Semi-rational directed evolution of a Deepsea－derived P450_S18 for Phenazines Construction

Deep sea-derived microbes are great sources for novel enzymes due to their extreme living environment, such as high pressure, high salinity, etc. CYP152 family P450s have the ability to directly use H₂O₂ instead of complex redox partners, which have high biocatalytic values in biofuels and fine chemical synthesis. Therefore, in this project, we mined a CYP152 family P450_S18 from a deep sea-derived bacteria and improved its catalytic ability toward 1,2-phenylenediamine (OPD, 1). In vitro assays showed that P450_S18 can directly transform OPD (1) to 2,3-diaminophenazine (2) through C‒N bond construction, in the presence of H₂O₂. We then performed protein structure prediction and molecular docking of OPD with P450_S18, and selected eight binding-related residues for further study. Alanine scanning and crude enzyme activity screening indicated F295A, P246A, F176A, F292A, F82A, and Q88A can effectively increase the catalytic activity. Finally, we purified the mutants and accurately measured their in vitro catalytic efficiency toward OPD (1). All of the mutants exhibited 1.5~3.5-fold increased activity with F292A as the optimal mutant. Our study provides foundation for further engineering of P450_S18 to obtain phenazine derivatives.