title: Engineering of an artificial glycosylation pathway blocked in core oligosaccharide assembly in the yeast Pichia pastoris: production of complex humanized glycoproteins with terminal galactose.
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 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 beta-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.
authors.affiliation: GlycoFi, Inc., 21 Lafayette Street, Suite 200, Lebanon, NH 03766, USA.
authors.forename: Piotr
authors.initials: P
authors.lastname: Bobrowicz
authors.forename: Robert C
authors.initials: RC
authors.lastname: Davidson
authors.forename: Huijuan
authors.initials: H
authors.lastname: Li
authors.forename: Thomas I
authors.initials: TI
authors.lastname: Potgieter
authors.forename: Juergen H
authors.initials: JH
authors.lastname: Nett
authors.forename: Stephen R
authors.initials: SR
authors.lastname: Hamilton
authors.forename: Terrance A
authors.initials: TA
authors.lastname: Stadheim
authors.forename: Robert G
authors.initials: RG
authors.lastname: Miele
authors.forename: Beata
authors.initials: B
authors.lastname: Bobrowicz
authors.forename: Teresa
authors.initials: T
authors.lastname: Mitchell
authors.forename: Sebastian
authors.initials: S
authors.lastname: Rausch
authors.forename: Eduard
authors.initials: E
authors.lastname: Renfer
authors.forename: Stefan
authors.initials: S
authors.lastname: Wildt
mesh_terms: D001483:Base Sequence; D017931:DNA Primers; D005690:Galactose / Q000378:metabolism*; D006023:Glycoproteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D006031:Glycosylation; D006801:Humans; D009844:Oligosaccharides / Q000737:chemistry*; D010843:Pichia / Q000378:metabolism*; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
chemical_list: D017931:DNA Primers; D006023:Glycoproteins; D009844:Oligosaccharides; D011994:Recombinant Proteins; D005690:Galactose