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Glycobiology Advance Access originally published online on June 9, 2004
Glycobiology 2004 14(9):757-766; doi:10.1093/glycob/cwh104
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Glycobiology vol. 14 no. 9 © Oxford University Press 2004; all rights reserved.

Engineering of an artificial glycosylation pathway blocked in core oligosaccharide assembly in the yeast Pichia pastoris: production of complex humanized glycoproteins with terminal galactose

Piotr Bobrowicz1, Robert C. Davidson1, Huijuan Li, Thomas I. Potgieter, Juergen H. Nett, Stephen R. Hamilton, Terrance A. Stadheim, Robert G. Miele, Beata Bobrowicz, Teresa Mitchell, Sebastian Rausch, Eduard Renfer and Stefan Wildt2

GlycoFi, Inc., 21 Lafayette Street, Suite 200, Lebanon, NH 03766

Received on January 20, 2004; revised on May 25, 2004; accepted on May 26, 2004

A significant percentage of eukaryotic proteins contain posttranslational 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:Man5GlcNAc2-PP-Dol mannosyltransferase was deleted in a strain that was previously engineered to produce hybrid GlcNAcMan5GlcNAc2 human N-glycans. Employing this approach, combined with the use of combinatorial genetic libraries, we engineered P. pastoris strains that synthesize complex GlcNAc2Man3GlcNAc2 N-glycans with striking homogeneity. Furthermore, through expression of a Golgi-localized fusion protein comprising UDP-glucose 4-epimerase and ß-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.

1 These authors contributed equally to this work.

2 To whom correspondence should be addressed; e-mail: swildt{at}glycofi.com


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