Systematic enzyme and cofactor engineering for efficient ursolic acid biosynthesis in Yarrowia lipolytica.

Ursolic acid (UA) is a pharmaceutically valuable pentacyclic triterpenoid, but its microbial production is constrained by inefficient cytochrome P450 catalysis and limited cofactor availability. Here, we engineered an efficient α-amyrin-producing Yarrowia lipolytica chassis for UA biosynthesis through integrated enzyme engineering, cofactor optimization, and metabolic flux balancing. Screening of heterologous plant cytochrome P450 monooxygenases identified CYP716A48 from Olea europaea as the most efficient α-amyrin oxidase in Y. lipolytica. Fusion of OeCYP716A48 with its redox partner AtCPR1 using optimized flexible linkers enhanced intramolecular electron transfer and significantly increased UA titers. The OeCYP716A48 D114Q/L211F variant enlarged the substrate-access tunnel and improved catalytic efficiency, resulting in a 5-fold increase in UA production. To support high OeCYP716A48 activity, intracellular FAD, heme, and iron availability were systematically enhanced, leading to a 14.4-fold increase in UA production. Multicopy integration of CrMAS, OeCYP716A48 D114Q/L211F, and AtCPR1 at rDNA loci further balanced pathway flux. In 5-L fed-batch fermentation, the engineered strain produced 813 ± 24 mg/L UA, representing the highest titer reported in Y. lipolytica to date. This study establishes a scalable and broadly applicable engineering strategy to overcome cytochrome P450 limitations and enable efficient triterpenoid biosynthesis in yeast.