title: A combined system for engineering glycosylation efficiency and glycan structure in Saccharomyces cerevisiae.
abstract: We describe a novel synthetic N-glycosylation pathway to produce recombinant proteins carrying human-like N-glycans in Saccharomyces cerevisiae, at the same time addressing glycoform and glycosylation efficiency. The Δalg3 Δalg11 double mutant strain, in which the N-glycans are not matured to their native high-mannose structure, was used. In this mutant strain, lipid-linked Man(3)GlcNAc(2) is built up on the cytoplasmic side of the endoplasmic reticulum, flipped by an artificial flippase into the ER lumen, and then transferred with high efficiency to the nascent polypeptide by a protozoan oligosaccharyltransferase. Protein-bound Man(3)GlcNAc(2) serves directly as a substrate for Golgi apparatus-targeted human N-acetylglucosaminyltransferases I and II. Our results confirmed the presence of the complex human-like N-glycan structure GlcNAc(2)Man(3)GlcNAc(2) on the secreted monoclonal antibody HyHEL-10. However, due to the interference of Golgi apparatus-localized mannosyltransferases, heterogeneity of N-linked glycans was observed.
authors.affiliation: Institute of Microbiology, ETH Zürich, Zürich, Switzerland.
authors.forename: Farnoush
authors.initials: F
authors.lastname: Parsaie Nasab
authors.forename: Markus
authors.initials: M
authors.lastname: Aebi
authors.forename: Gesche
authors.initials: G
authors.lastname: Bernhard
authors.forename: Alexander Daniel
authors.initials: AD
authors.lastname: Frey
mesh_terms: D004721:Endoplasmic Reticulum / Q000378:metabolism; D017353:Gene Deletion; D006031:Glycosylation; D060847:Metabolic Engineering; D053858:Metabolic Networks and Pathways / Q000235:genetics*; D011134:Polysaccharides / Q000378:metabolism*; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000235:genetics* / Q000378:metabolism*
chemical_list: D011134:Polysaccharides; D011994:Recombinant Proteins