Succinic acid (SA) is an important C4-dicarboxylic acid. Microbial production of SA at low pH results in low purification costs and hence good overall process economics. However, redox imbalances limited SA biosynthesis from glucose via the reductive tricarboxylic acid (TCA) cycle in yeast. Here, we engineer the strictly aerobic yeast Yarrowia lipolytica for efficient SA production without pH control. Introduction of the reductive TCA cycle into the cytosol of a succinate dehydrogenase-disrupted yeast strain causes arrested cell growth. Although adaptive laboratory evolution restores cell growth, limited NADH supply restricts SA production. Reconfiguration of the reductive SA biosynthesis pathway in the mitochondria through coupling the oxidative and reductive TCA cycle for NADH regeneration results in improved SA production. In pilot-scale fermentation, the engineered strain produces 111.9 g/L SA with a yield of 0.79 g/g glucose within 62 h. This study paves the way for industrial production of biobased SA.

琥珀酸（SA）是一种重要的C4-二羧酸。在低 pH 值下微生物生产 SA 可以降低纯化成本，从而实现良好的整体工艺经济性。然而，氧化还原失衡限制了酵母中通过还原三羧酸（TCA）循环从葡萄糖生物合成SA。在这里，我们设计了严格需氧的解脂耶氏酵母，无需控制 pH 即可高效生产 SA。将还原性 TCA 循环引入琥珀酸脱氢酶破坏的酵母菌株的细胞质中会导致细胞生长停滞。尽管适应性实验室进化恢复了细胞生长，但有限的 NADH 供应限制了 SA 的生产。通过耦合用于 NADH 再生的氧化和还原 TCA 循环，重新配置线粒体中还原性 SA 生物合成途径，从而提高 SA 产量。在中试规模发酵中，工程菌株在 62 小时内产生 111.9 g/L SA，产量为 0.79 g/g 葡萄糖。该研究为生物基SA的工业化生产铺平了道路。