Engineering O-glycosylation in modified N-linked oligosaccharide (Man12GlcNAc2∼Man16GlcNAc2) Pichia pastoris strains

Abstract

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 (Man₁₂GlcNAc₂∼Man₁₆GlcNAc₂) 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.