Establishment of a cell-free multi-enzyme cascade for the synthesis of UDP-GalNAc

UDP-N-acetylgalactosamine (UDP-GalNAc) is an essential building block in the synthesis of glycans including O-glycans and glycosaminoglycans. For the latter, enzymatic synthesis is often a promising approach for producing multi-gram amounts. However, the high cost of UDP-GalNAc has limited this approach. This study reports on the development and optimization of a multi-enzyme cascade for synthesizing UDP-GalNAc from inexpensive substrates. Consisting of six recombinant enzymes, the cascade converts uridine (Uri) and GalNAc to UDP-GalNAc with in situ ATP regeneration using polyphosphate (PolyP_n). Two rounds of Design of Experiments (DoE) optimization were performed to systematically evaluate and optimize reaction parameters including pH, temperature, MgCl₂, ATP, and PolyP_n concentrations. The established cascade achieved a percentage yield of 95 % and an UDP-GalNAc titer of 46.1 mM (28 g/L). This represented a 19-fold improvement over the initial conditions. Purification by anion-exchange chromatography yielded a maximum recovery of 89 % and a purity of 90 %. Overall, this scalable, low-cost enzymatic synthesis of UDP-GalNAc overcomes current limitations in availability and cost, potentially enabling new applications in the field of glycobiotechnology.