Abstract
Alkaloids represent a vast class of natural products with significant pharmaceutical value, yet their structural complexity often hinders scalable production and diversification. Cytochrome P450 enzymes play pivotal roles in alkaloid biosynthesis, catalyzing regioselective and stereoselective oxidations. Here, we report a modular P450 engineering strategy to produce non-natural alkaloids in Saccharomyces cerevisiae. By combining directed evolution and rational design, we generated chimeric P450s with altered substrate specificity and enhanced catalytic efficiency. A library of 24 engineered P450 variants was constructed and screened against a panel of 12 non-natural alkaloid precursors. Three variants, designated P450-7, P450-12, and P450-19, exhibited high activity toward precursors bearing halogen and methyl substitutions. The best variant, P450-12, showed a 15-fold improvement in turnover number compared to the wild-type enzyme. Pathway optimization enabled de novo biosynthesis of 6-fluororetinine and 8-methylcorydaline at titers of 45 mg/L and 32 mg/L, respectively. Our results demonstrate that modular P450 engineering can expand the biosynthetic repertoire to access non-natural alkaloids, providing a platform for drug discovery and metabolic engineering.