Photoinduced electron transfer enables cytochrome P450 enzyme-catalyzed reaction cycling.

Cytochrome P450 enzymes (CYPs), the members of the largest superfamily of enzymes in plant kingdom, catalyze a variety of functional group transformations involved in metabolite biosynthesis, end-product derivatization, and exogeneous molecule detoxification. Nevertheless, CYPs' functional characterization and practically industrial application have been largely encumbered by their critical dependency on the reducing equivalent for the catalytic cycling, driven by the tedious electron relay mediated by CYP reductase (CPR). Here, we report a photoinduced electron transfer system that initiates and sustains the CYP-catalyzed reaction cycling. Using Camptotheca acuminata CYP72A565-catalyzed carbon-carbon cleavage reaction, a key biosynthetic reaction in the biosynthesis of plant-derived antitumor monoterpene indole alkaloid camptothecin, as a representative CYP-catalyzed reaction model, we identified eosin Y (EY) and triethanolamine (TEOA) as an efficient photosensitizer/sacrificial reagent pair for the photoinduced electron generating system. The C. acuminata camptothecin 10-hydroxylase-catalyzed regioselective C10-hydroxylation of camptothecin into 10-hydroxycamptothecin could be enabled by the photoinduced electron transfer system, demonstrating that the EY/TEOA pair serves as an efficient surrogate for membranous CPR and can be expanded to other CYP-catalyzed reaction cycling. The catalytic efficiency of the photoinduced electron transfer-driven CYP-catalyzed cycling exceeds that of the native NADPH-dependent CPR-supported CYP-catalyzed reaction, thereby circumventing the dependency on both NADPH and the reductase CPR. The present study provides a photoinduced electron generating and transferring system as an efficient and facile alternative to membranous NADPH-dependent CPR, offering a new avenue for CYP-mediated conversion of complex bioactive natural products using synthetic biology approaches.