Systematic enzyme and cofactor engineering for efficient ursolic acid biosynthesis in

Source: PubMed Central Open Access, NCBI / U.S. National Library of Medicine

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–producingchassis for UA biosynthesis through integrated enzyme engineering, cofactor optimization, and metabolic flux balancing. Screening of heterologous plant cytochrome P450 monooxygenases identifiedfromas the most efficient α-amyrin oxidase in. Fusion of Oewith its redox partnerusing optimized flexible linkers enhanced intramolecular electron transfer and significantly increased UA titers. The Oevariant enlarged the substrate-access tunnel and improved catalytic efficiency, resulting in a 5-fold increase in UA production. To support high Oeactivity, intracellular FAD, heme, and iron availability were systematically enhanced, leading to a 14.4-fold increase in UA production. Multicopy integration of,, andat 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 into date. This study establishes a scalable and broadly applicable engineering strategy to overcome cytochrome P450 limitations and enable efficient triterpenoid biosynthesis in yeast. abs0010