Identification of functional cytochrome P450 and ferredoxin from Streptomyces sp. EAS-AB2608 by transcriptional analysis and their heterologous expression

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 Δsaverm3882 Δsaverm7246 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.

Actinobacteria as Microbial Cell Factories and Biocatalysts in The Synthesis of Chiral Intermediates and Bioactive Molecules; Insights and Applications

“…The latter could catalyze the selective hydroxylation at C4 producing the R ‐isomer of the corresponding hydroxylated form (Scheme 5). [3b] It is noteworthy that CPD‐1 plays a role in selectively modulating metabotropic glutamate receptors involved in developing a drug for Alzheimer's disease [95] . Furthermore, the C4‐hydroxylated form, CPD‐2 was isolated and purified via reverse phase HPLC, which is a helpful form in understanding the SAR of CPD‐1.…”

“…Furthermore, S. avermitilis ES_SUKA_63 exhibited oxidative activities towards other tested substrates, such as indomethacin without side reactions like the glycosylated form produced upon using the native host. Such an approach can be applied to the identification of other genes encoding metabolic enzymes of interest [3b] …”