Directed evolution of P450cin for mediated electron transfer

Directed evolution is a powerful method to optimize enzyme properties for application demands. Interesting targets are P450 monooxygenases which catalyze the stereo- and regiospecific hydroxylation of chemically inert C–H bonds. Synthesis employing P450s under cell-free reaction conditions is limited by low total turnover numbers, enzyme instability, low product yields and the requirement of the expensive co-factor NADPH. Bioelectrocatalysis is an alternative to replace NADPH in cell-free P450-catalyzed reactions. However, natural enzymes are often not suitable for using non-natural electron delivery systems. Here we report the directed evolution of a previously engineered P450 CinA-10aa-CinC fusion protein (named P450cin-ADD-CinC) to use zinc/cobalt(III)sepulchrate as electron delivery system for an increased hydroxylation activity of 1,8-cineole. Two rounds of Sequence Saturation Mutagenesis (SeSaM) each followed by one round of multiple site-saturation mutagenesis of the P450 CinA-10aa-CinC fusion protein generated a variant (Gln385His, Val386Ser, Thr77Asn, Leu88Arg; named KB8) with a 3.8-fold increase in catalytic efficiency (28 µM−1 min−1) compared to P450cin-ADD-CinC (7 µM−1 min−1). Furthermore, variant KB8 exhibited a 1.5-fold higher product formation (500 µM µM−1 P450) compared to the equimolar mixture of CinA, CinC and Fpr using NADPH as co-factor (315 µM µM−1 P450). In addition, electrochemical experiments with the electron delivery system platinum/cobalt(III)sepulchrate showed that the KB8 variant had a 4-fold higher product formation rate (0.16 nmol (nmol) P450−1 min−1 cm−2) than the P450cin-ADD-CinC (0.04 nmol (nmol) P450−1 min−1 cm−2). In summary, the current work shows prospects of using directed evolution to generate P450 enzymes suitable for use with alternative electron delivery systems.