Schmitz, BenjaminBenjaminSchmitzSharma, ShubhangShubhangSharmaOhde, DanielDanielOhdeLiese, AndreasAndreasLieseElling, LotharLotharElling2026-07-172026-07-172026-06-26New biotechnology 94: 253-263 (2026)https://hdl.handle.net/11420/63924UDP-GlcNAc and UDP-GalNAc are well-demanded nucleotide sugars and serve as precursors for the enzymatic synthesis of N- and O-glycans as well as glycosaminoglycans. Although the enzymatic synthesis cascade has been extensively studied, limitations remain for an efficient large-scale production. Unfavorable inhibitions by intermediate accumulation, precipitation of the byproduct phosphate with the needed cofactor magnesium, and efficient enzyme dosage still pose significant challenges. These challenges were investigated in an enzyme cascade including N -acetyl-hexosamine kinase from Bifidobacterium longum ( Bl NahK), UDP- N -acetylgalactosamine diphosphorylase from Homo sapiens ( Hs AGX1), and inorganic pyrophosphatase from Pasteurella multocida ( Pm PpA) for UDP-GlcNAc and UDP-GalNAc synthesis. ATP supply started from AMP and polyphosphate by polyphosphate kinase from Cytophaga hutchinsonii ( Ch PPK), and UTP was generated from UMP using cytidine/uridine monophosphate kinase from Escherichia coli ( Ec CMPK) and cytidine/uridine diphosphate kinase from Saccharomyces cerevisiae ( Sc CDPK). To reach a high productivity, enzyme cascade parameters were determined and optimized using Multiplex capillary electrophoresis. By kinetic modelling of the enzyme cascade and fine-tuning of the uncoupled UTP/ATP generation/regeneration system as well as observing the magnesium polyphosphate interplay with enzyme activity we were able to scale the production up to a molarity of 100 mM in 200 mL, yielding 10 g of UDP-GlcNAc with a conversion of 82.4% in 48 h and 12 g of UDP-GalNAc with full conversion in 29 h. The key limitations for successful scale-up, namely the promiscuity of Bl NahK towards UTP and interplay of magnesium ions with phosphate species, were overcome. Overall, the presented insights are expected to be transferable to similar enzyme cascades.en1871-67842026253263Elsevierhttps://creativecommons.org/licenses/by/4.0/Nucleotide sugarsMulti-enzyme cascadeCofactor regenerationUDP-GlcNAc synthesisUDP-GalNAc synthesisNatural Sciences and Mathematics::572: BiochemistryJournal Article2026-07-16doi:10.1016/j.nbt.2026.06.00810.15480/882.17490