过量表达苹果酸脱氢酶对大肠杆菌NZN111产丁二酸的影响

Effect of overexpression of malate dehydrogenase on succinic acid production in Escherichia coli NZN111

摘要:

大肠杆菌NZN111是敲除了乳酸脱氢酶的编码基因 (ldhA) 和丙酮酸-甲酸裂解酶的编码基因 (pflB) 的工程菌，厌氧条件下由于辅酶NAD(H) 的不平衡导致其丧失了代谢葡萄糖的能力。构建了苹果酸脱氢酶的重组菌大肠杆菌NZN111/pTrc99a-mdh，在厌氧摇瓶发酵过程中通过0.3 mmol/L的IPTG诱导后重组菌的苹果酸脱氢酶 (Malate dehydrogenase，MDH) 酶活较出发菌株提高了14.8倍，NADH/NAD+的比例从0.64下降到0.26，同时NAD+和NADH浓度分别

Abstract:

Escherichia coli NZN111 is a double mutant with lactate dehydrogenase (ldhA) and pyruvate formate-lyase (pflB) inactivated. Under anaerobic conditions, disequilibrium of coenzyme NADH and NAD+ causes Escherichia coli NZN111 losing the glucose utilizing capability. In this study, we constructed a recombinant strain E. coli NZN111/pTrc99a-mdh and overexpressed the mdh gene with 0.3 mmol/L of IPTG under anaerobic fermentation condition in sealed bottles. The specific malate dehydrogenase (MDH) activity in the recombinant strain was 14.8-fold higher than that in E. coli NZN111. The NADH/ NAD+ ratio decreased from 0.64 to 0.26 and the concentration of NAD+ and NADH increased 1.5-fold and 0.2-fold respectively. Under anaerobic conditions, the recombinant strain possessed the capability of growth and glucose absorption. We took dual-phase fermentation for succinate production. After the dry cell weight (DCW) reached 6.4 g/L under aerobic conditions, the cell culture was changed to anaerobic conditions. After 15 h, 14.75 g/L glucose was consumed and succinic acid reached 15.18 g/L. The yield of succinic acid was 1.03 g/g Glu and the productivity of succinic acid was 1.012 g/(L·h).