Strategies for modulating transglycosylation activity, substrate specificity, and product polymerization degree of engineered transglycosylases

Glycosides are widely used in many fields due to their favorable biological activity. The traditional plant extractions and chemical methods for glycosides production are limited by environmentally unfriendly, laborious protecting group strategies and low yields. Alternatively, enzymatic glycosylation has drawn special attention due to its mild reaction conditions, high catalytic efficiency, and specific stereo-/regioselectivity. Glycosyltransferases (GTs) and retaining glycoside hydrolases (rGHs) are two major enzymes for the formation of glycosidic linkages. Therein GTs generally use nucleotide phosphate activated donors. In contrast, GHs can use broader simple and affordable glycosyl donors, showing great potential in industrial applications. However, most rGHs mainly show hydrolysis activity and only a few rGHs, namely non-Leloir transglycosylases (TGs), innately present strong transglycosylation activities. To address this problem, various strategies have recently been developed to successfully tailor rGHs to alleviate their hydrolysis activity and obtain the engineered TGs. This review summarizes the current modification strategies in TGs engineering, with a special focus on transglycosylation activity enhancement, substrate specificity modulation, and product polymerization degree distribution, which provides a reference for exploiting the transglycosylation potentials of rGHs.