Bacterial Acyl Homoserine Lactones Triggered Non‐Native Substrate Hydroxylation Catalyzed by Directed‐Evolution‐Derived Cytochrome P450BM3 Mutants

Abstract We report the directed evolution of cytochrome P450BM3 to efficiently utilize the bacterial quorum sensing signalling molecule N ‐decanoyl homoserine lactone (C10‐HSL) as an effective decoy molecule. This represents the first important step in our endeavor to develop of a self‐sufficient decoy‐molecule system in whole‐cells that only necessitates the addition of culture medium and substrate to realize the hydroxylation of non‐native substrates. Following five rounds of directed evolution, mutant P450BM3, in the presence of C10‐HSL, catalyzed the hydroxylation of benzene at a rate of 475 min −1 , the highest turnover rate recorded for any P450 enzyme, and achieving a 46% yield in a whole‐cell reaction system. High‐resolution X‐ray crystal structure analysis of a series of mutants narrates the directed evolution process, revealing how C10‐HSL is fixed in the binding pocket to permit binding of non‐native substrates. Finally, introduction of the C10‐HSL synthase gene ExpI into Escherichia coli , enabled the in situ production of C10‐HSL, realizing, for the first time, the hydroxylation of non‐native substrates without the need for the laborious synthesis and addition of decoy molecules.