State Key Laboratory of Materials-Oriented Chemical Engineering, College of Biotechnology and Pharmaceutical Engineering, Nanjing University of Technology, Nanjing 211816, People's Republic of China.
Enzyme Microb Technol. 2012 Oct 10;51(5):286-93. doi: 10.1016/j.enzmictec.2012.07.011. Epub 2012 Jul 28.
Succinic acid is not the dominant fermentation product from glucose in wild-type Escherichia coli W1485. To reduce byproduct formation and increase succinic acid accumulation, pyruvate formate-lyase and lactate dehydrogenase, encoded by pflB and ldhA genes, were inactivated. However, E. coli NZN111, the ldhA and pflB deletion strain, could not utilize glucose anaerobically due to the block of NAD(+) regeneration. To restore glucose utilization, overexpression of nicotinic acid phosphoribosyltransferase, a rate limiting enzyme of NAD(H) synthesis encoded by the pncB gene, resulted in a significant increase in cell mass and succinic acid production. Furthermore, the results indicated a significant increase in NAD(H) pool size, and decrease in the NADH/NAD(+) ratio from 0.64 to 0.13, in particular, the concentration of NAD(+) increased 6.2-fold during anaerobic fermentation. In other words, the supply of enough NAD(+) for NADH oxidation by regulation of NAD(H) salvage synthesis mechanism could improve the cell growth and glucose utilization anaerobically. In addition, the low NADH/NAD(+) ratio also change the metabolite distribution during the dual-phase fermentation. As a result, there was a significant increase in succinic acid production, and it is provided further evidence that regulation of NAD(H) pool and NADH/NAD(+) ratio was very important for succinic acid production.
琥珀酸不是野生型大肠杆菌 W1485 从葡萄糖产生的主要发酵产物。为了减少副产物的形成和增加琥珀酸的积累，通过失活编码丙酮酸甲酸裂解酶和乳酸脱氢酶的 pflB 和 ldhA 基因来实现。然而，由于 NAD(+)再生受阻，ldhA 和 pflB 缺失菌株 E. coli NZN111 无法在无氧条件下利用葡萄糖。为了恢复葡萄糖的利用，过表达烟酰胺磷酸核糖基转移酶（pncB 基因编码的 NAD(H)合成的限速酶），导致细胞质量和琥珀酸产量显著增加。此外，结果表明 NAD(H)池的大小显著增加，NADH/NAD(+)的比值从 0.64 降低到 0.13，特别是 NAD(+)的浓度在厌氧发酵过程中增加了 6.2 倍。换句话说，通过调节 NAD(H)回收合成机制为 NADH 氧化提供足够的 NAD(+)可以改善厌氧条件下的细胞生长和葡萄糖利用。此外，低 NADH/NAD(+)比值也改变了双相发酵过程中的代谢物分布。结果，琥珀酸的产量显著增加，这进一步证明了调节 NAD(H)池和 NADH/NAD(+)比值对琥珀酸生产非常重要。
