Efficient steroid hydroxylation using whole-cell biocatalysts

Abstract

Hydroxylated steroids enjoy high priority in the pharmaceutical industry, requiring highly selective hydroxylation processes. This thesis investigates the self-sufficient Cytochrome P450 monooxygenase BM3 variant KSA14m in recombinant Escherichia coli whole-cell biocatalysts for testosterone hydroxylation. Limited substrate uptake through the outer membrane was overcome by co-expression of the hydrophobic pore AlkL, increasing activity up to unprecedented levels. Further studies revealed enzyme instability, product inhibition, and poor substrate solubility as key factors limiting productivity. Resting cells showed the best performance and were successfully applied in a stirred-tank reactor. Product titers reached the gram-per-liter scale, with a maximum of 3.6 g L-1 obtained within 4 h. This work demonstrates the potential of BM3/AlkL-based whole-cell biocatalysts for efficient steroid hydroxylation and provides a basis for future process optimization and industrial application.