Constructing an Intensified UDP Recycling System for the Glycosylation of Natural Products by Phosphorylation of Byproduct Fructose.

UDP-glucosyltransferase can be coupled with sucrose synthase to construct a two-enzyme UDP (UDP-2E) recycling system for glucosylation of natural products with inexpensive sucrose as the consumed substrate. However, sucrose hydrolysis leads to the accumulation of fructose as a byproduct, which decreases the atom economy of sucrose and suppresses in situ UDP recycling. In this study, a polyphosphate-dependent glucokinase was demonstrated to convert fructose to fructose-6-phosphate independent of expensive ATP for the first time. Then the glucokinase was introduced into the UDP-2E recycling system to construct a modified three-enzyme UDP (UDP-3E) recycling system, which showed enhanced glucosylation efficiency of triterpenoids by fructose phosphorylation to accelerate sucrose hydrolysis and UDP recycling. Finally, by further introducing a phosphofructokinase into the UDP-3E recycling system, we transformed fructose-6-phosphate into fructose-1,6-diphosphate, demonstrating that the UDP-3E recycling system can be coupled with extra enzymes to obtain final products with high added-value without compromising the glycosylation efficiency.