Protein Engineering of Glucosylglycerol Phosphorylase Facilitating Efficient and Highly Regio- and Stereoselective Glycosylation of Polyols in a Synthetic System

O-Glycosylation of polyols attracts great interest in manufacturing chiral polyol glucosides available in cosmetics and detergent industries. Achieving synchronously controlled regio- and stereoselectivities in glycosylation of polyols remains challenging. Here, we discovered that the glucosylglycerol phosphorylase from Marinobacter adhaerens (MaGGP) presented broad catalytic promiscuity in glycosylation of various polyols, particularly for high carbon 1,2-diols, with excellent regioselectivity and stereoselectivity. Then, we overcame the well-known activity–stability trade-off in enzyme engineering and simultaneously improved thermostability and catalytic efficiency of MaGGP by 1200- and 13.7-fold, respectively. Crystal structure analysis and molecular dynamics simulation revealed the origin of enhanced thermostability and catalytic efficiency. Furthermore, we presented an ATP-free and thermodynamically favorable multienzyme system for polyol glycosylation with inexpensive starch as a glycosyl donor and successfully synthesized several polyol glucosides with high conversion yield. This efficient synthetic platform overcomes the reaction equilibrium and low atom economies of conventional biocatalytic methods and provides the promise in manufacturing other diversified valuable chiral 1,2-cis-glycosides.