Establishing an Efficient Electron Transfer System for P450 Enzyme OleP to Improve the Biosynthesis of...

The electron transfer from NAD(P)H to heme is a rate-limiting step in the redox partner-mediated catalysis of P450 enzyme. However, due to the lack of efficient engineering strategies, it is difficult to improve the properties of redox partner. Herein, we construct an effective approach to modify the redox partner for a typical P450 enzyme (OleP) that can catalyze the stereoselective conversion of lithocholic acid to murideoxycholic acid. First, the combination of computational modeling and experimental validation was performed to rapidly identify the most suitable redox partner (PetH/PetF). Next, the interactions between PetF and OleP were investigated and the engineering on PetF was conducted to enhance the efficiency of electron transfer. Using a novel microplate screening method, a superior mutant (PetF_F64D) was efficiently selected, which exhibited a significant enhancement in MDCA conversion yield from 32.5% to 80.9% and total turnover number (TTN) from 406.2 to 1617.9. Finally, through a combination of molecular dynamics simulations, the analysis of electron transfer pathway, and the calculations of electron transfer rate, the mechanism of electron transfer was investigated. The applied engineering strategies, high-throughput screening methods, and analytical approaches provide a feasible way to construct an ideal redox partner for other P450 enzymes.