State Key Laboratory of Materials-Oriented Chemical Engineering, College of Biotechnology and Pharmaceutical Engineering, Nanjing University of Technology, Nanjing, 211816, Jiangsu, China.
Appl Microbiol Biotechnol. 2013 Aug;97(15):6739-47. doi: 10.1007/s00253-013-4910-1. Epub 2013 Jun 6.
Escherichia coli BA002, in which the ldhA and pflB genes are deleted, cannot utilize glucose anaerobically due to the inability to regenerate NAD(+). To restore glucose utilization, overexpression of nicotinic acid phosphoribosyltransferase (NAPRTase) encoded by the pncB gene, a rate-limiting enzyme of NAD(H) synthesis pathway, resulted in a significant increase in cell mass and succinate production under anaerobic conditions. However, a high concentration of pyruvate accumulated. Thus, co-expression of NAPRTase and the heterologous pyruvate carboxylase (PYC) of Lactococcus lactis subsp. cremoris NZ9000 in recombinant E. coli BA016 was investigated. The total concentration of NAD(H) was 9.8-fold higher in BA016 than in BA002, and the NADH/NAD(+) ratio decreased from 0.60 to 0.04. Under anaerobic conditions, BA016 consumed 17.50 g l(-1) glucose and produced 14.08 g l(-1) succinate with a small quantity of pyruvate. Furthermore, when the reducing agent dithiothreitol or reduced carbon source sorbitol was added, the cell growth and carbon source consumption rate of BA016 was reasonably enhanced and succinate productivity increased.
缺失了 ldhA 和 pflB 基因的大肠杆菌 BA002 由于无法再生 NAD(+)，因此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 pncB 基因（NAD(H)合成途径的限速酶）编码的烟酸磷酸核糖基转移酶（NAPRTase），导致在厌氧条件下细胞质量和琥珀酸产量显著增加。然而，丙酮酸的浓度却很高。因此，研究了在重组大肠杆菌 BA016 中同时表达 NAPRTase 和乳球菌乳亚种 cremoris NZ9000 的异源丙酮酸羧化酶（PYC）。与 BA002 相比，BA016 中的 NAD(H)总量高 9.8 倍，NADH/NAD(+) 比值从 0.60 降低至 0.04。在厌氧条件下，BA016 消耗了 17.50 g/L 的葡萄糖，产生了 14.08 g/L 的琥珀酸，同时产生了少量的丙酮酸。此外，当添加还原剂二硫苏糖醇或还原碳源山梨醇时，BA016 的细胞生长和碳源消耗速率得到了合理的提高，琥珀酸的生产力也随之提高。
