Identification of functional cytochrome P450 and ferredoxin from Streptomyces sp. EAS-AB2608 by transcriptional analysis and their heterologous expression
| Autor: | Akifumi Okuda, Eri Ena, Kazuo Shin-ya, Manabu Fujie, Yoshie Nishitsuji, Noriyuki Satoh, Ikuko Kozone, Junko Hashimoto, Haruo Ikeda, Shinya Okubo |
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| Rok vydání: | 2021 |
| Předmět: |
Bioconversion
education Applied Microbiology and Biotechnology Streptomyces Hydroxylation 03 medical and health sciences chemistry.chemical_compound Cytochrome P-450 Enzyme System Japan Gene Ferredoxin 030304 developmental biology 0303 health sciences biology 030306 microbiology Cytochrome P450 General Medicine biology.organism_classification Biochemistry chemistry biology.protein Ferredoxins Heterologous expression Streptomyces avermitilis Biotechnology |
| Zdroj: | Applied Microbiology and Biotechnology. 105:4177-4187 |
| ISSN: | 1432-0614 0175-7598 |
| Popis: | Bioconversion using microorganisms and their enzymes is an important tool in many industrial fields. The discovery of useful new microbial enzymes contributes to the development of industries utilizing bioprocesses. Streptomyces sp. EAS-AB2608, isolated from a soil sample collected in Japan, can convert the tetrahydrobenzotriazole CPD-1 (a selective positive allosteric modulator of metabotropic glutamate receptor 5) to its hydroxylated form at the C4-(R) position. The current study was performed to identify the genes encoding the enzymes involved in CPD-1 bioconversion and to verify their function. To identify gene products responsible for the conversion of CPD-1, we used RNA sequencing to analyze EAS-AB2608; from its 8333 coding sequences, we selected two genes, one encoding cytochrome P450 (easab2608_00800) and the other encoding ferredoxin (easab2608_00799), as encoding desirable gene products involved in the bioconversion of CPD-1. The validity of this selection was tested by using a heterologous expression approach. A bioconversion assay using genetically engineered Streptomyces avermitilis SUKA24 ∆saverm3882 ∆saverm7246 co-expressing the two selected genes (strain ES_SUKA_63) confirmed that these gene products had hydroxylation activity with respect to CPD-1, indicating that they are responsible for the conversion of CPD-1. Strain ES_SUKA_63 also showed oxidative activity toward other compounds and therefore might be useful not only for bioconversion of CPD-1 but also as a tool for synthesis of drug metabolites and in optimization studies of various pharmaceutical lead compounds. We expect that this approach will be useful for bridging the gap between the latest enzyme optimization technologies and conventional enzyme screening using microorganisms. KEY POINTS: • Genes easab2608_00800 (cyp) and easab2608_00799 (fdx) were selected by RNA-Seq. • Selection validity was evaluated by an engineered S. avermitilis expression system. • Strain ES_SUKA_63 showed oxidative activity toward CPD-1 and other compounds. |
| Databáze: | OpenAIRE |
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