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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Acknowledgements
We thank Dr. Tadashi Kadowaki (DENSO Corporation) for instruction and support with regard to RNA-Seq analysis, which is an important aspect of this study. This work was supported in part by the Japan Agency for Medical Research and Development (AMED) under grant number JP20ae0101045 to K.S.
Availability of data and materials
Streptomyces sp. EAS-AB2608 was deposited with National Institute of Technology and Evaluation, Biological Resource Center (www.nite.go.jp/en/nbrc/) as NBRC 114648. The genome sequence information of Streptomyces sp. EAS-AB2608 is available in the DDBJ/EMBL/GenBank databases under accession number AP024135. The RNA-Seq raw data obtained in this study is also available in the DDBJ Sequence Read Archive under accession number DRA011062.
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Strand NGS v3.1 (www.strand-ngs.com/) was used for RNA-Seq analysis.
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SO, HI, and KS conceived the experiments. YN, MF, and NS contributed genome sequencing. HI contributed development of expression system (S. avermitilis SUKA24 ∆saverm3882 ∆saverm7426 and expression vector pKU565bla-tsr::Psav2794-fld-fpr-ter). IK and JH performed heterologous expression. SO performed RNA-Seq analysis, bioconversion, and LC-PDA-MS analyses. AO performed purification of CPD-2. EE performed NMR analyses. SO wrote the manuscript. All authors discussed the results and contributed to the final manuscript.
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Shinya Okubo now belongs to the Global Health Research Section, hhc Data Creation Center, Eisai Co., Ltd.
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Okubo, S., Ena, E., Okuda, A. et al. Identification of functional cytochrome P450 and ferredoxin from Streptomyces sp. EAS-AB2608 by transcriptional analysis and their heterologous expression. Appl Microbiol Biotechnol 105, 4177–4187 (2021). https://doi.org/10.1007/s00253-021-11304-z
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DOI: https://doi.org/10.1007/s00253-021-11304-z