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Article Abstract
Glycoside phosphorylases (GPases) enable oligosaccharide assembly using sugar-1-phosphate donors, but ATP dependency for kinase-mediated phosphorylation limits practicality. Here, a polyphosphate kinase (PPK)-coupled ATP regeneration system is introduced, requiring only <0.05 equiv of AMP to synthesize diverse oligosaccharides (40-92% yields) from monosaccharides and polyphosphate. By integrating PPK with GPases and sugar 1-kinases, lacto--biose I, galacto--biose, -glycan core trisaccharides, and β-1,2/3/4-mannosides were efficiently produced in one-pot reactions. This ATP-free strategy eliminates exogenous nucleotide costs, circumvents product inhibition, and demonstrates broad compatibility with GPases targeting galactosides, glucosaminides, and mannosides, offering a scalable and cost-efficient enzymatic platform.
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