| Summary: | <p>Abstract</p> <p>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 <it>k</it><sub>cat</sub>/K<sub>m</sub> value of 16.8 μM<sup>-1</sup> min<sup>-1</sup>, which was about 24-fold higher than that of the 3’-DH-camA/B reconstituted enzyme. Finally, a recombinant <it>Streptomyces avermitilis</it> 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.</p>
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