title: In vivo synthesis of mammalian-like, hybrid-type N-glycans in Pichia pastoris.
abstract: The Pichia pastoris N-glycosylation pathway is only partially homologous to the pathway in human cells. In the Golgi apparatus, human cells synthesize complex oligosaccharides, whereas Pichia cells form mannose structures that can contain up to 40 mannose residues. This hypermannosylation of secreted glycoproteins hampers the downstream processing of heterologously expressed glycoproteins and leads to the production of protein-based therapeutic agents that are rapidly cleared from the blood because of the presence of terminal mannose residues. Here, we describe engineering of the P. pastoris N-glycosylation pathway to produce nonhyperglycosylated hybrid glycans. This was accomplished by inactivation of OCH1 and overexpression of an alpha-1,2-mannosidase retained in the endoplasmic reticulum and N-acetylglucosaminyltransferase I and beta-1,4-galactosyltransferase retained in the Golgi apparatus. The engineered strain synthesized a nonsialylated hybrid-type N-linked oligosaccharide structure on its glycoproteins. The procedures which we developed allow glycan engineering of any P. pastoris expression strain and can yield up to 90% homogeneous protein-linked oligosaccharides.
authors.affiliation: Department of Molecular Biomedical Research, Ghent University and Flanders Interuniversity Institute for Biotechnology, Ghent, Belgium.
authors.forename: Wouter
authors.initials: W
authors.lastname: Vervecken
authors.forename: Vladimir
authors.initials: V
authors.lastname: Kaigorodov
authors.forename: Nico
authors.initials: N
authors.lastname: Callewaert
authors.forename: Steven
authors.initials: S
authors.lastname: Geysens
authors.forename: Kristof
authors.initials: K
authors.lastname: De Vusser
authors.forename: Roland
authors.initials: R
authors.lastname: Contreras
mesh_terms: D001709:Biotechnology / Q000379:methods; D004721:Endoplasmic Reticulum / Q000201:enzymology; D005700:Galactosyltransferases / Q000235:genetics / Q000378:metabolism; D005818:Genetic Engineering / Q000379:methods*; D006031:Glycosylation; D006056:Golgi Apparatus / Q000201:enzymology; D006801:Humans; D008361:Mannosidases / Q000235:genetics / Q000378:metabolism; D017351:N-Acetylglucosaminyltransferases / Q000235:genetics / Q000378:metabolism; D010843:Pichia / Q000235:genetics / Q000254:growth & development / Q000378:metabolism*; D011134:Polysaccharides / Q000096:biosynthesis* / Q000737:chemistry; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism
chemical_list: D011134:Polysaccharides; D011993:Recombinant Fusion Proteins; D005700:Galactosyltransferases; D017351:N-Acetylglucosaminyltransferases; C024850:alpha-1,3-mannosyl-glycoprotein beta-1,2-N-acetylglucosaminyltransferase I; D008361:Mannosidases