Metabolic Engineering Of Corynebacterium Glutamicum For Efficient Synthesis Of L-histidine

L-histidine is an essential protein amino acid,which is very important for the growth of infants and animals,and is an urgently needed amino acid variety in the market.L-histidine is a synthetic precursor for a variety of drugs that can be used to treat ulcers,kidney failure,rheumatoid arthritis,senile dementia and other diseases.Methods for producing L-histidine include proteolysis,chemical synthesis and microbial fermentation.Compared with the former two,microbial fermentation has advantages,making it the preferred method for L-histidine production.Therefore,this study used a food-safe strain,Corynebacterium glutamicum ATCC13032,as the starting strain.The study used protein engineering and metabolic engineering strategies to semi-rationally modify rate-limiting enzymes,regulate metabolic flow of product synthesis,and improve energy supply.This resulted in the construction of a C.glutamicum strain,CG3-7,with a high yield of L-histidine.The main research contents are as follows:（1）ATP-phosphoribosyl transferases(His G_WT),a rate-limiting enzyme in the L-histidine synthesis pathway of C.glutamicum ATCC 13032,were taken as the research object.The potential binding site of L-histidine and His G_WT was saturated mutated according to the result of molecular docking analysis.A mutant His G_T235P was screened by the high-throughput screening platform,and the yield of L-histidine was 0.42 g·L^-1.By comparing the free energy changes between the wild type and mutant and conducting molecular dynamics simulation analysis,the His GT235P exhibited enhanced stability.（2）Combined with the prediction results of molecular docking and Ohm platform,alanine scanning was used to screen the mutation sites of catalytic domains.A saturated mutation library of key sites was constructed to screen mutants.The results showed that the Y56M mutation played an important role in the production of L-histidine,the hydrophobicity and folding stability of protein,and the catalytic activity.The L-histidine production of the mutant His G_T235P-Y56M reached 0.83 g·L^-1.（3）The L-histidine metabolic pathway was divided into three modules（L-histidine synthesis module,pentose phosphate module and glycolysis module）by using modular metabolic engineering strategy.By increasing the copy number of mutants His GT235P-Y56M and phosphoribose pyrophosphate kinase（Prs）,replacing the promoter of the pentose phosphate pathway,and knocking out glucose 6-glucose isomerase（PGI）,the production of L-histidine reached 1.21 g·L^-1.（4）The study optimized the supply of adenosine triphosphate（ATP）to promote L-histidine synthesis.This was done by knocking out the frd1 and frd2 genes encoding flavin reductase,AMP nucleosidase（AMN）,and amidophosphoribosyltransferase（Pur F）,to reduce the reactive oxygen species（ROS）level and promote ATP synthesis.The L-histidine shake flask production of CG3-7 was increased to 3.10 g·L^-1,and the titer of L-histidine was increased to 5.07 g·L^-1by fed batch fermentation.