title: Knockout of an endogenous mannosyltransferase increases the homogeneity of glycoproteins produced in Pichia pastoris.
abstract: The yeast Pichia pastoris is a common host for the recombinant production of biopharmaceuticals, capable of performing posttranslational modifications like glycosylation of secreted proteins. However, the activity of the OCH1 encoded α-1,6-mannosyltransferase triggers hypermannosylation of secreted proteins at great heterogeneity, considerably hampering downstream processing and reproducibility. Horseradish peroxidases are versatile enzymes with applications in diagnostics, bioremediation and cancer treatment. Despite the importance of these enzymes, they are still isolated from plant at low yields with different biochemical properties. Here we show the production of homogeneous glycoprotein species of recombinant horseradish peroxidase by using a P. pastoris platform strain in which OCH1 was deleted. This och1 knockout strain showed a growth impaired phenotype and considerable rearrangements of cell wall components, but nevertheless secreted more homogeneously glycosylated protein carrying mainly Man8 instead of Man10 N-glycans as a dominant core glycan structure at a volumetric productivity of 70% of the wildtype strain.
authors.affiliation: Graz University of Technology, Institute of Molecular Biotechnology, Graz, Austria.
authors.forename: Florian W
authors.initials: FW
authors.lastname: Krainer
authors.forename: Christoph
authors.initials: C
authors.lastname: Gmeiner
authors.forename: Lukas
authors.initials: L
authors.lastname: Neutsch
authors.forename: Markus
authors.initials: M
authors.lastname: Windwarder
authors.forename: Robert
authors.initials: R
authors.lastname: Pletzenauer
authors.forename: Christoph
authors.initials: C
authors.lastname: Herwig
authors.forename: Friedrich
authors.initials: F
authors.lastname: Altmann
authors.forename: Anton
authors.initials: A
authors.lastname: Glieder
authors.forename: Oliver
authors.initials: O
authors.lastname: Spadiut
mesh_terms: D061249:Batch Cell Culture Techniques; D019149:Bioreactors; D002455:Cell Division / Q000235:genetics; D002853:Chromatography, Liquid; D004789:Enzyme Activation; D005656:Fungal Proteins / Q000235:genetics* / Q000378:metabolism*; D055786:Gene Knockout Techniques; D023061:Gene Order; D018390:Gene Targeting; D006023:Glycoproteins / Q000737:chemistry / Q000378:metabolism*; D037241:Mannose-Binding Lectins / Q000378:metabolism; D008364:Mannosyltransferases / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D013058:Mass Spectrometry; D010641:Phenotype; D010843:Pichia / Q000235:genetics* / Q000254:growth & development / Q000378:metabolism*; D011134:Polysaccharides / Q000737:chemistry / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D013312:Stress, Physiological
chemical_list: D005656:Fungal Proteins; D006023:Glycoproteins; D037241:Mannose-Binding Lectins; D011134:Polysaccharides; D011994:Recombinant Proteins; D008364:Mannosyltransferases