Engineering of the yeast Yarrowia lipolytica for the production of glycoproteins lacking the outer-chain mannose residues of N-glycans

Appl Environ Microbiol. 2007 Jul;73(14):4446-54. doi: 10.1128/AEM.02058-06. Epub 2007 May 18.

Abstract

In an attempt to engineer a Yarrowia lipolytica strain to produce glycoproteins lacking the outer-chain mannose residues of N-linked oligosaccharides, we investigated the functions of the OCH1 gene encoding a putative alpha-1,6-mannosyltransferase in Y. lipolytica. The complementation of the Saccharomyces cerevisiae och1 mutation by the expression of YlOCH1 and the lack of in vitro alpha-1,6-mannosyltransferase activity in the Yloch1 null mutant indicated that YlOCH1 is a functional ortholog of S. cerevisiae OCH1. The oligosaccharides assembled on two secretory glycoproteins, the Trichoderma reesei endoglucanase I and the endogenous Y. lipolytica lipase, from the Yloch1 null mutant contained a single predominant species, the core oligosaccharide Man8GlcNAc2, whereas those from the wild-type strain consisted of oligosaccharides with heterogeneous sizes, Man8GlcNAc2 to Man12GlcNAc2. Digestion with alpha-1,2- and alpha-1,6-mannosidase of the oligosaccharides from the wild-type and Yloch1 mutant strains strongly supported the possibility that the Yloch1 mutant strain has a defect in adding the first alpha-1,6-linked mannose to the core oligosaccharide. Taken together, these results indicate that YlOCH1 plays a key role in the outer-chain mannosylation of N-linked oligosaccharides in Y. lipolytica. Therefore, the Yloch1 mutant strain can be used as a host to produce glycoproteins lacking the outer-chain mannoses and further developed for the production of therapeutic glycoproteins containing human-compatible oligosaccharides.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Cellulase / chemistry
  • Cellulase / genetics
  • Gene Deletion
  • Genetic Complementation Test
  • Genetic Engineering
  • Glycoproteins / biosynthesis*
  • Glycoproteins / chemistry
  • Glycoproteins / genetics*
  • Lipase / chemistry
  • Lipase / genetics
  • Mannosidases / metabolism
  • Mannosyltransferases / genetics
  • Mannosyltransferases / metabolism
  • Membrane Glycoproteins / genetics
  • Membrane Glycoproteins / metabolism
  • Oligosaccharides / analysis
  • Oligosaccharides / metabolism
  • Saccharomyces cerevisiae / genetics
  • Saccharomyces cerevisiae / metabolism
  • Saccharomyces cerevisiae Proteins / genetics
  • Saccharomyces cerevisiae Proteins / metabolism
  • Yarrowia / genetics*
  • Yarrowia / metabolism*

Substances

  • Glycoproteins
  • Membrane Glycoproteins
  • OCH1 protein, S cerevisiae
  • Oligosaccharides
  • Saccharomyces cerevisiae Proteins
  • Mannosyltransferases
  • alpha 1,6-mannosyltransferase
  • Lipase
  • Mannosidases
  • alpha(1-6)mannosidase
  • mannosyl-oligosaccharide 1,2-alpha-mannosidase
  • Cellulase