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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.<\/jats:p>","DOI":"10.1093\/jimb\/kuab069","type":"journal-article","created":{"date-parts":[[2021,9,16]],"date-time":"2021-09-16T07:08:28Z","timestamp":1631776108000},"source":"Crossref","is-referenced-by-count":10,"title":["Engineering sequence and selectivity of late-stage C-H oxidation in the MycG iterative cytochrome P450"],"prefix":"10.1093","volume":"49","author":[{"ORCID":"https:\/\/orcid.org\/0000-0002-5951-4511","authenticated-orcid":false,"given":"Yohei","family":"Iizaka","sequence":"first","affiliation":[{"name":"Faculty of Pharmaceutical Sciences, Toho University, 2-2-1 Miyama, Funabashi, Chiba 274-8510, Japan"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Ryusei","family":"Arai","sequence":"additional","affiliation":[{"name":"Faculty of Pharmaceutical Sciences, Toho University, 2-2-1 Miyama, 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