Effect Of TCA Cycle Modification On L-arginine Production And Metabolic Engineering In Corynebacterium Crenatum

L-arginine is widely used in the fields of medicine, food, cosmetics and animal husbandry for its physiological and biochemical function of diversity. A high L-arginine production strain C. crenatum SYPA 5-5 was obtained by screening and multi-level mutagenesis and the L-arginine production level was up to 36.1 g/L. In this study, the central metabolic key enzymes involved in TCA cycle were cloned and expressioned and NADPH level were regulated to metabolic engineer the L-arginine biosynthesis to increase the L-arginine production using C. crenatum SYPA 5-5 as the original strain. The main contents are as follows:(1) The prp C2 gene coding citrate synthase(CS) and icd gene coding isocitrate dehydrogenase(ICD) in TCA cycle were cloned and expressed in C. crenatum SYPA 5-5 respectively. The CS specific activity of CS in C. crenatum SYPA 5-5/p DXW-10-prp C2 increased 2.41 fold compared with C. crenatum SYPA 5-5, L-arginine production of flask fermentation was 34.5 g/L, increased about 10.2 % than C. crenatum SYPA 5-5. C. crenatum SYPA 5-5/p DXW-10-prp C2 enhanced the L-arginine production to 41.8 g/L about 15.2% in 5 L fermenter, as compared to C. crenatum SYPA 5-5. The ICD specific activity increased more than 4.50 fold in the icd-overexpressing strain C. crenatum SYPA 5-5/p DXW-10-icd, at the same time the NADPH level increased to 147 pmol/OD562, L-arginine production was 38.6 g/L in the 250 m L flask, while the L-arginine production could reach 47.0 g/L in 5 L fermenter, increased 23.3 % and 29.4 % compared with C. crenatum SYPA 5-5, respectively. Amino acid metabolism pathways was strengthened by the increased intracellular NADPH levels induced through the overexpression of icd resulting in proline accumulated to 3.55 g /L, increased 3.5 g/L compared with 0.05 g/L of SYPA 5-5. The recombinant plasmid p K18 mobsac B-odh A’ was electrotransformed into C. crenatum SYPA 5-5, the odh A gene was deleted to obtain C. crenatum SYPA 5-5(Δodh A). The cell growth of the resulted odh A gene deleted strain was reduced seriously.(2) The recombinant plasmid p DXW-10-icd was electrotransformed into C. crenatum SYPA 5-5(Δpro B) to construct SYPA 5-5(Δpro B)/p DXW-10-icd. L-arginine yield of flask fermentation was 41.8 g/L, increase 33.5 % than C. crenatum SYPA 5-5, L-arginine production was up to 51.8 g/L in 5 L fermenter, about 42.7% higher compared with C. crenatum SYPA 5-5 and proline content is very low.(3) N-acetyl glatamyl phosphate reductase(NAGSD) is the NADPH-dependent enzyme in L-arginine biosynthesis. The icd gene and arg C gene coding NAGSD were coexpressed in SYPA 5-5(Δpro B)/p DXW-10-icd-tac-arg C. The intracellular ICD and NAGSD specific activity were 140 m U/mg and 8.12 U/mg improved 3.50 fold and 1.75 fold compared with C. crenatum SYPA 5-5 respectively and the NADPH level was 115 pmol/OD562. L-arginine level of flask fermentation was 44.9 g/L, about 43.5 % increased compared with C. crenatum SYPA 5-5. L-arginine was accumulated up to 54.5 g/L in the 5 L fermentor, 50.1% increased than C. crenatum SYPA 5-5 and proline and lysine levels decreased.