Stereoselective oxyfunctionalization of steroids by designed peroxygenase mutants: Bridging batch screening and continuous operation

Engineered peroxygenase-mode P450s that use H2O2 as the terminal oxidant offer a simplified route to stereoselective C-H oxyfunctionalization without redox partner proteins or nicotinamide cofactors. Here, we report the development of a hydrogen peroxide-driven cytochrome P450-based process for the stereoselective C16α-hydroxylation of progesterone, combining enzyme engineering, solubility optimization, and continuous operation in an enzyme membrane reactor (EMR). Among several engineered CYP154C8 variants, the QE double variant exhibited superior catalytic performance and remarkable stability under elevated hydrogen peroxide concentrations in batch experiments, enabling product yields of up to ∼90% at substrate concentrations limited only by aqueous solubility. Translation of the optimised conditions to continuous flow demonstrated stable operation over extended residence times, highlighting the advantages of controlled hydrogen peroxide dosing, short residence times, and physical enzyme retention in mitigating oxidative deactivation. To overcome substrate solubility limitations, β-cyclodextrin and 2-hydroxypropyl-β-cyclodextrin (HP-β-CD) were evaluated as solubilizing agents, yielding enhanced progesterone solubility via 1:1 inclusion complex formation. Overall, this study demonstrates a robust and scalable strategy for continuous peroxygenase-catalyzed steroid hydroxylation, providing a foundation for further process intensification and industrial implementation.