Multi-enzyme cascade developed for UDP-GalNAc synthesis from inexpensive substrates.

Design of Experiments approach optimized reaction parameters in two stages.

Percentage yield increased from 5 % to 95 %, with final titer of 46.1 mM (28 g/L).

19-fold improvement in UDP-GalNAc yield achieved through systematic optimization.

Anion exchange chromatography purification yielded up to 89 % recovery, 90 % purity.

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 (PolyPn). Two rounds of Design of Experiments (DoE) optimization were performed to systematically evaluate and optimize reaction parameters including pH, temperature, MgCl2, ATP, and PolyPn 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.

UDP-GalNAc synthesis

Multi-enzyme cascade

Design of Experiments (DoE)

Nucleotide sugar production

Glycobiotechnology

© 2025 The Author(s). Published by Elsevier B.V.