The cytochrome P450 monooxygenase RosC catalyzes the three-step oxidation reactions, which leads to the formation of a hydroxy, formyl, and carboxy group at C-20 during rosamicin biosynthesis in Micromonospora rosaria IFO13697. To determine if amino acid substitutions in RosC could allow for the control of the multistep oxidation reactions, we screened RosC random mutants. The RosC mutant RM30, with five amino acid substitutions (P107S, L176Q, S254N, V277A, and I319N), catalyzed only the first step of the oxidation reaction. Whole-cell assays using Escherichia coli cells expressing RosC mutants with single and double amino acid substitutions derived from RM30 indicated that P107S/L176Q, P107S/V277A, P107S/I319N, L176Q/V277A, L176Q/I319N, and S254N/V277A significantly reduced the catalytic activity of the second reaction, which is alcohol oxidation. Of the previously mentioned mutants, double mutants containing L176Q, which was presumed to occur in the FG loop region, lost the total catalytic activity of the third reaction (aldehyde oxidation). Additionally, an engineered M. rosaria strain with rosC disruption, which introduced the gene encoding the RosC mutants P107S/L176Q and P107S/V277A preferentially produced 20-dihydrorosamicin, which is formed after the first oxidation reaction of RosC.
Keywords: Cytochrome P450 RosC; Engineered biosynthesis; Multistep oxidation; Rosamicin; Whole-cell assays.