Engineering of daidzein 3’-hydroxylase P450 enzyme into catalytically self-sufficient cytochrome P450
1 School of Chemical and Biological Engineering, Institute of Bioengineering, Seoul National University, Seoul, South Korea
2 School of Biotechnology, Yeungnam University, Gyeongsan, Gyeongbuk, South Korea
3 Interdisciplinary Program for Bioengineering, Seoul National University, Seoul, 151-744, Republic of Korea
4 Korea Bio-Hub Center, Bio-MAX Institute, Seoul National University, Seoul, South Korea
Microbial Cell Factories 2012, 11:81 doi:10.1186/1475-2859-11-81Published: 14 June 2012
A cytochrome P450 (CYP) enzyme, 3’-daidzein hydroxylase, CYP105D7 (3’-DH), responsible for daidzein hydroxylation at the 3’-position, was recently reported. CYP105D7 (3’-DH) is a class I type of CYP that requires electrons provided through electron transfer proteins such as ferredoxin and ferredoxin reductase. Presently, we constructed an artificial CYP in order to develop a reaction host for the production of a hydroxylated product. Fusion-mediated construction with the reductase domain from self-sufficient CYP102D1 was done to increase electron transfer efficiency and coupling with the oxidative process. An artificial self-sufficient daidzein hydroxylase (3’-ASDH) displayed distinct spectral properties of both flavoprotein and CYP. The fusion enzyme catalyzed hydroxylation of daidzein more efficiently, with a kcat/Km value of 16.8 μM-1 min-1, which was about 24-fold higher than that of the 3’-DH-camA/B reconstituted enzyme. Finally, a recombinant Streptomyces avermitilis host for the expression of 3’-ASDH and production of the hydroxylated product was developed. The conversion that was attained (34.6%) was 5.2-fold higher than that of the wild-type.