Regioselective hydroxylation of daidzein using P450 (CYP105D7) from Streptomyces avermitilis MA4680.
Regiospecific 3'-hydroxylation reaction of daidzein was performed with CYP105D7 from Streptomyces avermitilis MA4680 expressed in Escherichia coli. The apparent K(m) and k(cat) values of CYP105D7 for daidzein were 21.83 +/- 6.3 microM and 15.01 +/- 0.6 min(-1) in the presence of 1 microM of CYP105D7, putidaredoxin (CamB) and putidaredoxin reductase (CamA), respectively. When CYP105D7 was expressed in S. avermitilis MA4680, its cytochrome P450 activity was confirmed by the CO-difference spectra at 450 nm using the whole cell extract. When the whole-cell reaction for the 3'-hydroxylation reaction of daidzein was carried out with 100 microM of daidzein in 100 mM of phosphate buffer (pH 7.5), the recombinant S. avermitilis grown in R2YE media overexpressing CYP105D7 and ferredoxin FdxH (SAV7470) showed a 3.6-fold higher conversion yield (24%) than the corresponding wild type cell (6.7%). In a 7 L (working volume 3 L) jar fermentor, the recombinants S. avermitilis grown in R2YE media produced 112.5 mg of 7,3',4'-trihydroxyisoflavone (i.e., 29.5% conversion yield) from 381 mg of daidzein in 15 h.
School of Chemical and Biological Engineering, Institute of Molecular Biology and Genetics, Institute of Bioengineering, Seoul National University, Seoul 151-742, South Korea.
Bishnu Prasad
BP
Pandey
Changhyun
C
Roh
Kwon-Young
KY
Choi
Nahum
N
Lee
Eun Jung
EJ
Kim
Sungghi
S
Ko
Taejin
T
Kim
Hyundon
H
Yun
Byung-Gee
BG
Kim
D001709:Biotechnology / Q000379:methods; D003577:Cytochrome P-450 Enzyme System / Q000032:analysis / Q000235:genetics* / Q000378:metabolism*; D004926:Escherichia coli / Q000235:genetics; D015870:Gene Expression; D005798:Genes, Bacterial; D006900:Hydroxylation; D007529:Isoflavones / Q000378:metabolism*; D019906:Nuclear Magnetic Resonance, Biomolecular; D011994:Recombinant Proteins / Q000032:analysis / Q000235:genetics / Q000378:metabolism; D013302:Streptomyces / Q000201:enzymology* / Q000235:genetics
D007529:Isoflavones; D011994:Recombinant Proteins; C004742:daidzein; D003577:Cytochrome P-450 Enzyme System