Tran Vinh G, Tan Shih-I, Xu Hao, Weilandt Daniel R, Li Xi, Bhagwat Sarang S, Zhu Zhixin, Guest Jeremy S, Rabinowitz Joshua D, Zhao Huimin
Department of Chemical and Biomolecular Engineering, University of Illinois Urbana-Champaign, Urbana, IL, USA.
Carl R. Woese Institute for Genomic Biology, University of Illinois Urbana-Champaign, Urbana, IL, USA.
Nat Commun. 2025 Aug 2;16(1):7110. doi: 10.1038/s41467-025-62304-w.
Microbial production of chemicals may suffer from inadequate cofactor provision, a challenge further exacerbated in yeasts due to compartmentalized cofactor metabolism. Here, we perform cofactor engineering through the decompartmentalization of mitochondrial metabolism to improve succinic acid (SA) production in Issatchenkia orientalis. We localize the reducing equivalents of mitochondrial NADH to the cytosol through cytosolic expression of its pyruvate dehydrogenase (PDH) complex and couple a reductive tricarboxylic acid pathway with a glyoxylate shunt, partially bypassing an NADH-dependent malate dehydrogenase to conserve NADH. Cytosolic SA production reaches a titer of 104 g/L and a yield of 0.85 g/g glucose, surpassing the yield of 0.66 g/g glucose constrained by cytosolic NADH availability. Additionally, expressing cytosolic PDH, we expand our I. orientalis platform to enhance acetyl-CoA-derived citramalic acid and triacetic acid lactone production by 1.22- and 4.35-fold, respectively. Our work establishes I. orientalis as a versatile platform to produce markedly reduced and acetyl-CoA-derived chemicals.
微生物生产化学品可能会受到辅因子供应不足的影响，由于酵母中辅因子代谢的区室化，这一挑战在酵母中进一步加剧。在这里，我们通过线粒体代谢的去区室化进行辅因子工程改造，以提高东方伊萨酵母中琥珀酸（SA）的产量。我们通过丙酮酸脱氢酶（PDH）复合体的胞质表达将线粒体NADH的还原当量定位到胞质溶胶中，并将还原性三羧酸途径与乙醛酸循环支路耦合，部分绕过依赖NADH的苹果酸脱氢酶以保存NADH。胞质溶胶中SA的产量达到104 g/L，葡萄糖产率为0.85 g/g，超过了受胞质溶胶中NADH可用性限制的0.66 g/g葡萄糖产率。此外，通过表达胞质PDH，我们扩展了东方伊萨酵母平台，使源自乙酰辅酶A的柠苹酸和三乙酸内酯的产量分别提高了1.22倍和4.35倍。我们的工作将东方伊萨酵母确立为一个多功能平台，用于生产显著减少的和源自乙酰辅酶A的化学品。
