earticle

영어

We have previously shown that the glycoengineered methylotrophic yeast Hansenula polymorpha strains, the Δoch1 and Δoch1Δalg3 double deletion strains, with the targeted expression of Aspergillus saitoi α-1,2-mannosidase in the ER, were able to produce human mannose-type N-glycans (Man5GlcNAc2 or core Man3GlcNAc2)1, 2. Here we report the further modification of yeast glycosylation pathway to synthesize the complex-type N-glycans with a terminal N-acetyl glucosamine in both the glycoengineered ΔHpoch1 and ΔHpoch1 ΔHpalg3 strains, respectively. First, several combinatorial synthetic leaders were constructed to localize efficiently active human β -1,2 N-acetyl glucosaminyl transferase I (GnT I) in the Golgi apparatus of yeast. The short, medium, and long N-terminal leader sequences with the various length of stem region of yeast type II membrane proteins (ScMnn9, HpOch1, and HpOcr1), located in the early Golgi compartment, were fused in-frame to the catalytic domain of human GnT I lacking its own N-terminal leader sequence. The GnT I constructs combined with various yeast N-terminal leader sequences were introduced into the H. polymorpha och1 and och1 alg3 strains carrying the ER-targeted α-1,2 mannosidase, and the obtained transformants were screened for proper localization of GnT1 into the Golgi compartment by the size fractional centrifugation and Western blotting. Subsequently, the production of the complex-type glycans with monoantennary N-acetyl glucosamine was analyzed by a capillary electrophoresis of ATPS-labeled cell wall glycans. Our data strongly suggested that the ΔHpoch1 single deletion strain would be a better host for the production of human complex-type N-glycans than the ΔHpoch1 ΔHpalg3 double deletion strain in the respect of the glycosylation site occupancy and the byproduct Hex6GlcNAcs formation.