title: Enhanced oxidative activities of cytochrome P450Rhf from Rhodococcus sp. expressed using the hyper-inducible expression system.
abstract: Bacterial cytochrome P450 enzymes catalyze the oxidative biotransformation of various types of compounds. Although the functional expression of Actinomycetes P450 in a closely related heterologous host can serve as a useful biocatalyst in whole-cell biotransformation assays, the co-expression of an electron transfer partner is required. To overcome this limitation, P450Rhf from Rhodococcus sp. NCIMB 9784 is an ideal candidate, because it is fused to a reductase domain at the C terminus and does not require an electron transfer partner. Here, we cloned P450Rhf into the hyper-inducible expression vector pSH19 in Streptomyces lividans TK24 for developing an efficient whole-cell biotransformation system with bacterial P450. The recombinant strain displayed high conversion activity (79.1%) of 7-ethoxycoumarin to 7-hydroxycoumarin after 48 h, and the observed activity was markedly higher than those for 7-methoxycoumarin and 7-propoxycoumarin used as substrates. We next screened several commercially available substrates possessing an ethyl phenyl ether moiety, which is also present in 7-ethoxycoumarin, and found that 4'-ethoxy-2'-hydroxyacetphenone was almost completely dealkylated (95.0%), and that 7-ethoxy-4-methylcoumarin was converted to two products, 7-hydroxy-4-methylcoumarin and 7-ethoxy-4-hydroxymethyl-coumarin. Our research suggests that enhancement of heterologous P450 expression using the pSH19 system in whole-cell biotransformation assays is valuable for identifying novel substrates of P450, as well as for obtaining high yields of conversion products.
authors.affiliation: Pharmacology Research Laboratories, Astellas Pharma Inc., 21 Miyukigaoka, Tsukuba-shi, Ibaraki 305-8585, Japan. Electronic address: motoi.ueno@astellas.com.
authors.forename: Motoi
authors.initials: M
authors.lastname: Ueno
authors.affiliation: Pharmacology Research Laboratories, Astellas Pharma Inc., 21 Miyukigaoka, Tsukuba-shi, Ibaraki 305-8585, Japan.
authors.forename: Midori
authors.initials: M
authors.lastname: Yamashita
authors.affiliation: Analysis & Pharmacokinetics Research Laboratories, Astellas Pharma Inc., 5-2-3 Tokodai, Tsukuba-shi, Ibaraki 300-2698, Japan.
authors.forename: Shigehiro
authors.initials: S
authors.lastname: Takase
authors.affiliation: Fermentation and Biotechnology Laboratories, Astellas Pharma Inc., 156 Nakagawara, Kiyosu-shi, Aichi 452-0915, Japan.
authors.forename: Motohiro
authors.initials: M
authors.lastname: Hino
authors.affiliation: Institute of Applied Biochemistry and Graduate School of Life and Environmental Sciences, The University of Tsukuba, 1-1-1 Tennodai, Tsukuba, Ibaraki 305-8572, Japan.
authors.forename: Michihiko
authors.initials: M
authors.lastname: Kobayashi
authors.affiliation: Research Management, Astellas Pharma Inc., 21 Miyukigaoka, Tsukuba-shi, Ibaraki 305-8585, Japan.
authors.forename: Akihiko
authors.initials: A
authors.lastname: Fujie
mesh_terms: D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism*; D001711:Biotransformation; D003374:Coumarins / Q000737:chemistry* / Q000378:metabolism; D003577:Cytochrome P-450 Enzyme System / Q000235:genetics / Q000378:metabolism*; D004798:Enzymes; D015394:Molecular Structure; D010084:Oxidation-Reduction; D010957:Plasmids / Q000235:genetics*; D012240:Rhodococcus / Q000201:enzymology* / Q000235:genetics / Q000254:growth & development; D048170:Streptomyces lividans / Q000235:genetics / Q000254:growth & development / Q000378:metabolism; D013379:Substrate Specificity; D015533:Transcriptional Activation; D014468:Umbelliferones / Q000737:chemistry* / Q000378:metabolism
chemical_list: D001426:Bacterial Proteins; D003374:Coumarins; D004798:Enzymes; D014468:Umbelliferones; C017299:7-ethoxycoumarin; C031477:7-hydroxycoumarin; C115983:4-methyl-7-ethoxycoumarin; D003577:Cytochrome P-450 Enzyme System; C580890:7-methylcoumarin
keywords: 4′-Ethoxy-2′-hydroxyacetphenone; Biotransformation; Monooxygenation; P450Rhf; Streptomyces; pSH19