Reconfiguration of the reductive TCA cycle enables high-level succinic acid production by Yarrowia lipolytica.

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.