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 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.
Bobrowicz
Piotr
P
GlycoFi, Inc., 21 Lafayette Street, Suite 200, Lebanon, NH 03766, USA.
Davidson
Robert C
RC
Li
Huijuan
H
Potgieter
Thomas I
TI
Nett
Juergen H
JH
Hamilton
Stephen R
SR
Stadheim
Terrance A
TA
Miele
Robert G
RG
Bobrowicz
Beata
B
Mitchell
Teresa
T
Rausch
Sebastian
S
Renfer
Eduard
E
Wildt
Stefan
S
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
D017931:DNA Primers; D006023:Glycoproteins; D009844:Oligosaccharides; D011994:Recombinant Proteins; D005690:Galactose