title: [Reconstruction of N-glycosylation pathway for producing human glycoproteins in Saccharomyces cerevisiae].
abstract: OBJECTIVE
  To produce human glycoproteins in Saccharomyces cerevisiae, human N-glycosylation pathway must be genetically engineered into the yeast cell. We tried to construct a strain, which can be used to introduce human N-glycosylation reactions, by disrupting several special glycosyltransferases in yeast N-glycosylation pathway. Furthermore, this mutant cell was applied for adaptive evolution to overcome its growth-defect phenotype.
  
  
  METHODS
  Three yeast genes ALG3, OCH1 and MNN1 were disrupted. The N-linked oligosaccharides from the mutant cells were analyzed by the activity staining of invertase, and their structure was further confirmed by high-performance liquid chromatography (HPLC) and the treatment with glycosidase. Mutant cells were cultured under a high temperature for their adaptive evolution of growth.
  
  
  CONCLUSIONS
  We obtained a delta och1 delta alg3 delta mnn1 strain that produces Man5 GlcNAc2 intermediate of human N-glycosylation. Our approach for adaptive evolution resulted a remarkable improvement on the growth phenotype of delta och1 delta alg3 delta mnn1 strain. In addition, we also confirmed a small amount of unexpected Man6GlcNAc2 intermediate from delta och1delta alg3 delta mnnl strain. Treatment with alpha-1,2-mannosidase converted both Man,GlcNAc2 and Man,GlcNAc2 products to a single Man3GlcNAc, form, indicating that the additional mannose on Man,GlcNAc, product comes from an alpha-1, 2 modification. Our results demonstrate that delta och1 delta alg3 deltamnn1 triple mutant can be used as an initial strain to construct an yeast therapeutic glycoprotein-expression system by introducing various enzymes that are involved in human N-glycosylation pathway.
authors.forename: Tiefan
authors.initials: T
authors.lastname: He
authors.forename: Sha
authors.initials: S
authors.lastname: Xu
authors.forename: Geyuan
authors.initials: G
authors.lastname: Zhang
authors.forename: Hideki
authors.initials: H
authors.lastname: Nakanishi
authors.forename: Xiaodong
authors.initials: X
authors.lastname: Gao
mesh_terms: D053898:Biosynthetic Pathways; D005818:Genetic Engineering; D006023:Glycoproteins / Q000235:genetics* / Q000378:metabolism; D006026:Glycoside Hydrolases / Q000235:genetics / Q000378:metabolism; D006031:Glycosylation; D016695:Glycosyltransferases / Q000235:genetics / Q000378:metabolism; D006801:Humans; D008562:Membrane Glycoproteins / Q000235:genetics / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000235:genetics* / Q000378:metabolism; D043324:beta-Fructofuranosidase
chemical_list: D006023:Glycoproteins; D008562:Membrane Glycoproteins; D016695:Glycosyltransferases; D006026:Glycoside Hydrolases; D043324:beta-Fructofuranosidase