ABSTRACT

In Saccharomyces cerevisiae, ethanol can serve as both a carbon source and NADH donor for the production of acetyl-CoA derivatives. Here we investigated the metabolic regulation of ethanol utilization for itaconic acid production by S. cerevisiae. To understand the interconnection between the TCA cycle and the glyoxylate pathway, mitochondrial membrane transporter proteins SFC1, YHM2, CTP1, DIC1 and MPC1 were knocked out and results showed that SFC1 functions as an important entrance of the glyoxylate pathway into the TCA cycle, and YHM2 is helpful to IA production but not the primary pathway for citric acid supply. To decrease the accumulation of acetic acid, the major ADP/ATP carrier of the mitochondrial inner membrane, AAC2, was upregulated and determined to accelerate ethanol utilization and itaconic acid production. RNA sequencing results showed that AAC2 overexpression enhanced IA titer by upregulating the ethanol-acetyl-CoA pathway and NADH oxidase in the mitochondrial membrane. RNA-seq analysis also suggested that aconitase ACO1 may be a rate-limiting step of IA production. However, the expression of exogenous aconitase didn't increase IA production but enhanced the rate of ethanol utilization and decreased cell growth.

中文翻译：

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工程化酿酒酵母利用乙醇合成衣康酸

摘要

在酿酒酵母中，乙醇可以作为碳源和 NADH 供体，用于生产乙酰辅酶 A 衍生物。在这里，我们研究了酿酒酵母生产衣康酸的乙醇利用的代谢调节. 为了解 TCA 循环与乙醛酸途径之间的相互关系，我们敲除线粒体膜转运蛋白 SFC1、YHM2、CTP1、DIC1 和 MPC1，结果表明 SFC1 是乙醛酸途径进入 TCA 循环的重要入口，而 YHM2有助于 IA 生产，但不是柠檬酸供应的主要途径。为了减少乙酸的积累，线粒体内膜的主要 ADP/ATP 载体 AAC2 被上调并确定加速乙醇利用和衣康酸生产。RNA测序结果表明，AAC2过表达通过上调线粒体膜中的乙醇-乙酰-CoA途径和NADH氧化酶来增强IA滴度。RNA-seq 分析还表明，乌头酸酶 ACO1 可能是 IA 产生的限速步骤。