(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.
