Stability engineering of sucrose synthase for robust UDP-glucose regeneration
Glycosyltransferase-driven glycosylation enables environmentally mild synthesis of high-value chemicals, but industrial implementation is constrained by the cost of UDP-glucose. Sucrose synthase (SuSy) offers an attractive route for UDP-glucose recycling, yet inadequate operational stability has limited its use. Here, we report an integrated engineering workflow that overcomes the longstanding trade-off between efficiency and robustness in glycosyl donor recycling enzymes. We engineered GmSuSy wild-type and obtained variants combining supra-wildtype activity (178%) with enhanced thermostability ({Delta}Tmapp = 13.3 {degrees}C), solvent tolerance (70% retained activity in 25% DMSO) and a 123-fold longer half-life. The variants achieved total turnover numbers of [~]1 million (60 {degrees}C), supporting their industrial relevance. Mechanistic analyses revealed that long-range residue communication networks couple oligomeric interfaces with active sites, shifting conformational populations toward stable, catalytically competent states while increasing hydrophobic packing and reducing solvent accessibility. This enhanced robustness enabled significant process-level gains, including >90% conversion yields in indoxyl and MANT glycosylation. Techno-economic and life-cycle assessments indicate the potential to halve reaction costs and reduce environmental impacts threefold, establishing SuSy robustness as a key lever for sustainable industrial glycosylation.