Xylose Metabolism Perturbation in <i>Yarrowia lipolytica</i> for Efficient Succinic Acid Bioproduction from Lignocellulosic Biomass

Lignocellulosic biomass is a sustainable feedstock for biorefineries, but inefficient xylose utilization limits microbial bioproduction. Here, the oleaginous yeast Yarrowia lipolytica was engineered to produce succinic acid (SA) from xylose by resolving metabolic and regulatory conflicts. Initial overexpression of xylose catabolic genes (XR, XDH, XK) in an SA-hyperproducing strain did not activate xylose utilization, indicating underlying cryptic constraints. Adaptive evolution identified critical mutations (Snf1^R78W, Scp1^delGTC) that globally downregulated downstream pathways, including glycolysis and β-oxidation, restoring growth using xylose but reducing SA production. To overcome this trade-off, a random expression library strategy incorporating multi-copy amplification of XR, XDH, and XK genes via nonhomologous end joining (NHEJ) was employed. This approach significantly enhanced xylose utilization and SA production, achieving 83.78 g L^-1 SA from corn stover hydrolysate at pH 3.5 (yield: 0.66 g g^-1 mixed sugars; productivity: 1.21 g L^-1 h^-1). Mechanistic studies revealed that fatty acid metabolism drives a futile cycle converting cytosolic NADPH to mitochondrial NADH, essential for SA biosynthesis via the reductive TCA pathway. This cycle competitively inhibits xylose catabolism unless pathway genes are amplified to balance cofactor demand. This work highlights the importance of fatty acid metabolism in Y. lipolytica for SA biosynthesis, cofactor rebalancing, and pathway cross-talks.