We demonstrate the use of two-stage dynamic metabolic control to manipulate feedback regulation in central metabolism and improve stationary phase biosynthesis in engineered E. coli. Specifically, we report the impact of dynamic control over two enzymes: citrate synthase, and glucose-6-phosphate dehydrogenase, on stationary phase fluxes. Firstly, reduced citrate synthase levels lead to a reduction in alpha-ketoglutarate, which is an inhibitor of sugar transport, resulting in increased stationary phase glucose uptake and glycolytic fluxes. Reduced glucose-6-phosphate dehydrogenase activity activates the SoxRS regulon and expression of pyruvate-ferredoxin oxidoreductase, which is in turn responsible for large increases in acetyl-CoA production. The combined reduction in citrate synthase and glucose-6-phosphate dehydrogenase, leads to greatly enhanced stationary phase metabolism and the improved production of citramalic acid enabling titers of 126+/-7g/L. These results identify pyruvate oxidation via the pyruvate-ferredoxin oxidoreductase as a central metabolic pathway in stationary phase E. coli, which coupled with ferredoxin reductase comprise a pathway whose physiologic role is maintaining NADPH levels.

中文翻译：

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对反馈调节的动态控制将丙酮酸-铁氧还蛋白氧化还原酶鉴定为固定相大肠杆菌中的中心代谢酶。

我们展示了使用两阶段动态代谢控制来操纵中央代谢中的反馈调节并改善工程化大肠杆菌中的固定相生物合成。具体来说，我们报告了动态控制两种酶（柠檬酸合酶和葡萄糖-6-磷酸脱氢酶）对固定相通量的影响。首先，降低的柠檬酸合酶水平导致α-酮戊二酸的减少，α-酮戊二酸是糖转运的抑制剂，导致固定相葡萄糖摄取和糖酵解通量增加。降低的6-磷酸葡萄糖脱氢酶活性会激活SoxRS调节剂和丙酮酸-铁氧还蛋白氧化还原酶的表达，进而导致乙酰辅酶A产量大幅增加。柠檬酸合酶和6-磷酸葡萄糖脱氢酶的联合降低，大大提高了固定相的新陈代谢，并改善了柠檬酸的生产，效价达到126 +/- 7g / L。这些结果表明丙酮酸通过丙酮酸-铁氧还蛋白氧化还原酶的氧化是固定相大肠杆菌中的主要代谢途径，再加上铁氧还蛋白还原酶构成了其生理作用是维持NADPH水平的途径。