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Engineering sequence and selectivity of late-stage C-H oxidation in the MycG iterative cytochrome P450
Journal of Industrial Microbiology & Biotechnology ( IF 3.2 ) Pub Date : 2021-09-16 , DOI: 10.1093/jimb/kuab069
Yohei Iizaka 1 , Ryusei Arai 1 , Akari Takahashi 1 , Mikino Ito 1 , Miho Sakai 1 , Atsushi Fukumoto 1 , David H Sherman 2 , Yojiro Anzai 1
Affiliation  

MycG is a multifunctional P450 monooxygenase that catalyzes sequential hydroxylation and epoxidation or a single epoxidation in mycinamicin biosynthesis. In the mycinamicin-producing strain Micromonospora griseorubida A11725, very low-level accumulation of mycinamicin V generated by the initial C-14 allylic hydroxylation of MycG is observed due to its subsequent epoxidation to generate mycinamicin II, the terminal metabolite in this pathway. Herein, we investigated whether MycG can be engineered for production of the mycinamicin II intermediate as the predominant metabolite. Thus, mycG was subject to random mutagenesis and screening was conducted in Escherichia coli whole-cell assays. This enabled efficient identification of amino acid residues involved in reaction profile alterations, which included MycG R111Q/V358L, W44R, and V135G/E355K with enhanced monohydroxylation to accumulate mycinamicin V. The MycG V135G/E355K mutant generated 40-fold higher levels of mycinamicin V compared to wild-type M. griseorubida A11725. In addition, the E355K mutation showed improved ability to catalyze sequential hydroxylation and epoxidation with minimal mono-epoxidation product mycinamicin I compared to the wild-type enzyme. These approaches demonstrate the ability to selectively coordinate the catalytic activity of multifunctional P450s and efficiently produce the desired compounds.

中文翻译:

MycG迭代细胞色素P450中后期CH氧化的工程序列和选择性

MycG 是一种多功能 P450 单加氧酶,可催化霉素生物合成中的顺序羟基化和环氧化或单次环氧化。在产生霉素的菌株 Micromonospora griseorubida A11725 中,观察到由 MycG 的初始 C-14 烯丙基羟基化产生的霉素 V 的非常低水平的积累,这是由于其随后环氧化生成霉素 II,该途径中的末端代谢物。在这里,我们研究了 MycG 是否可以被设计用于生产作为主要代谢物的霉素 II 中间体。因此,mycG 受到随机诱变,并在大肠杆菌全细胞测定中进行筛选。这使得能够有效识别参与反应谱改变的氨基酸残基,其中包括 MycG R111Q/V358L、W44R、和 V135G/E355K 具有增强的单羟基化以积累霉素 V。与野生型 M. griseorubida A11725 相比,MycG V135G/E355K 突变体产生的霉素 V 水平高 40 倍。此外,与野生型酶相比,E355K 突变显示出改进的催化连续羟基化和环氧化的能力,其单环氧化产物霉素 I 最少。这些方法证明了选择性协调多功能 P450 的催化活性并有效生产所需化合物的能力。与野生型酶相比,E355K 突变显示出改进的催化连续羟基化和环氧化的能力,其单环氧化产物霉素 I 最少。这些方法证明了选择性协调多功能 P450 的催化活性并有效生产所需化合物的能力。与野生型酶相比,E355K 突变显示出改进的催化连续羟基化和环氧化的能力,其单环氧化产物霉素 I 最少。这些方法证明了选择性协调多功能 P450 的催化活性并有效生产所需化合物的能力。
更新日期:2021-09-16
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