We report improved NADPH flux and xylitol biosynthesis in engineered E. coli. Xylitol is produced from xylose via an NADPH dependent reductase. We utilize 2-stage dynamic metabolic control to compare two approaches to optimize xylitol biosynthesis, a stoichiometric approach, wherein competitive fluxes are decreased, and a regulatory approach wherein the levels of key regulatory metabolites are reduced. The stoichiometric and regulatory approaches lead to a 20-fold and 90-fold improvement in xylitol production, respectively. Strains with reduced levels of enoyl-ACP reductase and glucose-6-phosphate dehydrogenase, led to altered metabolite pools resulting in the activation of the membrane bound transhydrogenase and an NADPH generation pathway, consisting of pyruvate ferredoxin oxidoreductase coupled with NADPH dependent ferredoxin reductase, leading to increased NADPH fluxes, despite a reduction in NADPH pools. These strains produced titers of 200 g/L of xylitol from xylose at 86% of theoretical yield in instrumented bioreactors. We expect dynamic control over the regulation of the membrane bound transhydrogenase as well as NADPH production through pyruvate ferredoxin oxidoreductase to broadly enable improved NADPH dependent bioconversions or production via NADPH dependent metabolic pathways.

我们报告了工程大肠杆菌中NADPH 通量和木糖醇生物合成的改善. 木糖醇是通过 NADPH 依赖性还原酶从木糖中产生的。我们利用 2 阶段动态代谢控制来比较两种优化木糖醇生物合成的方法，一种化学计量方法，其中竞争性通量降低，以及一种调节方法，其中关键调节代谢物的水平降低。化学计量和调节方法分别使木糖醇产量提高了 20 倍和 90 倍。烯酰-ACP 还原酶和葡萄糖-6-磷酸脱氢酶水平降低的菌株导致代谢物库改变，导致膜结合转氢酶和 NADPH 生成途径的激活，由丙酮酸铁氧还蛋白氧化还原酶与 NADPH 依赖性铁氧还蛋白还原酶组成，导致增加 NADPH 通量，尽管 NADPH 池减少。这些菌株在仪器化生物反应器中以 86% 的理论产量从木糖中产生 200 g/L 的木糖醇。我们期望通过丙酮酸铁氧还蛋白氧化还原酶对膜结合转氢酶的调节以及 NADPH 生产的动态控制，以广泛地改善 NADPH 依赖性生物转化或通过 NADPH 依赖性代谢途径的生产。