The Coupling of a Universal Polyphosphate Kinase and Glycosyltransferases Enabled In Situ Regeneration of Sugar Nucleotides for Efficient Synthesis of Functional Glycans

Chemoenzymatic assembly of complex glycans is hindered by the high cost of sugar nucleotides (SNs) and product inhibition from released nucleoside phosphates. Here, we report a versatile polyphosphate-driven regeneration system that overcomes these limitations. Central to this system is the class III polyphosphate kinase EbPPK, which converts all four canonical nucleoside monophosphates (NMPs) into nucleoside triphosphates (NTPs) in situ, permitting one-pot cascades that start from low-cost NMPs supplied at either catalytic or near-stoichiometric levels. Except for GDP-Fuc and UDP-GlcA, regeneration of UDP-Gal, UDP-GalNAc, UDP-GlcNAc and CMP-Sia demands only uridine monophosphates (UMP) or cytidine monophosphate (CMP) as a sole cofactor, thereby eliminating the need for adenine nucleotides. Coupling EbPPK with salvage enzymes and Leloir glycosyltransferases created a three-module platform that furnished gram quantities of tumor-associated globo-series glycosphingolipids and five representative human-milk oligosaccharides in isolated yields of 71–98% with cofactor total turnover numbers up to 76 and space-time yields approaching 5.1 g L –1 h –1 . The same strategy was extended to medium-molecular-weight hyaluronic acid, delivering 1.2 g at 5.9 g L –1 . The approach relies on inexpensive polyphosphate and monosaccharide inputs, avoids extra kinases and is readily scalable, offering a cost-effective route to complex carbohydrates for biomedical and nutritional applications.