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

Periodic Reporting for period 1 - Glycoli (Design of glycan epitope toolbox: a novel pathway for protein N-glycosylation in the E. coli cytosol)

Reporting period: 2016-11-01 to 2018-10-31

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.
(1) Design of a modular glycoengineering toolbox
Based on earlier results obtained in the host lab generating a polysialic acid epitope on various carrier proteins, the range of glycan epitopes was expanded. In this project the notion of creating a glycoengineering toolbox was taken a step further, by creating a modular system. Based on well-established synthetic biology principles of biobricking genes, GTBbs were created. These are sequences of DNA encoding for a glycosyltransferase, the enzyme attaching a certain glycan to a certain epitope. One GTBb harbors the genetic material encoding one glycosyltransferase, but also all regulatory elements (inducible promoter, ribosome binding site, terminator) to express this gene, as well as a His6 tag allowing the easy purification of the enzyme. The GTBb is flanked by isocaudameric restriction sites, allowing the simple and straightforward cloning and combination of different GTBbs in one vector system, thus gathering all genetic material encoding for one glycosylation pathway. To also make the vector system as modular as possible, the pSEVA vector system was used, which is a vector system specifically designed to be modular, by allowing the easy exchange of the origin of replication, antibiotic resistance cassette and cargo (here the GTBbs). The application of principles of synthetic biology and metabolic engineering to the generation of a diverse range of glycoproteins resulted in the successful realization of a modular glycoengineering toolbox. Several diverse glycans can be specifically attached to a protein, thus illustrating the successful design and implementation of the Glycoli toolbox.

(2) Substrate engineering
To further enhance the power of the glycoengineering toolbox, the target substrate, i.e. protein was also redesigned. Many lectins and antibodies in nature recognize multivalent glycans, i.e. not one glycan epitope, but a certain combination or repetition of glycan epitopes. Most glycoprotein design efforts focus on generating proteins carrying one specific glycan epitope. To enable the production of glycoproteins carrying multiple repeats of a glycan and thus making them a better ‘fit’ for recognition and binding by existing lectins and glycan-recognizing antibodies, protein tags were designed harboring from one up to five glycosylation sites. Experiments showed that these five sites can be efficiently modified by the glycosylation toolbox designed in part one of the project, thus illustrating the synergistic power of the here-created glycoprotein engineering toolbox, Glycoli.

The results of this work will be subject of an upcoming publication and patent, and were already shared with peers during conferences. To inform the broader audience about this project, a workshop was designed on glycobiology, which is taught to youngsters in the framework of the Ekoli NGO.
This project has advanced the field of glycoengineering massively, by not only making the production of defined glycan epitopes on proteins modular, but also by providing tags that can be multivalent glycosylated. The results of this project implicate that glycan epitopes can now be readily designed, produced and used to studied fundamental processes as well as applied towards novel biotherapeutics. The generated toolbox and knowledge of this project can be further applied and expanded to create any desired glycan in a site-specific manner attached to a protein. As an added bonus, this can also be done in a multivalent manner, relying on the multivalent glycosylation tags that can be added to virtual any protein. This means that the here-generated knowledge has provided the knowledge and tools to glycosylate any protein with any glycan in any desired valency. Compared to the state of the art, the production of free oligosaccharides, the Glycoli project dramatically advanced the field of glycoengineering, tackling both glycan and substrate engineering in a modular manner. This project has brought the tailored design and production of glycoproteins for biotherapeutics within reach.
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