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

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

Context To fully understand the mechanisms underlying essential processes in living organisms, it is necessary to go down to the molecular level. Glycoconjugates, which are all molecules modified with a sugar structure, are ubiquitously present in all domains of life and are involved in a myriad of processes central to every living organism. Glycans bring an extra level of structural and functional diversity to molecules and provide an extremely diverse range of very specific ligands. These glycans form the basis of extensive sweet crosstalk between and within living systems. In this project, the focus lies on glycoproteins, i.e. proteins modified with a glycan structure, as it is estimated that more than half of all proteins are glycosylated. Glycoproteins have been shown to play a role in communication, pathogenicity of bacteria, diverse physiological processes and in human health and disease. However, the inability to readily synthesize proteins with a defined glycan epitope makes this an extremely challenging topic of research. In contrast to other approaches where eukaryotic cells can only be engineered to produce a number of defined eukaryotic glycoproteins, this project uses the bacterium Escherichia coli as a living factory to produce defined glycoproteins. Benefits include the absence of an endogenous N-glycosylation system in E. coli, its fast growth, availability of genetic tools and low fermentation cost. In the past E. coli was already used to produce free oligosaccharides, but in nature these glycan structures are often present on a protein. Objectives The project aims to tackle the major challenge of the site-specific synthesis of oligosaccharides directly onto proteins in the E. coli cytosol. By metabolically engineering E. coli glycoproteins with defined structures will be produced that are of importance to both basic research and commercial applications, like vaccines and other biotherapeutics. Starting point is a family of cytosolic N-glycosyltransferases (NGT), which transfer a single glucose residue onto proteins at asparagine (N) residues in an N-X-S/T sequon. Further elongation of this N-Glucose is possible by heterologous expression of a galactosyltransferase, thus yielding N-linked lactose. This N-linked lactose was used as the starting point for the further expansion of this technology towards a modular glycoengineering toolbox and thus more diverse glycans. Two objectives were postulated: (1) the screening of bacterial glycosyltransferases for their potential to extend N-lactose with defined sugars, thus generating a diverse repertoire of glycans directly synthesized on proteins and (2) the expansion of the range of protein substrates that can be utilized by the proposed glycoengineering toolbox. The overall aim is to generate a well-characterized glycoengineering toolbox enabling the bottom-up production of defined glycoprotein structures in E. coli.

Текст от CORDIS, на английски · Данни: CORDIS, © Европейски съюз