Cell-Free Biocatalytic Processes for the Synthesis and Purification of Nucleotide Sugars

Cell-Free Biocatalytic Processes for the Synthesis and Purification of Nucleotide Sugars

Motivation

The presence and structures of glycans play a crucial role in cellular life and functioning of glycoproteins, e.g. cell-cell recognition, pharmacokinetics, physical stability and immunogenicity. However, there is a lack of thorough understanding of the structure-function relationships of glycans due to macro- and micro-heterogeneities of biological samples. In vitro biosynthetic techniques have the potential to overcome challenges associated with cell culture methods to produce proteins with defined glycoforms. To in vitro glycoengineer oligosaccharides and to build glycopeptides and glycoproteins with defined glycan structures, nucleotide sugars are needed as building blocks. However, the availability of sugar nucleotides is limited by expensive synthesis processes, i.e. purchase prices for 1 gram typically exceed 1000 €. Accordingly, the cost-efficient synthesis of sugar nucleotides is essential for the development synthetic glycoengineering platforms.

Aim of the project

Aim of the project is the efficient synthesis and purification of nucleotide sugars, such as UDP-GlcNAc and UDP-Glucose.  Various production systems such as E. coli and B. subtilis are used for the synthesis of biocatalysts. Moreover, various bioreactor systems are setup and studied for their efficacy for nucleotide sugar production. Downstream processing methods based on ion and reversed chromatography are established for robust and scalable sugar nucleotide purification.

References

Mahour, R.; Marichal-Gallardo, P.; Rexer, T.; Reichl, U.: Multi-enzyme cascades for the in vitro synthesis of guanosine diphosphate L-fucose. ChemCatChem 13 (8), pp. 1981 - 1989 (2021)
Mahour, R.; Klapproth, J.; Rexer, T.; Schildbach, A.; Klamt, S.; Pietzsch, M.; Rapp, E.; Reichl, U.: Establishment of a five-enzyme cell-free cascade for the synthesis of uridine diphosphate N-acetylglucosamine. Journal of Biotechnology 283, pp. 120 - 129 (2018)
Rexer, T.; Schildbach, A.; Klapproth, J.; Schierhorn, A.; Mahour, R.; Pietzsch, M.; Rapp, E.; Reichl, U.: One pot synthesis of GDP-mannose by a multi-enzyme cascade for enzymatic assembly of lipid-linked oligosaccharides. Biotechnology and Bioengineering 115 (1), pp. 192 - 205 (2018)

Patents

Rexer, T.; Mahour, R.: Enzymatic method for preparation of GDP-fucose. EP 3719135A1 (2020)
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