Exploitation of transglycosylation activities of bifidobacterial glycoside hydrolases for the synthesis of human milk oligosaccharide (HMO) structures

Background.

Bifidobacteria play an important role in the eco-physiology of the colonic microbiota. These species are considered to be important in maintaining human health as they contribute to carbohydrate fermentations in the colon; their diversity and number provide a marker for the stability of the human intestinal microflora. Human milk is known as the sole source of nourishment for breast-fed infants and for its promotion of a healthy development of newborns. Human milk oligosaccharides (HMO) are prominent among the functional components of human milk and selectively support the growth and activity of desired bacteria in the infant intestine, thus they have prebiotic or bifidogenic effects (Bode, 2012). Infant-associated Bifidobacterium species are equipped with genetic and enzymatic sets dedicated to the assimilation of HMO, and they appear to have developed different strategies for degrading HMO, which are then utilized as growth substrates (LoCascio et al., 2007; Sela and Mills, 2010; Zivkovic et al., 2011). Thus, it will be interesting and also challenging to exploit transglycosylation activities of the glycoside hydrolases (GHs) of these species, which are involved in HMO degradation, to synthesize HMO structures.

Aims and methods.

The proposed project aims: (i) to investigate transglycosylation activities and the extent of glycosyl transfer of two selected glycoside hydrolases from the infant gut isolate Bifidobacterium breve, which are a GH20 β-N-acetylhexosaminidase and a GH42 β-galactosidase; (ii) to identify the function roles of the key residues in the active sites of these selected glycoside hydrolases and to engineer these enzymes towards higher transglycosylation activity; (iii) to synthesize the core, the precursor and the backbone structures of HMO, namely lacto-N-biose (LNB; Gal-β-1,3-GlcNAc), N-acetyl-lactosamine (LacNAc; Gal-β-1,4-GlcNAc), lacto-N-triose II (LNT2; GlcNAc-β-1,3-Gal-β-1,4-Glc), lacto-N-tetraose (LNT; Gal-β-1,3-GlcNAc-β-1,3-Gal-β-1,4-Glc) and lacto-N-neo-tetraose (LNnT; Gal-β-1,4-GlcNAc-β-1,3-Gal-β-1,4-Glc) using these glycoside hydrolases.

Figure 1. Structures of the tetrasaccharides lacto-N-tetraose and lacto-N-neo-tetraose

The genes encoding the selected enzymes will be cloned and overexpressed in Escherichia coli. The recombinant enzymes will be characterized pertaining to biochemical properties that are relevant to the application of the enzymes in biocatalytic transformation. Three-dimensional structures of the GH20 β-N-acetylhexosaminidase and the GH42 β-galactosidase from B. breve will be constructed. Based on the modeled structures, catalytically active residues and potential residues involved in transglycosylation will be identified from molecular docking. Rationally designed mutants will be generated based on structural modeling and characterized. Detailed investigations of the transglycosylation activities and the analysis of glycosyl transfer of the selected enzymes to the substrates and different glycosyl acceptors for the formation of certain HMO structures will be performed. The core, precursor and backbone structures of HMOs will be synthesized, analyzed and structurally elucidated. Overall, the scientific goal of the project is to obtain engineered biocatalysts showing strong transglycosylation activity, which have the potential to synthesize some of the core structures of HMOs.

Collaborations within this thesis will include Roland Ludwig (enzyme kinetics), the core facility BmCA (DSC, crystallography), as well as Prof. Lubbert Dijkhuizen, Faculty of Science and Engineering, University of Groningen, Groningen, the Netherlands (analysis of HMO structures).

Bode, L. (2012). "Human milk oligosaccharides: Every baby needs a sugar mama." Glycobiology 22: 1147-1162.
LoCascio, R. G., et al. (2007). "Glycoprofiling of bifidobacterial consumption of human milk oligosaccharides demonstrates strain specific, preferential consumption of small chain glycans secreted in early human lactation." Journal of Agricultural and Food Chemistry 55:8914-8919.
Sela, D. A.; Mills, D. A. (2010). "Nursing our microbiota: molecular linkages between bifidobacteria and milk oligosaccharides." Trends in Microbiology 18:298-307.
Zivkovic, A. M., et al. (2011) "Human milk glycobiome and its impact on the infant gastrointestinal microbiota." Proceedings of the National Academy of Sciences 108:4653-4658.