Engineering of a Fungal Steroid 11α-hydroxylase and Construction of Recombinant Yeast for Improved Production of 11α-hydroxyprogesterone

Cytochrome P450 enzyme CYP68J5 from filamentous fungus Aspergillus ochraceus is industrially used for selective C11α-hydroxylation of canrenone and progesterone. To improve its selectivity of C11α-hydroxylation for relevant steroid substrates, a sequence-based targeted mutagenesis combined with saturation mutagenesis was conducted to search for variants with improved hydroxylation reaction specificity toward progesterone and D-ethylgonendione. Recombinant yeast expressing triple mutant V64F/E65G/N66T showed significantly increased C11α-hydroxylation selectivity (85 % VS WT 69.7 %). Saturation mutagenesis of V64, E65 and N66 resulted in the identification of single mutant V64K with greatly enhanced 11α-hydroxylation specificity toward progesterone (90.6 % VS WT 69.7 %). Furthermore, mutant N66D showed significant enhanced selectivity of C11α-hydroxylation toward D-ethylgonendione (70.8 % VS WT 58 %). Evaluation of recombinant yeast over-expressing V64K for progesterone transformation in 50 mL scale resulted in product 11α-OH progesterone concentrations of 432.5 mg/L, a 30.2 % increase compared with the CYP68J5 control. Our results also reveal that V64, E65 and N66 are key residues of CYP68J5 influencing its selectivity of C11α-hydroxylation, thus offering opportunities for further engineering of CYP68J5 for expanded industrial applications.