Glucosylglycerol is a powerful osmolyte that has attracted attention as a useful moisturizing ingredient in the cosmetic industry. This study demonstrates two artificially designed synthetic routes for manufacturing glucosylglycerol by combining phosphorolysis and transglycosylation reactions. The overall Gibbs energy change of the synthetic routes was negative, indicating that they are thermodynamically favorable. In vitro biosystems were constructed through combining the phosphorolysis ability of sucrose/maltose phosphorylase and the transglycosylation capacity of glucosylglycerol phosphorylases from different organisms. A near-stoichiometric conversion of sucrose and glycerol with a high product yield of 98% was achieved under optimal reaction conditions. The large-scale glucosylglycerol production of this biosystem was investigated under a high concentration of substrates (2 mol/L sucrose and 2.4 mol/L glycerol), and the titer reached 1.78 mol/L (452 g/L) with a productivity of 24.3 g/L/h. To the best of our knowledge, this value presented the highest glucosylglycerol production level until now, which indicated a great industrial application potential for glucosylglycerol manufacturing.

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.