Expanding Steroid Glycodiversity: Tandem Steroid Glucosylation and Acetylation via Enzymatic Cascade

Steroid glycosides constitute an important class of bioactive molecules, yet their selective synthesis remains challenging. Here, we established a screening platform for nucleotide sugar-dependent glycosyltransferases (GTs) coupled with sucrose synthase (SuSy) for in situ UDP-glucose regeneration, enabling cost-efficient steroid glucosylation. A library of GTs comprising literature-derived enzymes and newly mined archaeal and fungal candidates was constructed using sequence filtering, AlphaFold3 modeling, and docking-guided prioritization. The resulting panel was screened against 31 structurally diverse steroids (androgens, estrogens, pregnanes, and corticosteroids) using crude Escherichia coli lysates as catalysts and UPLC-DAD, LC-MS and NMR analytics. YjiC and OleD glycosyltransferases emerged as the most promiscuous biocatalysts, while Sbaic7OGT and SgUGT74AC1_M7 displayed greater selectivity toward estrogens and selected testosterone derivatives. Product assignment for representative reactions was validated using authenticated reference standards or NMR (1D/2D) analysis, confirming regioisomeric estradiol monoglucosides (3-O- and 17-O-), estrone 3-O-glucoside, and an unexpected product diversification for 17α-testosterone by endogenous E. coli enzyme, where the major product was identified as a 6'-O-acetylated glucoside. Finally, SuSy-coupled cascades were applied in semi-preparative scale and evaluated under optimized conditions and co-immobilization formats.