Glycoengineering of the methylotrophic yeast Hansenula polymorpha for the production of glycoproteins with trimannosyl core N-glycan by blocking core oligosaccharide assembly.
The initial lipid-linked oligosaccharide Glc(3)Man(9)GlcNAc(2)-dolichyl pyrophosphate (Dol-PP) for N-glycan is synthesized and assembled at the membrane of the endoplasmic reticulum (ER) and subsequently transferred to a nascent polypeptide by the oligosaccharide transferase complex. We have identified an ALG3 homolog (HpALG3) coding for a dolichyl-phosphate-mannose dependent alpha-1,3-mannosyltransferase in the methylotrophic yeast Hansenula polymorpha. The detailed analysis of glycan structure by linkage-specific mannosidase digestion showed that HpALG3 is responsible for the conversion of Man5GlcNAc(2)-Dol-PP to Man(6)GlcNAc(2)-Dol-PP, the first step to attach a mannose to the lipid-linked oligosaccharide in the ER. The N-glycosylation pathway of H. polymorpha has been remodeled by deleting the HpALG3 gene in the Hpoch1 null mutant strain blocked in the yeast-specific outer mannose chain synthesis and by introducing an ER-targeted Aspergillus saitoi alpha-1,2-mannosidase gene. This glycoengineered H. polymorpha strain produced glycoproteins mainly containing trimannosyl core N-glycan (Man(3)GlcNAc(2)), which is the common core backbone of various human-type N-glycans. The results demonstrate the high potential of H. polymorpha to be developed as an efficient expression system for the production of glycoproteins with humanized glycans.
Oh
Doo-Byoung
DB
Omics and Integration Research Center, Korea Research Institute of Bioscience and Biotechnology, Daejeon, Korea.
Park
Jeong-Seok
JS
Kim
Moo Woong
MW
Cheon
Seon Ah
SA
Kim
Eun Jung
EJ
Moon
Hye Yun
HY
Kwon
Ohsuk
O
Rhee
Sang Ki
SK
Kang
Hyun Ah
HA
D024861:Genetic Enhancement / Q000379:methods*; D006023:Glycoproteins / Q000235:genetics / Q000378:metabolism*; D008364:Mannosyltransferases / Q000378:metabolism*; D008565:Membrane Proteins / Q000378:metabolism*; D009844:Oligosaccharides / Q000378:metabolism*; D010843:Pichia / Q000502:physiology*; D011134:Polysaccharides / Q000378:metabolism*; D015202:Protein Engineering / Q000379:methods*; D029701:Saccharomyces cerevisiae Proteins / Q000378:metabolism*
D006023:Glycoproteins; D008565:Membrane Proteins; D009844:Oligosaccharides; D011134:Polysaccharides; D029701:Saccharomyces cerevisiae Proteins; C104466:ALG3 protein, S cerevisiae; D008364:Mannosyltransferases