Flow Cytometer-Based High-Throughput Screening System for Accelerated Directed Evolution of P450 Monooxygenases

Flow cytometry-based screening systems have successfully been used in directed evolution experiments. Herein, we report the first whole-cell, high-throughput screening platform for P450 monooxygenases based on flow cytometry. O-dealkylation of 7-benzoxy-3-carboxycoumarin ethyl ester (BCCE) by P450 BM3 generates a fluorescence coumarin derivative. After one round of directed evolution, P450 BM3 variants with up to 7-fold increased activity (P450 M3 DM-1: R255H) could be identified at a sampling rate of 500 events s(-1). The reported screening platform can likely be applied to directed evolution campaigns of any P450 monooxygenase that catalyzes the O-dealkylation of BCCE.