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

Abstract

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.

Enhanced Thermostability of an l-Rhamnose Isomerase for d-Allose Synthesis by Computation-Based Rational Redesign of Flexible Regions

“…These results indicated that the G57H mutation resulted in an enhancement of hydrogen-bonding interactions, which was conducive to increasing the rigidity of this part of the enzyme. The distinctive conformation of the proline conformation is attributed to the pyrrolidine ring with low conformation freedom. − The G238P mutation therefore also enhanced the rigidity of the local loop, which would be conducive to stabilizing its conformation. Additionally, given the higher space steric hindrance of proline, it induced a change in the conformation of the surrounding residue.…”

Directed Evolution of the UDP-Glycosyltransferase UGTBL1 for Highly Regioselective and Efficient Biosynthesis of Natural Phenolic Glycosides

“…Simultaneous improvements in the catalytic efficiency and regioselectivity of UGTs remain a challenge. The insufficiently clear catalytic mechanism of UGTs and the poor regioselectivity limit the application of UGTs in the targeted synthesis of specific glycoside. − …”