Enhanced oxidative activities of cytochrome P450Rhf from Rhodococcus sp. expressed using the hyper-inducible expression system(GENETICS, MOLECULAR BIOLOGY, AND GENE ENGINEERING)
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概要
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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 P45ORhf 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.
著者
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Kobayashi Michihiko
Institute Of Applied Biochemistry And Graduate School Of Life And Environmental Sciences The Univers
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Hino Motohiro
Fermentation And Biotechnology Laboratories Astellas Pharma Inc.
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Ueno Motoi
Pharmacology Research Laboratories
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TAKASE Shigehiro
Analysis & Pharmacokinetics Research Laboratories, Astellas Pharma Inc.
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YAMASHITA Midori
Pharmacology Research Laboratories
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Fujie Akihiko
Research Planning & Administration
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Hino Motohiro
Fermentation and Biotechnology Laboratories, Astellas Pharma Inc.
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Fujie Akihiko
Research Management, Astellas Pharma Inc.
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Yamashita Midori
Pharmacology Research Laboratories, Astellas Pharma Inc.
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