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Identification of functional cytochrome P450 and ferredoxin from Streptomyces sp. EAS-AB2608 by transcriptional analysis and their heterologous expression.
Okubo, Shinya; Ena, Eri; Okuda, Akifumi; Kozone, Ikuko; Hashimoto, Junko; Nishitsuji, Yoshie; Fujie, Manabu; Satoh, Noriyuki; Ikeda, Haruo; Shin-Ya, Kazuo.
Afiliação
  • Okubo S; Eisai Co., Ltd., Tokodai 5-1-3, Tsukuba, Ibaraki, 300-2635, Japan. s5-okubo@hhc.eisai.co.jp.
  • Ena E; Eisai Co., Ltd., Tokodai 5-1-3, Tsukuba, Ibaraki, 300-2635, Japan.
  • Okuda A; Eisai Co., Ltd., Tokodai 5-1-3, Tsukuba, Ibaraki, 300-2635, Japan.
  • Kozone I; Japan Biological Informatics Consortium (JBIC), Aomi, Tokyo, Japan.
  • Hashimoto J; Technology Research Association for Next-Generation Natural Products Chemistry (N2PC), Aomi, Tokyo, Japan.
  • Nishitsuji Y; Japan Biological Informatics Consortium (JBIC), Aomi, Tokyo, Japan.
  • Fujie M; Technology Research Association for Next-Generation Natural Products Chemistry (N2PC), Aomi, Tokyo, Japan.
  • Satoh N; Technology Research Association for Next-Generation Natural Products Chemistry (N2PC), Aomi, Tokyo, Japan.
  • Ikeda H; Okinawa Institute of Science and Technology Graduate University (OIST), Onna-son, Okinawa, Japan.
  • Shin-Ya K; Okinawa Institute of Science and Technology Graduate University (OIST), Onna-son, Okinawa, Japan.
Appl Microbiol Biotechnol ; 105(10): 4177-4187, 2021 May.
Article em En | MEDLINE | ID: mdl-33944982
ABSTRACT
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.
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Texto completo: 1 Coleções: 01-internacional Base de dados: MEDLINE Assunto principal: Streptomyces / Ferredoxinas Tipo de estudo: Diagnostic_studies País/Região como assunto: Asia Idioma: En Revista: Appl Microbiol Biotechnol Ano de publicação: 2021 Tipo de documento: Article

Texto completo: 1 Coleções: 01-internacional Base de dados: MEDLINE Assunto principal: Streptomyces / Ferredoxinas Tipo de estudo: Diagnostic_studies País/Região como assunto: Asia Idioma: En Revista: Appl Microbiol Biotechnol Ano de publicação: 2021 Tipo de documento: Article