[2P-86*] Directed Evolution of Cytochrome P450BM3 for Responding N-Acyl Homoserine Lactones as Decoy Molecules
Cytochrome P450BM3 (P450BM3) isolated from Bacillus megaterium is a heme enzyme that catalyzes the efficient hydroxylation of long-chain fatty acids. We have reported that synthesized substrate mimics, called "decoy molecules", enable hydroxylation of non-native substrates by wild-type (WT) P450BM3. However, addition of chemically-synthesized decoy molecules are required in current reaction system. In this research, we attempt to develop the reaction system without addition of synthetic decoy molecules using C10-acyl homoserine lactone (C10-AHL), produced by a kind of bacteria, as a decoy molecule. Herein, we engineered P450BM3 mutants for responding C10-AHL and improving its activities by directed evolution. At first, we introduced random mutagenesis into the heme domain of WT P450BM3. As the results of about 1600 mutants screening through benzene hydroxylation with C10-AHL, we obtained a 1.5-fold active mutant. Then, we found that the mutant contains mutagenesis positions which are far from heme in the active site. Therefore, we made and screened second generation library in which these residues were subjected to single site-saturation mutagenesis. We will discuss detail effects of these residues and the mutants of further rounds.