过量表达烟酸转磷酸核糖激酶对大肠杆菌NZN111产丁二酸的影响

Effect of overexpression of nicotinic acid phosphoribosyl transferase on succinic acid production in Escherichia coli NZN111

大肠杆菌NZN111厌氧发酵的主要产物为丁二酸,是发酵生产丁二酸的潜力菌株。但是由于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),导致辅酶NADH/NAD+不平衡,厌氧条件下不能利用葡萄糖生长代谢。构建烟酸转磷酸核糖激酶的重组菌Escherichia coli NZN111/pTrc99a-pncB,在厌氧摇瓶发酵过程中通过添加0.5 mmol/L的烟酸、0.3 mmol/L的IPTG诱导后重组菌的烟酸转磷酸核糖激酶(Nicotinic acid phosphoribosyl transferase)酶活较出发菌株提高了11倍,辅酶NAD(H)的总量提高了3.85倍,特别是NAD+的浓度提高了5.17倍,NADH/NAD+的比例从0.640下降到0.125,使重组菌株在厌氧条件下具有生长和代谢葡萄糖的能力。

Escherichia coli strain NZN111 is a promising candidate for the fermentative production of succinate.However,because lactate dehydrogenase and pyruvate formate lyase were inactivated in NZN111,this strain had an unbalanced NADH/NAD＋ ratio and could not use glucose under anaerobic conditions.In this study,a recombinant strain E.coli NZN111/pTrc99a-pncB was constructed to overexpress the nicotinic acid phosphoribosyl transferase gene（pncB）.Under anaerobic conditions with the addition of 0.5 mmol/L nicotinic acid and 0.3 mmol/L isopropyl beta-D-thiogalactopyranoside（IPTG）,the specific nicotinic acid phosphoribosyl transferase（NAPRTase,EC 2.4.2.11） activity in the recombinant strain was 11-fold higher than that in E.coli NZN111,the concentration of NAD（H） was increased by 3.85-fold,especially the concentration of NAD＋ was increased by 5.17-fold and NADH/NAD＋ was decreased from 0.640 to 0.125.The recombinant strain regained the capability of growth and glucose utilization under anaerobic conditions.