Metabolic Engineering Of Escherichia Coli To Enhance The Synthesis Of L-arginine By Acetyl-CoA

L-Arginine is an important amino acid with significant applications in the pharmaceutical,food and animal husbandry industries,especially in disease treatment.Therefore,in this study,E.coli was selected as the host strain to enhance the carbon metabolic flow of L-arginine synthesis pathway,knocking out the metabolic pathway that consumes L-arginine,enhancing the acetyl coenzyme conversion and glyoxylate cycle pathway,and increasing the metabolic modification of precursors to achieve the accumulation of L-arginine in E.coli.（1）Using CRISPR/Cas9 gene editing technology,the arg R gene（encoding arginine repressor protein）was knocked out,and the feedback repression effect of the blocking protein on the L-arginine synthesis pathway was removed to achieve L-arginine accumulation.After48 h of shake flask,the accumulation of L-arginine reached 1.2 g·L^-1.Knockdown of the genes encoding the L-arginine branched pathway,adi A,spe A,spe F,spe C,ast A,gad A,gad B,weakened the competing metabolic branched for L-arginine synthesis so that the central carbon source flowed more to the target product,weakened the consumption of the precursors for arginine synthesis,and the accumulated co-precursors were synthesized toward L-arginine.Shake flask fermentation of ARG-8,L-arginine production was found to be 3.1 g·L^-1,which was 139.06%higher compared to ARG-1.（2）The recombinant strain was designed to release the feedback inhibition of N-acetylglutamate synthase（NAGS）by targeted mutation and overexpress the arg A～H of the main L-arginine synthesis pathway,to increase the carbon metabolic flow of the L-arginine synthesis pathway of ARG-13,resulting in an L-arginine yield of 7.9 g·L^-1,which was 160.79%higher than that of ARG-8,respectively.The recombinant strain was sequentially integrated with the heterologous genes The acid production capacity of ARG-14 and ARG-15 reached 8.0and 8.8 g·L^-1 of shake flask,respectively.In order to enhance the TCA cycle supplementation pathway glyoxylate pathway flux,the icl R gene was knocked out and ARG-16 recombinant strain was constructed,and the L-arginine yield was 9.0 g·L^-1 after 48 h of shake flask.（3）In order to effectively utilize acetic acid to reduce the side effects of strain growth and to improve the flux of acetyl coenzyme A metabolism at a high level,the gene encoding acetyl coenzyme A synthase acs was heterologously expressed in the recombinant strain.acs from four different bacterial sources were cloned and purified in E.coli,and by comparing the enzymatic properties and kinetic parameters,it was found that Acetobacter pasteurianus.The highest enzymatic activity of Ap ACS1 from Acetobacter pasteurianus ATCC 33445 was 784.59U·m L^-1,the optimum reaction p H was 7.0,and the optimum reaction temperature was 37℃,and Ap ACS1 had better p H and temperature stability compared with the other three ACS sources.Thus,acs₁ was selected for integration into recombinant E.coli to enhance the acetyl coenzyme A metabolic flux.After 48 h of shake flask,9.5 g·L^-1 L-arginine could be produced in ARG-17,which was 6.3%higher than the L-arginine accumulation in ARG-16,respectively,indicating that Ap-ACS has a great role in enhancing acetyl coenzyme A metabolic flux for L-arginine production A metabolic flux for L-arginine production.（4）The gene encoding 6-phosphoglucose dehydrogenase zwf was integrated in ARG-17 to improve the supply of the arginine coenzyme NADPH in the recombinant strain while saving ATP consumption,resulting in intracellular NADPH and ATP levels of 410.34 and 1550.64nmol·g^-1 DCW^-1 in ARG-18 that were 193.38%and 602.67%higher than the starting strain,respectively.After 48 h of shake flask fermentation,9.8 g·L^-1 L-arginine could be produced in ARG-18.The recombinant E.coli ARG-18 was subjected to a 5-L fermenter study,and the fermentation coefficients were examined during 48 h of fermentation.From the experimental results,it was found that the L-arginine yield was 32.2 g·L^-1,the sugar-acid ratio was 0.31 g·g^-1 glucose,the production intensity was 0.74 g·L^-1·h^-1 at 48 h of fermentation,which provided some theoretical basis for industrial amino acid production.