Overflow metabolism in Escherichia coli during steady-state growth:: Transcriptional regulation and effect of the redox ratio
Overflow metabolism in Escherichia coli during steady-state growth:: Transcriptional regulation and effect of the redox ratio
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DOI：
10.1128/aem.72.5.3653-3661.2006
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发表时间：
2006-05-01
影响因子：
4.4
通讯作者：
Eiteman, M. A.
中科院分区：
文献类型：
--
作者：
Vemuri, G. N.;Altman, E.;Eiteman, M. A.
大肠杆菌以好氧乙酸排泄形式的溢流代谢是这种常见工业微生物的重要生理特征。虽然乙酸盐的形成发生在高葡萄糖消耗的条件下，但引发这种现象的遗传机制尚不清楚。我们报告的作用的NADH/NAD比（氧化还原比）在溢流代谢。我们调节了E.大肠杆菌通过表达肺炎链球菌（水形成）NADH氧化酶。使用稳态恒化培养，我们证明了乙酸形成和氧化还原比之间的强相关性。我们进一步完成了对照E. coli菌株和E.过量表达NADH氧化酶的大肠杆菌菌株。转录结果表明，在对照菌株中，随着葡萄糖消耗速率的增加，参与三羧酸（TCA）循环和呼吸的几个基因受到抑制。此外，这些基因的相对抑制，减轻了表达的NADH氧化酶和由此产生的减少氧化还原比。分析与氧化还原比相关的基因上游的启动子结合位点，发现与ArcA结合位点具有很强同源性的简并序列。arcA的缺失导致乙酸减少，并由于TCA循环和呼吸能力的增加而增加了生物量产量。乙酸形成完全消除通过减少氧化还原比通过在arcA突变体中的NADH氧化酶的表达，即使在一个非常高的葡萄糖消耗率。这些结果为研究在还原的NADH/NAD比率下普遍存在的新的调节机制以及设计更有效的生物过程提供了基础。
Overflow metabolism in the form of aerobic acetate excretion by Escherichia coli is an important physiological characteristic of this common industrial microorganism. Although acetate formation occurs under conditions of high glucose consumption, the genetic mechanisms that trigger this phenomenon are not clearly understood. We report on the role of the NADH/NAD ratio (redox ratio) in overflow metabolism. We modulated the redox ratio in E. coli through the expression of Streptococcus pneumoniae (water-forming) NADH oxidase. Using steady-state chemostat cultures, we demonstrated a strong correlation between acetate formation and this redox ratio. We furthermore completed genome-wide transcription analyses of a control E. coli strain and an E. coli strain overexpressing NADH oxidase. The transcription results showed that in the control strain, several genes involved in the tricarboxylic acid (TCA) cycle and respiration were repressed as the glucose consumption rate increased. Moreover, the relative repression of these genes was alleviated by expression of NADH oxidase and the resulting reduced redox ratio. Analysis of a promoter binding site upstream of the genes which correlated with redox ratio revealed a degenerate sequence with strong homology with the binding site for ArcA. Deletion of arcA resulted in acetate reduction and increased the biomass yield due to the increased capacities of the TCA cycle and respiration. Acetate formation was completely eliminated by reducing the redox ratio through expression of NADH oxidase in the arcA mutant, even at a very high glucose consumption rate. The results provide a basis for studying new regulatory mechanisms prevalent at reduced NADH/NAD ratios, as well as for designing more efficient bioprocesses.