Stability engineering of sucrose synthase for robust UDP-glucose regeneration

Sucrose synthase (SuSy) has been suggested as a key enabling enzyme for uridine diphosphate glucose (UDP-Glc) regeneration in glycosyltransferase-catalyzed biotransformations. However, its stability and efficiency in industrially relevant conditions have not been characterized or engineered, limiting its industrial readiness. Here, we combined enzyme discovery and characterization with comprehensive semi-rational enzyme engineering strategies, to optimize SuSys catalytic activity, thermostability, solvent tolerance, and soluble expression. The engineered variants were significantly more stable than wild-type, with up to 13.6 {degrees}C increase in melting temperature, over two orders of magnitude improvement in half-lives at elevated temperatures, and approximately three orders of magnitude increase in total turnover number. Additionally, the optimized variants retained up to 75% relative activity at 60 {degrees}C in the presence of 25% (v/v) DMSO, which the wild-type shows near complete loss of activity. Structural and molecular dynamics analyses reveal how mutations modulate conformational dynamics and hydrophobic packing, favoring catalytically competent conformations. Using methyl anthranilate glycosylation as a representative biotransformation, we demonstrate that the engineered SuSy variants consistently outperform both wild-type SuSy and stoichiometric UDP-Glc systems, enabling efficient UDP-Glc regeneration at reduced enzyme and sugar donor loadings. Finally, techno-economic and environmental assessments further indicate that implementation of engineered SuSy reduces reaction cost by approximately 6- and 2-fold relative to UDP-Glc and wild-type systems, respectively, while achieving average reductions of 3- and 2-fold in environmental end-point impacts. These results established SuSy engineering as a critical enabler for sustainable glycosylation reactions.