title: Engineering class I cytochrome P450 by gene fusion with NADPH-dependent reductase and S. avermitilis host development for daidzein biotransformation.
abstract: Daidzein C6 hydroxylase (6-DH, nfa12130), which is a class I type of cytochrome P450 enzyme, catalyzes a hydroxylation reaction at the C6-position of the daidzein A-ring and requires auxiliary electron transfer proteins. Current utilization of cytochrome P450 (CYP) enzymes is limited by low coupling efficiency, which necessitates extramolecular electron transfers, and low driving forces, which derive electron flows from tightly regulated NADPH redox balances into the heterogeneous CYP catalytic cycle. To overcome such limitations, the heme domain of the 6-DH enzyme was genetically fused with the NADPH-reductase domain of self-sufficient CYP102D1 to enhance electron transfer efficiencies through intramolecular electron transfer and switching cofactor preference from NADH into NADPH. 6-DH-reductase fusion enzyme displayed distinct spectral properties of both flavoprotein and heme proteins and catalyzed daidzein hydroxylation more efficiently with a k cat/K m value of 120.3 ± 11.5 [10(3) M(-1) s(-1)], which was about three times higher than that of the 6-DH-FdxC-FdrA reconstituted system. Moreover, to obtain a higher redox driving force, a Streptomyces avermitilis host system was developed for heterologous expression of fusion 6-DH enzyme and whole cell biotransformation of daidzein. The whole cell reaction using the final recombinant strain, S. avermitilisΔcyp105D7::fusion 6-DH (nfa12130), resulted in 8.3 ± 1.4 % of 6-OHD yield from 25.4 mg/L of daidzein.
authors.affiliation: Department of Environmental Engineering, College of Engineering, Ajou University, Suwon, Gyeonggi-do, 443-749, South Korea.
authors.forename: Kwon-Young
authors.initials: KY
authors.lastname: Choi
authors.forename: EunOk
authors.initials: E
authors.lastname: Jung
authors.forename: Hyungdon
authors.initials: H
authors.lastname: Yun
authors.forename: Yung-Hun
authors.initials: YH
authors.lastname: Yang
authors.forename: Byung-Gee
authors.initials: BG
authors.lastname: Kim
mesh_terms: D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D001711:Biotransformation; D007529:Isoflavones / Q000378:metabolism*; D007700:Kinetics; D060847:Metabolic Engineering; D006899:Mixed Function Oxygenases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D009251:NADPH-Ferrihemoprotein Reductase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D009615:Nocardia / Q000201:enzymology* / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D013302:Streptomyces / Q000235:genetics / Q000378:metabolism*
chemical_list: D001426:Bacterial Proteins; D007529:Isoflavones; D011993:Recombinant Fusion Proteins; C004742:daidzein; D006899:Mixed Function Oxygenases; D009251:NADPH-Ferrihemoprotein Reductase