Systems Metabolic Engineering Of Escherichia Coli For L-arginine Production

L-arginine,an industrially high-value amino acid,is extensively used as an essential ingredient to provide specialty nutrients in animal feed,health care products and pharmaceutics fields.With the dramatical increasing in the market demand of arginine,the traditional proteolysis method was gradually substituted by microbial fermentation in the industrial production of arginine.Currently,the large-scale industrial production process of arginine is restricted to lacking of high-yield strains.Consequently,we redistributed the arginine biosynthetic pathway flux of wild Escherichia coli W3110 to construct arginine-producing strain,involving chromosomal modifications by the CRlSPR-Cas9 mediated genome editing method.Firstly,speA,adiA(encoding arginine decarboxylase),astA(encoding arginine succinyltransferase)and argR(encoding arginine responsive repressor protein)were successively koncked out,and the gene argJ(encoding feedback inhibition-resistant NAGS from Corynebacterium glutamicum)was integrated into the yncI locus of E.coli to construct ARG2-2.Campared with the wild strain,which did not acccumulate any arginine during fermentation,the titer of ARG2-2 was 1.1 g/L.Furthermore,the blockading of arginine catabolism pathways did not result in a obvious growth retardation,this lays a significant foundation for improving the efficiency of arginine synthesis.Further,the carbon flux towards arginine biosynthetic pathway was stepwise optimized in the ARG2-2.(1)The arginine operon genes donated from C.glutamicum were introduced to construct ARG3-1 strain.(2)The argB gene,which is known to encode a feedback inhibition-resistant NAGK in the E.coli,was overexpressed in ARG3-1 to construct ARG3-2 strain.(3)The argJ gene of C.glutamicum was integrated into the argE locus of ARG3-2 for configurating the acetyl cycle of arginine biosynthesis,thus establishing strain ARG4.The results of fermentation showed that the titers of ARG3-1,ARG3-2,and ARG4 were 10.61,12.54,and 13.84 g/L,respectively.Campared with that of ARG3-2,the arginine yeild from glucose of ARG4 reeached 0.146 g/g and increased by 10.15%,indicating that strengthening of acetyl cycle pathway could remarkably channel more carbon flux into arginine biosynthetic pathway.Further,to increasing in the supply of carbamyl phosphate and NADPH,the synthesis of carbamyl phosphate and the NADPH regeneration system were modified.(1)The gene pyrAA/pyrABE949*,which encode a feedback resistant CPS in B.subtilis F126,was integrated into the yjiT locus of ARG4 to construct ARG5-1.This led to a 15.21%increase of arginine production reaching 15.90 g/L.(2)The gene pntA/pntB was overexpressed to construct ARG5-2.The results of fermentation showed that ARG5-2 resulted in a production of 17.67 g/L arginine,which was 11.13%greater than that produced by the ARG5-1.Further,the lysE and argO genes,both responsible for transporting intracellular arginine out cells,were respectively overexpressed in the ARG5-2 to construct the ARG6-1 and ARG6-2.The results of fermentation showed that ARG6-1 and ARG6-2 could accummulate 31.26 and 19.02 g/L arginine,respectively.Campared with that of ARG5-2,the arginine titer of ARG6-1 increased by 76.91%,while the ARG6-2 only led to a slight increase(7.64%)in the arginine titer.Finally,fed-batch culture of the ARG6-1 strain results in a production of 91.1 g/L with a yield of 0.423 g/g glucose and productivity of 2.53 g/L/h.This result indicate that the ARG6-1 strain has a good prospect of large-scale industrial application.