The C11α-hydroxyl group is essential for many steroid drugs, yet chemical synthesis suffers from complex routes and environmental issues. Here, we isolate CYP68N3ₘa, a fungal cytochrome P450 from Metarhizium anisopliae EEG016, which converts 17α-hydroxyprogesterone to 11α, 17α-dihydroxyprogesterone. Expressing CYP68N3ₘa in Saccharomyces cerevisiae enabled the highly specific bioconversion with a concentration of 0. 16 mM. Structure-guided iterative saturation mutagenesis yielded the triple mutant N3M3 (F111A/E374H/T115I), increasing the concentration 13. 8-fold to 2. 21 mM. Molecular dynamics and near-attack conformation analysis revealed cooperative modulation of steric hindrance and hydrogen bonding that fine-tunes substrate orientation and enhances the near-attack conformation (NAC) formation. Expression in Komagataella phaffii improved conversion 3. 8-fold over that in S. cerevisiae. Overexpression of heme synthesis enzyme HEM1, cytochrome b5, an ABC transporter, and knockout of HMX1 resulted in 10. 6 g/L in flask and 24. 8 g/L in high-cell density bioreactor. This study establishes a versatile, sustainable, and scalable biocatalytic route for steroid functionalization.
Wei et al. (Tue,) studied this question.