A one-pot multienzyme cofactors recycling (OPME-CR) system was designed for the synthesis of UDP-α-d-galactose, which was combined with LgtB, a β-(1,4) galactosyltransferase from Neisseria meningitidis, to modify various polyphenol glycosides. This system recycles one mole of ADP and one mole of UDP to regenerate one mole of UDP-α-d-galactose by consuming two moles of acetylphosphate and one mole of d-galactose in each cycle. The ATP additionally used to generate UDP from UMP was also recycled at the beginning of the reaction. The engineered cofactors recycling system with LgtB efficiently added a d-galactose unit to a variety of sugar units such as d-glucose, rutinose, and 2-deoxy-d-glucose. The temperature, pH, incubation time, and divalent metal ions for the OPME-CR system were optimized. The maximum number of UDP-α-d-galactose regeneration cycles (RCmax) was 18.24 by fed batch reaction. The engineered system generated natural and non-natural polyphenol saccharides efficiently and cost-effectively.
Keywords: cofactors recycling; glycosylation; lactoside derivatives; one-pot system.