Glycoengineering of yeasts from the perspective of glycosylation efficiency

Abstract

N-linked glycosylation of proteins is one of the most common posttranslational modifications. N-glycan structures and N-glycosylation efficiency are crucial parameters in the production of N-glycosylated proteins. Yeast cells can be seen as an attractive production host for therapeutic glycoproteins and pioneering work of glycoengineering was performed in Pichia pastoris, realizing yeast strains capable of producing defined, human-type N-glycans. Most strategies used for glycoengineering rely on the modification of the lipid-linked oligosaccharide biosynthesis for the generation of the substrate for Golgi-localized glycosyltransferases. However, modifications in the lipid-linked oligosaccharide biosynthesis often result in the accumulation of intermediate structures and cause hypoglycosylation of client proteins. In order to ensure complete N-glycosylation, the flow of lipid-linked oligosaccharide through the biosynthetic pathway and the transfer of the oligosaccharide from the donor lipid onto the protein have to be optimized. A promising tool to improve site occupancy is the expression of protozoan oligosaccharyltransferases, which possess altered specificities for the oligosaccharide and also for the protein acceptor site. Furthermore, flipping of the lipid-linked oligosaccharide into the ER lumen can be improved by overexpression of an artificial flippase. Improving the glycosylation efficiency ensures that not only homogeneous N-glycan structures are generated, but also client proteins are fully glycosylated.