Computation-aided redesign of C-glycosyltransferases facilitates sustainable biosynthesis of C-glycosides

C-Glycosides are ubiquitous natural products found in plants and microorganisms and are distinguished by diverse pharmacological activities and high metabolic stability, making them attractive scaffolds for drug discovery and development. C-Glycosyltransferases (CGTs) are key biocatalysts for catalyzing the biosynthesis of C-glycosides in a sustainable manner. However, most CGTs suffer from weak stability, which has significantly limited their industrial application. In this study, we developed a computationally assisted multidimensional strategy (CASMDS) to identify key residues governing the stability of C-glycosyltransferase UGT708B4. A hyperstable mutant, Mut10, harboring ten mutations was obtained, exhibiting a 15 056-fold increase in thermostability, associated with a significant improvement in organic solvent tolerance and acidic stability. Crystal structural analysis and residue interaction network analysis revealed that stepwise network expansion in Mut10 substantially increased the total number of residue interactions. Molecular dynamics (MD) simulations further demonstrated that the stable hydrogen bond network and enhanced rigidity in flexible regions were responsible for the improved stability of Mut10. Finally, by coupling the thermostable Mut10 enzyme with a UDP recycling system, we established a high-temperature biocatalytic C-glycosylation platform that achieved a record space–time yield per unit catalyst of 12.95 mM h⁻¹ g⁻¹ for the synthesis of phloretin di-C-glucoside, with 100% conversion, 97.12% selectivity and a markedly shortened synthesis cycle from 20 h to 3 h. This platform represents an efficient and sustainable biosynthetic route for phloretin di-C-glucoside and can be readily extended to the synthesis of other C-glycosides.