Tuan Hoang Son

Wiss. Mitarbeiter/-in

Lehrstuhl Bioprozesstechnik
Vita

Title: Biocatalytic synthesis of nucleotide sugars and functional oligosaccharides

Motivation

Human breast milk consists of various functional biomolecules including lactose, lipids and human milk oligosaccharides (HMOs). The latter are associated with a number of benefits for infants, such as a healthy gut flora or the promoting of brain development. HMOs also support the infant's immune system. While breastfeeding of babies provides the best source of nutrition, many infants require supplementation with formula containing HMOs. In case breastfeed is not possible, infant formula containing HMOs can be a healthy alternative. HMOs are typically produced by microbial fermentation or chemical synthesis.  Enzymatic synthesis of HMOs is a promising alternative to complement existing methods and expand the portfolio of available HMOs for functional studies. However, the enzymatic synthesis of functional oligosaccharides like HMOs is limited by the price and availability of their building blocks - nucleotide sugars (Rexer et al., 2021; Zeuner et al., 2019). To overcome these bottlenecks, we have successfully developed in-vitro multi-enzyme cascades consisting of up to six recombinant enzymes to produce efficiently the nucleotide sugars UDP-N-acetylglucosamine (UDP-GlcNAc), UDP-galactose (UDP-Gal) and UDP-N-acetylgalactosamine (UDP-GalNAc) from inexpensive precursors (Mahour et al., 2018; Mahour et al., 2022).

 

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Figure 1: Schematic process workflow for the synthesis of nucleotide sugars: Production of biocatalysts (enzymes), biotransformation for nucleotide sugar synthesis and downstream processing for purification. The pure building blocks can be used for the synthesis of functional oligosaccharides, like HMOs and glycoconjugates.

 

Aim of the project

This project aims to develop production processes for nucleotide sugars up to the provision in their pure form (Figure 1). So far, the synthesis of nucleotide sugars has been carried out at small scales in Eppendorf tubes to produce milligram quantities. In order to enable the production of gram to kilogram amounts, an optimization of the productivity of the multi-enzyme cascades and a scale-up into stirred tank bioreactors is targeted. E. coli strains are designed to simplify the biocatalyst production. Using mathematical models, the impact of key reaction parameters such as pH value, temperature, enzyme and cofactor concentrations on the biotransformation is systematically studied to identify optimal reaction conditions.

Moreover, an efficient purification process is developed for the removal of proteins, endotoxins, intermediates and salts to ensure a high purity and quality of the activated building blocks. Freeze-drying or crystallization are considered as a polishing step.

In parallel, different glycosyltransferases are screened for synthesis of specific HMOs. This includes multiple enzyme variants and different expression strategies to obtain active and soluble enzyme forms.

 

Outlook

The affordable provision of nucleotide sugars could pave the way towards an economically viable enzymatic synthesis of functional oligosaccharides for use in the fields of nutrition and biopharma. In addition to these building blocks, efficient and scalable (bio-)processes are required. Mathematical modelling approaches support the optimization of critical process steps, in particular the biotransformation, which is based on multi-enzyme cascades, where a multitude of parameters affect productivity and yields.

 

References

Mahour, R., Klapproth, J., Rexer, T. F. T., Schildbach, A., Klamt, S., Pietzsch, M., . . . Reichl, U. (2018). Establishment of a Five-Enzyme Cell-Free Cascade for the Synthesis of Uridine Diphosphate N-Acetylglucosamine. Journal of Biotechnology, 283, 120-129. doi:doi.org/10.1016/j.jbiotec.2018.07.027

 

Mahour, R., Lee, J. W., Grimpe, P., Boecker, S., Grote, V., Klamt, S., . . . Reichl, U. (2022). Cell-Free Multi-Enzyme Synthesis and Purification of Uridine Diphosphate Galactose. ChemBioChem, 23(2), e202100361. doi:doi.org/10.1002/cbic.202100361

 

Rexer, T., Laaf, D., Gottschalk, J., Frohnmeyer, H., Rapp, E., & Elling, L. (2021). Enzymatic Synthesis of Glycans and Glycoconjugates. In E. Rapp & U. Reichl (Eds.), Advances in Glycobiotechnology (pp. 231-280). Cham: Springer International Publishing.

 

Zeuner, B., Teze, D., Muschiol, J., & Meyer, A. S. (2019). Synthesis of Human Milk Oligosaccharides: Protein Engineering Strategies for Improved Enzymatic Transglycosylation. Molecules, 24(11). doi:10.3390/molecules24112033

Letzte Änderung: 17.01.2024 - Ansprechpartner: Webmaster