Glycoengineering of the methylotrophic yeast Hansenula polymorpha for the production of glycoproteins with trimannosyl core N-glycan by blocking core oligosaccharide assembly

Biotechnol J. 2008 May;3(5):659-68. doi: 10.1002/biot.200700252.


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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Genetic Enhancement / methods*
  • Glycoproteins / genetics
  • Glycoproteins / metabolism*
  • Mannosyltransferases / metabolism*
  • Membrane Proteins / metabolism*
  • Oligosaccharides / metabolism*
  • Pichia / physiology*
  • Polysaccharides / metabolism*
  • Protein Engineering / methods*
  • Saccharomyces cerevisiae Proteins / metabolism*


  • Glycoproteins
  • Membrane Proteins
  • Oligosaccharides
  • Polysaccharides
  • Saccharomyces cerevisiae Proteins
  • ALG3 protein, S cerevisiae
  • Mannosyltransferases