Engineered non-canonical reductive TCA pathway drives high-yield succinic acid biosynthesis in Yarrowia lipolytica

The reductive tricarboxylic acid (rTCA) cycle is a crucial metabolic pathway employed in the microbial production of C4-dicarboxylic acids, especially succinic acid (SA). However, the inherent redox constraints associated with this cycle pose significant limitations on the yields of SA. Here, we address this critical bottleneck by engineering a non-canonical reductive TCA (Nc-rTCA) pathway in oleaginous yeast Yarrowia lipolytica. Our approach substitutes the NADH-dependent conversion of oxaloacetate to fumarate in the native rTCA cycle with an engineered cascade utilizing aspartate aminotransferase, aspartate ammonia-lyase, and glutamate dehydrogenase, effectively decoupling SA synthesis from NADH limitations. This NADPH-dependent module resulted in a remarkable 1.28-fold increase in fumarate titer. Further metabolic optimization in the engineered strain Ncr12 minimized malate byproduct formation, achieving an SA titer of 98.16 g/L with a high yield of 0.91 g/g glucose in 5-L bioreactors. Importantly, the Nc-rTCA pathway demonstrated potential for industrial application, yielding 74.34 g/L SA at 0.98 g/g glucose from lignocellulosic hydrolysate and 117.74 g/L SA at 0.74 g/g from glycerol. Our findings address the longstanding redox imbalance issues that have challenged rTCA-based engineering and establish a scalable platform for bio-based C4-dicarboxylic acid production.