinant Corynebacterium glutamicum.(1)Block the GlcNAc depletion and degradation pathway in recombinant Corynebacterium glutamicum and open the GlcNAc transport system in recombinant Corynebacterium glutamicum.By knocking out the acetylglucosamine-specific phosphotransferase gene cgl2642 in the genome of C.glutamicum,the extracellular GlcNAc was blocked from being transported into the cell.Compared with strains without cgl2642,the longer the fermentation time,the larger the difference in GlcNAc accumulation concentration between strains C.g4-pec-cglS-ScA and C.g2-pec-cglS-ScA,At 48 h of fermentation,the GlcNAc concentration of C.g4-pec-cglS-ScA reached 2.310 g/L,an increase of 17.26%,the conversion rate was 0.039 g/g glucose;the GlcNAc transport system in recombinant Corynebacterium glutamicum was constructed by overexpressing the 6p-acetylglucosamine-specific phosphatase gene yqaB from E.coli.The concentration of GlcNAc synthesized by the recombinant strain C.g4-pec-cglS-ScA-PXMJ-B was increased to 4.037 g/L,which was 2.4 times that of the control strain C.g4-pec-cglS-ScA.(2)Promote GlcNAc synthesis by optimizing chassis strains and enhancing the GlcNAc synthesis pathway in recombinant Corynebacterium glutamicum.Constructed the GlcNAc synthesis pathway and strengthened the GlcNAc transport pathway in four chassis strains C.gl(?nagA/B?ldh),C.g2(?nagA/B?ldh/? poxB),C.g3(? nagA/B ? ldh/? cgl2642),C.g4(?nagA/B ?ldh/? poxB/?cgl2642)and verified the fermentation of each strain.It was found that the knockout of ?poxB(acetic acid pathway)is unnecessary.The chassis strain C.g3(?nagA/B?ldh/?cgl2642)had the best GlcNAc accumulation effect after constructing the GlcNAc synthesis pathway and strengthening the GlcNAc transport pathway.The concentration increased to 5.278g/L,which was 10.88%higher than that of strain C.g4(?nagA/B?ldh/?poxB/?cgl2642);Strengthening the GlcNAc synthesis pathway in recombinant Corynebacterium glutamicum by screening and optimizing the best sources of the two key enzymes GlmS and GNA1 in the GlcNAc synthesis pathway.The effect of overexpression of glmS genes from E.coli,Saccharomyces cerevisiae,Bacillus subtilis,and Corynebacterium glutamicum on the synthesis of GlcNAc by recombinant Corynebacterium glutamicum was shown.The overexpression of glmS derived from Bacillus subtilis is the best choice,making The concentration of GlcNAc synthesized by recombinant Corynebacterium glutamicum was increased to 16.776 g/L,an increase of 220.40%;Subsequently,the effect of overexpressing C.elegans and Saccharomyces cerevisiae-derived GNA1 genes on the synthesis of GlcNAc by recombinant Corynebacterium glutamicum was further studied.The results showed that the expression of GNA1 from C.elegans was slightly better in the recombinant C.glutamicum.The concentration of GlcNAc synthesized by the recombinant C.glutamicum increased to 17.084 g/L,which was a 1.84%increase.(3)Optimize the fermentation conditions for the synthesis of GlcNAc by recombinant Corynebacterium glutamicum,and confirm that the glucose concentration in the fermentation medium is 60 g/L and the corn slurry concentration is 10 g/L;the induction time of the IPTG inducer is optimized,It is determined that it is the best to add it at 5 hours of fermentation(OD600?2.5).At this time,the concentration of GlcNAc synthesis is increased to 17.711 g/L,which is 15.72%compared to the above.The concentration of the IPTG inducer is optimized.It was determined that the concentration of the IPTG inducer was 0.8 mM.At this time,the GlcNAc synthesis concentration was increased to 18.175 g/L,and the conversion rate was increased to 0.303 g/g glucose.Finally,the feed-batch fermentation mode was used to optimize the 5L tank fermentation of GlcNAc synthesis in recombinant coryneform strains,and the reasonable fermentation sugar concentration was controlled by glucose feedback flow addition method to improve the fermentation density of the strain and the GlcNAc synthesis concentration.The OD600 of Corynebacterium glutamicum strain was increased to 120,the concentration of strain synthesized GlcNAc was increased to 83.0 g/L,and the conversion rate was increased to 0.305 g/g glucose.