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Glycobiology Advance Access published online on June 9, 2004
Glycobiology, doi:10.1093/glycob/cwh104
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Received January 20, 2004
Revised May 25, 2004
Accepted May 26, 2004

ORIGINAL ARTICLES

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 Bobrowicz 1, Robert C. Davidson 1, Huijuan Li 1, Thomas I. Potgieter 1, Juergen H. Nett 1, Stephen R. Hamilton 1, Terrance A. Stadheim 1, Robert G. Miele 1, Beata Bobrowicz 1, Teresa Mitchell 1, Sebastian Rausch 1, Eduard Renfer 1, Stefan Wildt 1*

1 GlycoFi, Inc, 21 Lafayette St, Suite 200, Lebanon, NH 03766, USA

* To whom correspondence should be addressed. E-mail: swildt{at}glycofi.com.


  Abstract

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 micro-heterogeneity of glycosylation in mammalian produced proteins. Recently, we have 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 the yeast 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 {beta}-1,4-galactosyl transferase activities we demonstrate that this structure is a substrate for highly efficient in vivo galactose addition. Taken together, this 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.


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