Efficient One-Pot Multienzyme Synthesis of β-Glycosides

Phenolic glycoside derivatives hold a significant position in medicine and food science. However, traditional chemical methods for the glycosylation of phenolic compounds require intricate procedures. In this study, we constructed an efficient one-pot multienzyme cascade system for the synthesis of β-glucuronide compounds through directed evolution of glycosyltransferase and optimization of the reaction system. The synthesis system contains three modules, for UDP-sugar synthesis, UTP regeneration, and glycoside synthesis, respectively, which enables large-scale product formation using catalytic amounts of UTP. Our method achieved high yields, with 5.95 g/L of β-glucuronide produced from the model substrate <i>p</i>-nitrophenol, and other glycosides also yielded in the grams-per-liter range. To validate the versatility of this strategy for other types of glycoside compounds, we constructed a one-pot multienzyme catalytic system for the synthesis of β-glucosides by simply substituting the UDP-sugar synthase and glycosyltransferase within the catalytic system. For the model substrate p-nitrophenol, the yield of β-glucoside reached 8.13 g/L, with the yield of other glucoside compounds also in the grams-per-liter range. This research provides an environmentally friendly biosynthesis method for the production of phenolic glycoside compounds.