Cortexolone 11 β-hydroxylation in protoplasts of Curvularia lunata

Transformation of cortexolone to hydrocortisone by the filamentous fungus Curvularia lunata using protoplasts as a research tool was studied. A stable mutant of C. lunata (IM 2901/366) hydroxylated cortexolone at 11 β-position with significantly higher activity than the wild strain (IM 2901) at the similar rate of growth. Protoplasts released from the mycelium of the wild strain and the mutant transformed the steroid substrate with about four and over five times higher yield than the parental mycelium (respectively). Due to the higher hydroxylation ability of the mutant, the activity of mutant protoplasts was 24 times higher than the activity of the mycelium of the wild strain. Hydroxylation of cortexolone was blocked in both strains by the inhibitor of cytochrome P-450 (cyt P-450) activity, ketoconazole. Cyt P-450, as hemoprotein involved in steroid-hydroxylating system, was localized in microsomal fractions of both strains. Estimation of cyt P-450 content in the intact protoplasts revealed that the level of cyt P-450 in the mutant cells was higher and it increased more rapidly in steroid presence than in the wild strain cells. It is suggested that the higher amount of cyt P-450 and higher steroid transformation activity of the mutant result from mutation(s) of genes involved in steroid 11 β-hydroxylase synthesis regulation.