title: Remodeling of the glycosylation pathway in the methylotrophic yeast Hansenula polymorpha to produce human hybrid-type N-glycans.
abstract: As a step forward to achieve the generation of human complex-type N-glycans in the methylotrophic yeast Hansenula polymorpha, we here report the modification of the yeast glycosylation pathway by heterologous expression of the human gene encoding β-1,2-N-acetylglucosaminyltransferase I (GnTI). For the optimal expression of human GnTI in the yeast Golgi compartment, the catalytic domain of the GnTI was fused to various N-terminal leader sequences derived from the yeast type II membrane proteins. The vectors containing GnTI fusion constructs were introduced into the H. polymorpha och1Δ single and och1Δalg3Δ double mutant strains expressing the ER-targeted Aspergillus saitoi α-1,2 mannosidase, respectively. Both of the glycoengineered Hpoch1Δ and Hpoch1ΔHpalg3Δ strains were shown to produce successfully the hybrid-type glycans with a monoantennary N-acetylglucosamine (GlcNAc(1)Man(5)GlcNAc(2) and GlcNAc(1)Man(3)GlcNAc(2), respectively) by N-glycan profile analysis of cell wall proteins. Furthermore, by comparative analysis of byproduct formation and the glycosylation site occupancy, we propose that the Hpoch1Δ strain would be more suitable than the Hpoch1ΔHpalg3Δ strain as a host for the production of recombinant proteins with humanized glycans.
authors.affiliation: Research Center for Biomolecules and Biosystems, Chung-Ang University, Seoul, 156-756, Republic of Korea.
authors.forename: Seon Ah
authors.initials: SA
authors.lastname: Cheon
authors.forename: Hyunah
authors.initials: H
authors.lastname: Kim
authors.forename: Doo-Byoung
authors.initials: DB
authors.lastname: Oh
authors.forename: Ohsuk
authors.initials: O
authors.lastname: Kwon
authors.forename: Hyun Ah
authors.initials: HA
authors.lastname: Kang
mesh_terms: D053898:Biosynthetic Pathways; D005656:Fungal Proteins / Q000235:genetics / Q000378:metabolism; D005818:Genetic Engineering; D006031:Glycosylation; D006801:Humans; D017351:N-Acetylglucosaminyltransferases / Q000235:genetics / Q000378:metabolism; D010843:Pichia / Q000235:genetics* / Q000378:metabolism*; D011134:Polysaccharides / Q000378:metabolism*
chemical_list: D005656:Fungal Proteins; D011134:Polysaccharides; D017351:N-Acetylglucosaminyltransferases