Sánchez Ailen M, Bennett George N, San Ka-Yiu
Department of Bioengineering, Rice University, 6100 Main Street, MS-142, Houston, TX, USA.
J Biotechnol. 2005 Jun 29;117(4):395-405. doi: 10.1016/j.jbiotec.2005.02.006. Epub 2005 Apr 7.
Escherichia coli overexpressing a NAD(+)-dependent formate dehydrogenase (FDH) from Candida boidinii was grown in chemostat culture on various carbon sources at 0.05 h(-1) dilution rate, under anaerobic conditions using defined medium and compared to a control without the heterologous FDH pathway. Metabolic fluxes, NADH/NAD(+) ratios and NAD(H/(+)) levels were determined under a range of intracellular NADH availability. The effect of NADH manipulation on the distribution of metabolic fluxes in E. coli was assessed under steady-state conditions. The heterologous FDH pathway converts 1 mol of formate into 1 mol of NADH and carbon dioxide, in contrast with the native FDH where no cofactor involvement is present. Previously, we found that this NADH regeneration system doubled the maximum yield of NADH from 2 to 4 mol NADH/mol glucose consumed and reached 4.6 mol NADH/mol of substrate when sorbitol was used as a carbon source in a complex medium. In the current study, it was found that higher NADH yields and NADH/NAD(+) ratios were achieved with our in vivo NADH regeneration system compared to a control lacking the new FDH pathway in the three carbon sources (glucose, gluconate and sorbitol) examined suggesting a more reduced intracellular environment. The total NAD(H/(+)) amounts were very similar for all the combinations studied. It was also found that the ethanol to acetate ratio increased with increased NADH availability. This ratio increased from 1.05 for the control strain in glucose to 9.45 for the strain expressing the heterologous NAD(+)-dependent FDH in sorbitol.
过表达来自博伊丁假丝酵母的NAD(+)依赖性甲酸脱氢酶（FDH）的大肠杆菌在厌氧条件下，使用限定培养基，以0.05 h(-1)的稀释率在恒化器培养中在各种碳源上生长，并与没有异源FDH途径的对照进行比较。在一系列细胞内NADH可利用性条件下测定代谢通量、NADH/NAD(+)比率和NAD(H/(+))水平。在稳态条件下评估NADH操纵对大肠杆菌中代谢通量分布的影响。与不存在辅因子参与的天然FDH不同，异源FDH途径将1摩尔甲酸转化为1摩尔NADH和二氧化碳。此前，我们发现该NADH再生系统使NADH的最大产量从每消耗1摩尔葡萄糖产生2摩尔NADH增加一倍至4摩尔NADH，并且当在复杂培养基中使用山梨醇作为碳源时，达到每摩尔底物4.6摩尔NADH。在当前研究中，发现在所研究的三种碳源（葡萄糖、葡萄糖酸盐和山梨醇）中，与缺乏新FDH途径的对照相比，我们的体内NADH再生系统实现了更高的NADH产量和NADH/NAD(+)比率，表明细胞内环境更还原。对于所有研究的组合，总的NAD(H/(+))量非常相似。还发现乙醇与乙酸的比率随着NADH可利用性的增加而增加。该比率从葡萄糖中对照菌株的1.05增加到山梨醇中表达异源NAD(+)依赖性FDH的菌株的9.45。
