E.coli Biosynthesis Of N-acetyl-α-D-glucosamine

N-acetyl-α-D-glucosamine(GlcNAc)is an amino monosaccharide with multiple biological activities,which is widely used in medicine,health care,cosmetics and other fields.Compared with the traditional hydrolysis method,biosynthesis of GlcNAc has the advantages of environmental protection,pollution-free,high yield,high efficiency,and short production time.However,there still is a big challenge of growth and production competition resulting in low carbon source conversion rate by the current synthesis of GlcNAc using glucose as a carbon source.Here are two aspects of research were carried out to solve the problem:firstly,the GlcNAc biosynthesis pathway was optimized,and the yield of GlcNAc was increased.Secondly,the feasibility of acetic acid as a cheap carbon source for biosynthesis of GlcNAc was verified,and the utilization rate of glycerol was improved.The production of GlcNAc provides new guidance.The specific results obtained are as follows:1.Through overexpression of endogenous glmS and exogenous gna1 genes,explore the use of glycerol to provide acetyl groups needed for bacterial growth metabolism and product synthesis,transform glucose to synthesize GlcNAc,and realize the use of mixed carbon sources.De novo synthesis by E.coli of GlcNAc yields 0.78 g/L.2.By knocking out zwf,pfkA and pfkB to block the pentose phosphate and glycolytic pathways,knocking out the GlcNAc degradation genes nagABE and manXYZ,the construction of the chassis cell factory was completed.After the transformation,the yield of the strain GlcNAc increased by 77.05%to 3.39 g/L.3.Through media optimization,module optimization,overexpression of glucose-assisted diffusion system and adjustment of the added amount of mixed carbon sources,etc.,the production of GlcNAc and the conversion rate of mixed carbon sources are improved.The constructed strain WYH11 had the highest GlcNAc yield of 12.36 g/L,and the highest conversion rate of mixed carbon sources reached 50%.4.By introducing acetic acid to reduce the consumption of glycerol in the GlcNAc synthesis process,adding 3.75 g/L of acetic acid to the shake flask makes the GlcNAc production reach 6.6 g/L,and the glycerol utilization rate is increased to 1.32 g(GlcNAc)/g glycerol.The expression of ppc shortened the fermentation period from 72 h to 48 h.This study provides guidance in several aspects for the initial trial of the subsequent fermentation tank to expand the production scale of GlcNAc.One is that blocking the competitive shunt of glucose is beneficial to increase the production of GlcNAc,and the other is that the overexpression of glucose assisted diffusion system can effectively increase the production and mixing of GlcNAc.The utilization rate of carbon source,the third is that cheap carbon source acetic acid is expected to be used in the industrial production of GlcNAc to reduce the cost of its production.