期刊
ACS CATALYSIS
卷 2, 期 12, 页码 2724-2728出版社
AMER CHEMICAL SOC
DOI: 10.1021/cs300115d
关键词
directed evolution; coumarin; flow cytometer; P450 monooxygenase; high-throughput screening
资金
- European Union [212281]
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.
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