One-pot sustainable synthesis of glucosylglycerate from starch and glycerol through artificial in vitro enzymatic cascade

Glucosylglycerate (R-2-O-α-D-glucopyranosyl-glycerate, GG) is a negatively charged compatible solution with versatile functions. Here, an artificial in vitro enzymatic cascade was designed to feasibly and sustainably produce GG from affordable starch and glycerol. First, Spirochaeta thermophila glucosylglycerate phosphorylase (GGP) was carefully selected because of its excellent heterologous expression, specific activity, and thermostability. The optimized two-enzyme cascade, consisting of alpha-glucan phosphorylase (αGP) and GGP, achieved a remarkable 81 % conversion rate from maltodextrin and D-glycerate. Scaling up this cascade resulted in a practical concentration of 58 g/L GG with a 62 % conversion rate based on the added D-glycerate. Additionally, the production of GG from inexpensive starch and glycerol in one-pot using artificial four-enzyme cascade was successfully implemented, which integrates alditol oxidase and catalase with αGP and GGP. Collectively, this sustainable enzymatic cascade demonstrates the feasibility of the practical synthesis of GG and has the potential to produce other glycosides using the phosphorylase-and-phosphorylase paradigm.