Engineering of an Artificial Glycosylation Pathway Blocked in Core Oligosaccharide Assembly in the Yeast Pichia pastoris - Production of Complex Humanized Glycoproteins with Terminal Galactose

A significant percentage of eukaryotic proteins contain post-translational modifications, including glycosylation, which are required for biological function. However, the understanding of the structure-function relationships of N-glycans has lagged significantly due to the microheterogeneity of glycosylation in mammalian produced proteins. Recently we reported on the cellular engineering of yeast to replicate human N-glycosylation for the production of glycoproteins. Here we report the engineering of an artificial glycosylation pathway in Pichia pastoris blocked in dolichol oligosaccharide assembly. The PpALG3 gene encoding Dol-P-Man:-Man 5 GlcNAc 2 PP-Dol mannosyltransferase was deleted in a strain that was previously engineered to produce hybrid GlcNAcman 5 GlcNAc 2 human N-glycans. Employing this approach, combined with the use of combinatorial genetic libraries, we engineered P. pastoris strains that synthesize complex GlcNAc 2 man 3 GlcNAc 2 N-glycans with striking homogeneity. Furthermore, through expression of a Golgi-localized fusion protein comprising UDP-glucose 4-epimerase and β-1,4-galactosyl transferase activities we demonstrate that this structure is a substrate for highly efficient in vivo galactose addition. Taken together, these data demonstrate that the artificial in vivo glycoengineering of yeast represents a major advance in the production of glycoproteins and will emerge as a practical tool to systematically elucidate the structure-function relationship of N-glycans.