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Design of glycan epitope toolbox: a novel pathway for protein N-glycosylation in the E. coli cytosol

Objective

Metabolic engineering of E. coli has been highly successful in producing diverse free oligosaccharides for research and commercial applications. Currently, we are poised to address the next major challenge: the site-specific synthesis of oligosaccharides directly onto proteins in the E. coli cytosol.
In this project we aim at the metabolic engineering of an N-glycosylation pathway in the E. coli cytosol using heterologous glycosyltransferases (GTs) to produce defined glycoprotein structures. Starting point is the newly discovered family of cytosolic N-glycosyltransferases (NGT), which transfer a single glucose residue onto proteins at asparagine (N) residues in the N-X-S/T sequon. Co-expression of an NGT with a galactosyltransferase has been shown to yield N-linked lactose in the E. coli cytosol. This N-linked lactose is an ideal starting point for the design of cytosolic N-glycosylation pathways to synthesize important glycans on proteins.
Two goals are delineated. 1) Bacterial GTs will be screened, and potentially engineered, for their ability to extend the N-lactose with defined sugars, thereby generating a diverse repertoire of glycans directly synthesized on proteins. The focus will be on screening of fucosyltransferases and efficient production of fucosylated oligosaccharides, as these are central glycan epitopes in diverse physiological processes. 2) The newly developed glycosylation pathways will be applied to a range of protein substrates in order to explore and address possible limitations of the glycosylation system.
The result will be a well-characterized glycoengineering toolbox enabling the bottom-up production of defined glycoprotein structures in E. coli.

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Topic(s)

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MSCA-IF-EF-ST - Standard EF

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Call for proposal

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(opens in new window) H2020-MSCA-IF-2015

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Coordinator

EIDGENOESSISCHE TECHNISCHE HOCHSCHULE ZUERICH
Net EU contribution

Net EU financial contribution. The sum of money that the participant receives, deducted by the EU contribution to its linked third party. It considers the distribution of the EU financial contribution between direct beneficiaries of the project and other types of participants, like third-party participants.

€ 187 419,60
Address
Raemistrasse 101
8092 Zuerich
Switzerland

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Region
Schweiz/Suisse/Svizzera Zürich Zürich
Activity type
Higher or Secondary Education Establishments
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Total cost

The total costs incurred by this organisation to participate in the project, including direct and indirect costs. This amount is a subset of the overall project budget.

€ 187 419,60
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