In Silico Enzyme Engineering of Thermostable UDP-Glycosyltransferases and Sucrose Synthase for Industrial Glycosylation

Abstract Uridine diphosphate-dependent glycosyltransferases (UGTs) coupled with sucrose synthase (SuSy) offer a selective and highly efficient route to natural product glycosylation. However, harnessing the full potential of these biocatalysts at industrial scale is challenged by their low operational stability. Enzyme mining and engineering offer a powerful strategy to address this challenge. A vast array of in silico tools supports this effort, which use sequence, structure, and thermodynamic knowledge to discover, predict and screen thermostabilized variants. In this review, we discuss the recent applications of those tools to improve the thermostability of UGTs and SuSy jointly, and their resulting impact on UGT-SuSy glycosylation cascades. We highlight integrated methodological approaches that predict and filter candidate mutations, allowing the reduction of false positives without sacrificing activity. These efforts have substantially advanced UGT thermostability, with melting temperatures reaching up to 90 °C and half-lives extending to several days at 60 °C. Comparatively fewer studies, however, have applied similar strategies to SuSy, and none have yet achieved the magnitude of improvement reported for UGTs. This asymmetry positions SuSy as a comparatively underexplored target, emphasizing the need for further mining and engineering to obtain stable variants that perform alongside UGTs under industrial conditions.