Cloning and heterologous expression of P450Lent4B11, a novel bacterial P450 gene, for hydroxylation of an antifungal agent sordaricin

• Published in: Journal of antibiotics Vol. 73; no. 9; pp. 615 - 621
• Main Authors: Ueno, Motoi, Kobayashi, Motoo, Fujie, Akihiko, Shibata, Takashi
• Format: Journal Article
• Language: English
• Published: London Springer Science and Business Media LLC 01.09.2020; Nature Publishing Group UK; Nature Publishing Group
• Subjects: 38; 38/22; 38/23; 45/29; 631/1647/2234; 631/326/252; 82/80; Actinomycetales; Actinomycetales - genetics; Antifungal Agents; Antifungal Agents - pharmacology; Bacteriology; Biomedical and Life Sciences; Bioorganic Chemistry; Biotransformation; Biotransformation - genetics; Cloning; Cloning, Molecular; Cloning, Molecular - methods; Cytochrome; Cytochrome P-450 Enzyme System; Cytochrome P-450 Enzyme System - genetics; Diterpenes; Diterpenes - pharmacology; Fungi; Fungi - drug effects; Genes, Bacterial; Genes, Bacterial - genetics; Hydroxylation; Hydroxylation - genetics; Indenes; Indenes - pharmacology; Life Sciences; Medicinal Chemistry; Metyrapone; Metyrapone - pharmacology; Microbiology; Organic Chemistry; Oxidation-Reduction; Oxidation-Reduction - drug effects; Protein synthesis
• ISSN: 0021-8820; 1881-1469; 1881-1469
• DOI: 10.1038/s41429-020-0310-9

Microbial transformation is known to be one of promising options to add functional groups such as a hydroxyl moiety to active base compounds to generate their derivatives. Sordaricin, a diterpene aglycone of the natural product sordarin, is an antifungal agent to selectively inhibit fungal protein synthesis by stabilizing the ribosome/EF-2 (elongation factor 2) complex. We screened actinomycetes to catalyze hydroxylation of sordaricin on the basis that the hydroxyl moiety would make it easier to generate derivatives of sordaricin. As a result of the screening, 6-hydroxylation of sordaricin was found to be catalyzed by Lentzea sp. 7887. We found that the cytochrome P450 inhibitor metyrapone inhibited this reaction, suggesting that a cytochrome P450 may be responsible for the biotransformation. As a next step, we cloned multiple cytochrome P450 genes, one of which were named P450Lent4B11, using degenerate PCR primers. The expressed cytochrome P450 derived from the P450Lent4B11 gene provided a different absorbance spectrum pattern from original one when it was incubated with sordaricin. Moreover, in cell-free conditions, the corresponding cytochrome P450 displayed the 6-hydroxylation activity toward sordaricin. Taken together, these results indicate that P450Lent4B11, derived from Lentzea sp. 7887, should be responsible for catalyzing 6-hydroxylation of sordaricin.