Preliminary Study On Metabolic Engineering Of Corynebacterium Crenatum For L-Arginine Biosynthesis

L-arginine(L-Arg), a semi-essential basic amino acid, is an important precursor of creatine and cytoplasmic protein as well as nucleoprotein synthesis. In recent years,the L-Arg demand is continuing to increase because of its wide applications in food,medicine and cosmetics. With the development of genetic engineering technology,microbial fermentation for L-Arg production is very popular, and the developing L-Arg high-yield strain based on metabolic engineering has become the research focus.In the present study, a mutant strain of Corynebacterium crenatum NAGK M4(M4 strain) was chosen as a host strain for L-Arg production. The effects of medium components including carbon, organic nitrogen, inorganic nitrogen and inrotrogen ions, as well as the temperature, pH value of initial medium, contents of inoculum and volume of fermentation broth on L-Arg production were firstly investigated in shaking flask fermentation. The results indicated that the optimal medium for L-Arg production was composed of 12% Glucose, 4.5% ammonium sulfate, 2.5% corn steep liquor, 0.005% potassium dihydrogen phosphate, 0.01% seven water magnesium sulfate, 0.01% six water ferric chloride, 0.05% Manganese sulfate monohydrate and3% calcium carbonate. The optimal fermentation conditions were including 30℃ of culture temperature, 10% of inoculum concentration, 7.0 of initial pH and 15 mL/250 mL of loading volume. On the optimal fermentation medium and fermentation conditions, the L-Arg production increased by 28.87% and reached 11.23 g/L.Secondly, serB gene, encoding phosphoserine phosphatase in serine biosynthetic pathway, was deleted using markless knock-out technology in M4 strain. The fermentation results indicated that the growth and glucose consumption in M4 ?serB strain were significantly decreased with or without serine extra addition in medium.The L-Arg production was also dramatically decreased compared with that of the parental strain.Thirdly,the three key isozymes, encoding the glucose dehydrogenase(GDH)in gluconate pathway, were also deleted using markless knock-out technology for improving the nicotinamide adenine dinucleotide phosphate(NADPH)accumulation.The results showed that the NADPH concentrations were enriched by 27.4%,29.4%and 7.8% in the three different mutants, respectively. To improving the utilization rate of NADPH for the L-Arg biosynthesis, the argC gene was overexpressed by gene replacement method on the basis of the strains of M4 ?Cgl213?Cgl2674 and M4?Cgl213?Cgl2674 ?Cgl2135, respectively. Compared with M4 strain, L-Glu, L-Pro and L-Arg concentrations in M4 ?Cgl213?Cgl2674, M4 ?Cgl213?Cgl2674::argC,and M4 ?Cgl213?Cgl2674 ?Cgl2135::argC were increased, whereas the L-Lys and L-Gly concentrations were slightly decreased, and L-Cys concentrations showed no change. Furthermore, compared with the mutant strain of M4 ?Cgl213?Cgl2674,L-Glu concentrations in the strains of M4 ?Cgl213?Cgl2674::argC and M4?Cgl213?Cgl2674?Cgl2135::argC were decreased, while L-Pro and L-Arg concentrations were increased, respectively. The above results indicated that argC overexpression in C. crenatum was beneficial to L-Pro and L-Arg productions by enhancing utilization rate of L-Glu in L-Arg biosynthesis.