Combining an Enhanced\nPolyphosphate Kinase-Driven\nUDP-Glucose Regeneration System with the Screening of Key Glycosyltransferases\nfor Efficient <i>In Vitro</i> Synthesis of Nucleoside Disaccharides

Nucleoside disaccharides are essential glycosides that\nnaturally\noccur in specific living organisms. This study developed an enhanced\nUDP-glucose regeneration system to facilitate the <i>in vitro</i> multienzyme synthesis of nucleoside disaccharides by integrating\nit with nucleoside-specific glycosyltransferases. The system utilizes\nmaltodextrin and polyphosphate as cost-effective substrates for UDP-glucose\nsupply, catalyzed by α-glucan phosphorylase (αGP) and\nUDP-glucose pyrophosphorylase (UGP). To address the low activity of\nknown polyphosphate kinases (PPKs) in the UDP phosphorylation reaction,\na sequence-driven screening identified RhPPK with high activity against\nUDP (>1000 U/mg). Computational design further led to the creation\nof a double mutant with a 2566-fold increase in thermostability at\n50 °C. The enhanced UDP-glucose regeneration system increased\nthe production rate of nucleoside disaccharide synthesis by 25-fold.\nIn addition, our UDP-glucose regeneration system is expected to be\napplied to other glycosyl transfer reactions.