Engineering multivalent protein glycosylation in E. coli

The project Engineering multivalent protein glycosylation in Escherichia coli: Improved glycan binding interactions and next generation therapeutics, will advance an artificial protein glycosylation system, which operates in the cytoplasm of the biotechnological workhorse E. coli. The project aims to develop novel multivalent glycoconjugates with applications as anti-pathogenic agents. Multivalency is an essential feature of many glycan binding interactions and, thus, is pivotal to the generation of efficient future therapeutics. The basis of the glycosylation system is a recently discovered bacterial, asparagine-glycosyltransferase (NGT), which carries out the site-specific modification of engineered proteins with a single glucose residue. For this purpose multiple glycosylation sites will be introduced into selected targets to achieve controlled, multivalent glycan display. In the laboratory of Prof. Markus Aebi I will investigate proteins with regard to their potential for multivalent glycan display. Requirements for reliable and efficient NGT-mediated glucosylation at multiple sites will be defined and subsequently applied to therapeutically relevant proteins as well as to novel scaffolds and virus-like particles. Standard analytical analyses will be applied to monitor correct protein folding, and mass spectrometry will be used to determine the degree of glucosylation. A major goal of this project is the design of novel, artificial biosynthetic pathways. Genes will be introduced to synthesize either GM1-oligosaccharides (GM1os) or globopentaose (Gb5) structures in E. coli. GM1-os is the cell-surface ligand of the cholera toxin. Globopentaoses are exclusively present on the surface of cancer cells, making them promising targets for the design of anti-cancer vaccines. It is possible to produce GM1os and Gb5 by both chemical and chemo-enzymatic approaches, but these are costly and arduous and not suitable for large-scale production. The cytoplasmic pathway, as outlined in this project, is much simpler, cheaper and more scalable. The combination of artificial synthesis pathways and multivalent glycan display will result in the production of high-affinity cholera toxin inhibitors and multivalent Gb5 cancer vaccine candidates.

In the project "Engineering multivalent protein glycosylation in Escherichia coli: Improved glycan binding interactions and next generation therapeutics", an artificial protein glycosylation system could be advanced, which operates in the cytoplasm of the biotechnological workhorse E. coli. The project aimed to develop novel multivalent glycoconjugates with applications as anti-pathogen agents. Multivalency is an essential feature of many glycan binding interactions and will be essential for the generation of efficient future therapeutics. The basis of the glycosylation system is a recently discovered, bacterial, asparagine-glycosyltransferase, which carries out the site-specific modification of engineered proteins with a single glucose residue. In the laboratory of Prof. Markus Aebi (ETH Zurich), multiple glycosylation sites on various underlying protein carriers could be introduced and a controlled multivalent glycan display could be achieved. A major goal of this project was the design of novel artificial biosynthetic glycosylation pathways. Genes have been introduced into E. coli to synthesize fucosyllactose or globotriose structures within the cytoplasm of the bacterium. Fucosyllactose is a human cell-surface ligand of the cholera toxin, and globotriose is the known binding partner of the Shiga toxin. It is possible to produce fucosyllactose and globotriose by both chemical and chemo-enzymatic approaches, but these are costly and arduous and not suitable for large-scale production. The cytoplasmic pathway, as exploited in this project, is much simpler, cheaper and more scalable. The combination of the artificial synthesis pathways and the multivalent glycan display resulted in the production of high affinity Cholera and Shiga toxin inhibitors and might be applied as future treatment of both infections.