Enhanced Production Of L-arginine By Improving Carbamoyl Phosphate Supply In Metabolically Engineered Corynebacterium Crenatum

L-arginine is a semi-essential amino acid,which has been widely used in food,pharmaceutical,chemical industries.In recent years,with the increasing understanding of industrial microbial metabolism and pathway regulation,microbial fermentation has become the main method to produce L-arginine.Carbamoyl phosphate is a common precursor for the synthesis of arginine and pyrimidine in almost all microbial metabolic pathways.It is the"intersection"of the two pathways and is essential for many life forms.In view of this importance,the synthesis of carbamoyl phosphate should be enhanced in cell factories to increase the production of related compounds.In this study,we verified the importance of carbamoyl phosphate for the production of L-arginine by Corynebacterium crenatum SYPA5-5,and then promoted the synthesis of carbamoyl phosphate through a series of metabolic engineering to improve the production of L-arginine.The main research contents include the following points:（1）To knock out carbamoyl phosphate synthetase CPSⅡ large subunit encoding gene car B,5-5/Δcar B become L-arginine and pyrimidine auxotroph.The growth can be restored only if L-arginine and pyrimidine are added to the medium.This explains that carbamoyl phosphate for C.crenatum growth and L-arginine synthesis is very important.The tandem expression of CPSⅡ encoding gene car A and car B enhanced the synthesis rate of carbamoyl phosphate,and L-arginine yield of ARG-CP1 reached 29.9±0.4 g?L^-1 after 96 h of shake-flask fermentation,which was 14.1%higher than that of SYPA5-5.（2）The L-glutamine is a nitrogen-supplying substrate for the synthesis of carbamoyl phosphate by CPSⅡ,so the concentration of L-glutamine is a necessary condition for the effective synthesis of carbamoyl phosphate.Replacing the original promoter by the strong promoter P_sod,the expression level of gln A encoded L-glutamine synthetase GS was increased by 5.29-fold.The yield of L-glutamine and L-arginine were increased by 2.76-fold and 9.70%by shaking flask fermentation of ARG-CP2.（3）In order to reduce the consumption of intracellular organic nitrogen source L-glutamine,a new synthesis pathway of carbamoyl phosphate was introduced into C.crenatum SYPA5-5.Carbamoyl kinase CK uses inorganic ammonia as substrate instead of L-glutamine,four kinds of CK were screened by comparing the enzymatic properties,kinetic parameters and so on.We found that the Ef-CK derived from Enterococcus faecalis V583 shown excellent performance and the enzyme activity in the 5-5/Δcar B reach to 1.59 U?m L^-1,significantly higher than other CKs,and can replace the CPSⅡ to maintain normal cell growth and metabolism.Therefore,Ef-CK was selected to introduce into ARG-CP1.Under the combined action of CK and CPSⅡ synthesis pathways,the total carbamoyl phosphate synthesis rate of ARG-CP1/efarc C was10.35-fold higher than that of SYPA5-5.The Ef-CK was introduced into ARG-CP2 to form strain ARG-CP3,after 96h shake-flask fermentation,it could produce 38.1±0.9 g?L^-1 L-arginine,which was 16.16%and 45.26%higher than that of ARG-CP2 and SYPA5-5,respectively.It indicating that Ef-CK had great application potential in improving carbamoyl phosphate supply for L-arginine production.（4）In ARG-CP2,the lactate dehydrogenase encoding gene ldh was knocked out to block the lactate synthesis pathway and optimize the carbon flux,which increased the yield of L-arginine by 20.43%.At the same time,it can save NADH for ATP regeneration,the intracellular NADH,ATP levels of ARG-CP2/Δldh than ARG-CP2 increased by 67.94%and 52.75%respectively.The strain ARG-CP4 was obtained by expressing Ef-CK in ARG-CP2/Δldh and studied in 5 L fermentor.The sugar-acid ratio was 0.34±0.02 g?g^-1 glucose,and the production intensity was 0.71±0.01 g?L^-1?h^-1,and the yield of L-arginine was 68.6±1.2 g?L^-1,which was54.5%higher than that of SYPA5-5.It is suggested that increasing carbamoyl phosphate supply is an effective strategy to increase L-arginine production.