Synthesis of Trifluoromethylated Monoterpenes by an Engineered Cytochrome P450

Protein engineering of cytochrome P450s has enabled these biocatalysts to promote a variety of abiotic reactions beyond nature‘s repertoire. Integrating such non‐natural transformations with microbial biosynthetic pathways could allow sustainable enzymatic production of modified natural product derivatives. In particular, trifluoromethylation is a highly desirable modification in pharmaceutical research due to the positive effects of the trifluoromethyl group on drug potency, bioavailability, and metabolic stability. This study demonstrates the biosynthesis of non‐natural trifluoromethyl‐substituted cyclopropane derivatives of natural monoterpene scaffolds using an engineered cytochrome P450 variant, P411‐PFA. P411‐PFA successfully catalyzed the transfer of a trifluoromethyl carbene from 2‐diazo‐1,1,1‐trifluoroethane to the terminal alkenes of several monoterpenes, including L‐carveol, carvone, perilla alcohol, and perillartine, to generate the corresponding trifluoromethylated cyclopropane products. Furthermore, integration of this abiotic cyclopropanation reaction with a reconstructed metabolic pathway for L‐carveol production in Escherichia coli enabled one‐step biosynthesis of a trifluoromethylated L‐carveol derivative from limonene precursor. Overall, amalgamating synthetic enzymatic chemistry with established metabolic pathways represents a promising approach to sustainably produce bioactive natural product analogs. The carbene transfer reaction was realized in the biosynthetic pathway. Using L‐limonene as the precursor, L‐carveol was biotransformed by P411‐PFA to generate unnatural terpenoid derivatives with trifluoromethyl group.