Divergent evolution of fungal P450 monooxygenase unlocks simultaneous access to C12β and C15α oxyfunctionalization of steroids.

Steroidal C12β/15α-hydroxylation are pivotal in synthesizing steroid drugs but remain challenging via chemical and biological methods. To address this, structure-guided divergent evolution is applied to the fungal P450 monooxygenase CYP68J5_fg. Two optimized variants, W12M5 (F107S/Q112R/N295T/V299T/R368K) and W15M4 (Q112C/D126V/V299L/A362M) are created, achieving high selectivity (97.7% for C12β- and 99.6% for C15α-hydroxylation of progesterone) alongside enhanced catalytic efficiency, effectively overcoming the classic activity-selectivity trade-off. Molecular dynamics simulations reveal that key mutations reorient the substrate by reshaping the binding pocket's polarity and hydrogen-bonding network, enabling hydroxylation at distinct positions. High-density fermentation with engineered Pichia pastoris yields titers of 4.6 g/L 12β-OH progesterone, 10.9 g/L 15α-OH progesterone and 14.1 g/L 15α-OH androstenedione. These products serve as key intermediates for streamlined synthesis of C12-/C15-functionalized steroids such as drospirenone and C-nor-D-homo derivatives. Collectively, this study demonstrates the successful divergent evolution of a fungal P450, a strategy which has so far not been reported in the literature, highlights its broad applicability for the scalable synthesis of complex bioactive molecules.