Engineering the redox cofactor metabolism is known to be a key challenge in developing a platform strain for biosynthesis of valuable products. Hence, general strategies for manipulation of co-factor metabolism in industrially relevant hosts are of significance. Here, we demonstrate an improvement in α-ketoglutarate (AKG) production in S. cerevisiae using a novel approach based on synthetic rescue. Here, we first perturb the cytosolic NADPH metabolism via deletion of glucose-6-phosphate dehydrogenase (ZWF1). In parallel, we used a strain design algorithm to identify strategies for further improvement in AKG production. Implementation of the identified genetic targets, including disruption of succinyl-CoA Ligase (LSC2) and constitutive expression of NADP+-specific isocitrate dehydrogenases (IDP1 and IDP2) resulted in more than 3 fold improvement in AKG production as compared to the wild type. Our results demonstrate this improvement is due to a synthetic rescue mechanism in which the metabolic flux was redirected towards AKG production through the manipulation of redox cofactors. Disrupting lsc2 in zwf1 mutant improved specific growth rate more than 15% as compared to the zwf1 mutant. In addition, our result suggests that cytosolic isocitrate dehydrogenase (IDP2) may be regulated by isocitrate pools. Together, these results suggest the ability to improve metabolite production via a model guided synthetic rescue mechanism in S. cerevisiae and the potential for using IDP2 expression as a generalized strategy to effectively meet NADPH requirements in engineered strains.

中文翻译：

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合成救援将NADPH生成与酿酒酵母中代谢物的过量生产结合起来。

在开发用于有价值产品生物合成的平台菌株中，工程化氧化还原辅助因子代谢是已知的关键挑战。因此，在工业上相关的宿主中操纵辅因子代谢的一般策略是重要的。在这里，我们证明了使用基于合成拯救的新方法在酿酒酵母中提高α-酮戊二酸（AKG）的产量。在这里，我们首先通过删除6磷酸葡萄糖脱氢酶（ZWF1）来扰乱细胞质NADPH的代谢。同时，我们使用应变设计算法来确定进一步改善AKG生产的策略。实施确定的遗传目标，包括琥珀酰辅酶A连接酶（LSC2）的破坏和NADP +特异性异柠檬酸脱氢酶（IDP1和IDP2）的组成型表达，与野生型相比，AKG产量提高了3倍以上。我们的结果表明，这种改善归因于一种合成的拯救机制，其中代谢通量通过操纵氧化还原辅助因子而重新导向AKG生产。与zwf1突变体相比，破坏zwf1突变体中的lsc2可使比生长速率提高15％以上。此外，我们的结果表明胞质异柠檬酸脱氢酶（IDP2）可能受异柠檬酸池的调节。在一起，这些结果表明通过模型指导的S的合成拯救机制来改善代谢产物产生的能力。