The hydroxylations of the steroid skeleton structure are catalyzed by a family of enzymes, the cytochromes P450 (CYPs). In this study, the pCB1004-PgpdA plasmid was used for cloning the cytochrome P450 reductase (CPR) gene from Rhizopus oryzae into Rhizopus nigericans to strengthen the expression of CPR gene in R. nigericans with REMI (Restriction Enzyme Mediate Integration) mediated protoplast transformation. The conditions for the protoplast production of R. nigericans were optimized as follows: 75 mu g/mL yatalase, 50 mu g/mL lywallzyme, fungus age of 12 h, digestion time of 3 h and digestion temperature of 30 degrees C. REMI mediated protoplast transformation with plasmid pCB1004-PgpdA into R. nigericans was performed to construct the transformants. More than 30 transformants were successfully selected from the hygromycin B-resistant plates and 6 transformants had the abilities to improve the biotransformation of 16 alpha, 17-epoxyprogesterone. The highest biotransformation rate of the transformants was 65.38%, which was 7.06% higher than that of the original strain. (C) 2014 Elsevier Inc. All rights reserved.