The biotransformation of soy isoflavones into ortho-hydroxyisoflavones by CYP105D7 from Streptomyces avermitilis MA4680 is investigated through chimeric expression in Pichia pastoris. Using N-terminal fusion With the transmembrane domain of CYP57B3 from Aspergillus oryzae and C-terminal fusion with a P450 reductase from Saccharomyces cerevisiae, CYP105D7 was expressed in the form of reductase fusion and membrane anchoring in P. pastoris. Recombinant P. pastoris expressing the chimera catalyzed the biotransformation of both daidzein and genistein. This is the first study to show the catalyzing activity of CYP105D7 towards genistein. The major product from daidzein was identified as 6-hydroxydaidzein by comparing the results of the ultra-performance liquid chromatography analysis with the authentic standard. The major product from genistein was purified using preparative high-performance liquid chromatography and identified as 3'-hydroxygenistein based on nuclear magnetic resonance and mass data. The recombinant P. pastoris produced 6-hydroxydaidzein and 3'-hydroxygenistein in a 5-l fermenter, with maximal yields of 7.5 and 15.0 mg/l, respectively. The production of 3'-hydroxygenistein was higher than any previously reported in the literature. (C) 2015 Taiwan Institute of Chemical Engineers. Published by Elsevier B.V. All rights reserved.