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.

中文翻译：

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由细胞色素 P450 酶 RosC 催化的多步氧化反应的人工控制。

细胞色素 P450 单加氧酶 RosC 催化三步氧化反应，导致在玫瑰花小单孢菌 IFO13697 中的罗霉素生物合成过程中在 C-20 处形成羟基、甲酰基和羧基。为了确定 RosC 中的氨基酸取代是否可以控制多步氧化反应，我们筛选了 RosC 随机突变体。RosC 突变体 RM30 具有五个氨基酸取代（P107S、L176Q、S254N、V277A 和 I319N），仅催化氧化反应的第一步。使用表达来自 RM30 的具有单和双氨基酸取代的 RosC 突变体的大肠杆菌细胞的全细胞测定表明 P107S/L176Q、P107S/V277A、P107S/I319N、L176Q/V277A、L176Q/I319N 和 S277A催化活性第二个反应，即醇氧化。在前面提到的突变体中，含有 L176Q 的双突变体（推测发生在 FG 环区域）失去了第三个反应（醛氧化）的总催化活性。此外，引入了编码 RosC 突变体 P107S/L176Q 和 P107S/V277A 的基因的具有 rosC 破坏的工程化 M. rosaria 菌株优先产生 20-dihydrorosamicin，其在 RosC 的第一次氧化反应后形成。