Engineering O -glycosylation in modified N -linked oligosaccharide (Man 12 GlcNAc 2 ∼Man 16 GlcNAc 2 ) Pichia pastoris strains.

Yeast have been engineered for the production of therapeutic glycoproteins with humanized N -linked oligosaccharides. Both N - and O -linked oligosaccharides engineered yeast have been attractive prospects, since yeast-specific O -mannosylated proteins were reported to induce an aberrant immune response and alter pharmacokinetics in vivo . In the present study, we genetically manipulated O -glycosylation by disrupting O -mannosyltransferase PMT1 and PMT5 in a low-mannose type N -linked oligosaccharide (Man 12 GlcNAc 2 ∼Man 16 GlcNAc 2 ) engineered Pichia pastoris strain to produce therapeutic glycoproteins. The O -mannosyltransferase PMT1 mutant produces anti-Her-2 antibodies with reduced O -linked oligosaccharides and protein degradation, but this strain exhibited growth defects. However, the deletion of O -mannosyltransferase PMT5 individually has a minimal effect on O -glycosylation, degradation of the anti-Her-2 antibody, and strain growth. Thus, by disrupting O -mannosyltransferase PMT1 in an N -glycosylation engineered Pichia pastoris strain, we generated an effective glycoengineered Pichia pastoris strain to effectively produce therapeutic glycoproteins with both engineered N - and O -linked oligosaccharides.