Nucleotide sugars (NS) are highly requested products in research and industry but only of limited availability. Those sugars are mainly produced by chemical synthesis or in microorganisms. However, enzymatic synthesis by salvage pathway is an alternative. It allows the stereoselective synthesis of NS from a monosaccharide-1-P (Acceptor) and a nucleotide (Donor) after the C1 phosphorylation of a sugar by a kinase. The aim of this thesis was the establishment of enzyme cascades to produce NS in the multigram-scale. For that, enzymes from the salvage pathway were combined in one-pot cascades. For production, enzyme cascades were separated from the product by filtration and fed with new substrate to repeat the production (repetitive batch). The one-pot cascade reactions were optimized in a small scale (<600 μL) and applied to the repetitive batch (rep.-batch). By this procedure, 1.6 g GDP-Fuc, 2.6 g UDP-6-azido-GalNAc and 1.4 g GDP-Man were produced. The production of NS was further developed in a continuous fed-batch membrane reactor. Here, the enzyme cascades were optimized in small-scale batch reactions (<600 μL) to match the average residence time of the reactor (τ=24-30 min). By the continuous fed-batch membrane reactor, 8.2 g CMP-Neu5Ac, 14.6 g UDP-GalNAc, 2.5 g UDP-Gal, 1.4 g GDP-Man und 14.8 g UDP-GlcA were produced. This process was further successfully filed as an international patent (WO2025132321A1). For downstream processing of NS a precipitation method was established and the NS subsequently by the industrial cooperation partners. For that, orthophosphates (Pi) were precipitated in 40 % (v/v) isopropanol at -20 °C, while the NS remain in solution. Afterward, the content of isopropanol is increased to 80% (v/v) to precipitate the NS at -20 °C. The precipitation was applied to a GDP-Fuc and UDP-GlcA raw solution and gained 77% and 56% recovery, respectively. Finally, a procedure for the regeneration of ATP was developed and evaluated, using only sucrose and pyrophosphate (PPi), and applied to the production of UDP-GlcNAc, GDP-Fuc and CMP-Neu5Ac. The method for the ATP regeneration was filed as a international patent (WO2021219463A1).
Hannes Frohnmeyer (Wed,) studied this question.