Tailoring N-Glycan Biosynthesis for Production of Therapeutic Proteins in Saccharomyces cerevisiae

  • Mari A. Piirainen
  • Heidi Salminen
  • Alexander D. FreyEmail author
Part of the Methods in Molecular Biology book series (MIMB, volume 1923)


The ability to control and adjust the N-glycosylation pathway of Saccharomyces cerevisiae is a key step toward production of therapeutic glycoproteins such as antibodies or erythropoietin. The focus of this chapter is to describe the road from yeast-type N-glycosylation to human-type complex N-glycosylation. The chapter describes the cell engineering and provides the detailed analytical procedures required to perform glycan analysis using MALDI-TOF mass spectrometry.

Key words

S. cerevisiae Glycoengineering Glycosyltransferases Glycosylation efficiency Glycan analysis MALDI-TOF MS 


  1. 1.
    Walsh G, Jefferis R (2006) Post-translational modifications in the context of therapeutic proteins. Nat Biotechnol 24:1241–1252. CrossRefPubMedGoogle Scholar
  2. 2.
    Jacobs PP, Geysens S, Vervecken W et al (2009) Engineering complex-type N-glycosylation in Pichia pastoris using GlycoSwitch technology. Nat Protoc 4:58–70. CrossRefPubMedPubMedCentralGoogle Scholar
  3. 3.
    Hamilton SRR, Bobrowicz P, Bobrowicz B et al (2003) Production of complex human glycoproteins in yeast. Science 301(5637):1244–1246. CrossRefPubMedGoogle Scholar
  4. 4.
    Li H, Sethuraman N, Stadheim TA et al (2006) Optimization of humanized IgGs in glycoengineered Pichia pastoris. Nat Biotechnol 24:210–215. CrossRefPubMedGoogle Scholar
  5. 5.
    Parsaie Nasab F, Aebi M, Bernhard G, Frey AD (2013) A combined system for engineering glycosylation efficiency and glycan structure in Saccharomyces cerevisiae. Appl Environ Microbiol 79:997–1007. CrossRefPubMedPubMedCentralGoogle Scholar
  6. 6.
    Piirainen MA, Boer H, De Ruijter JC, Frey AD (2016) A dual approach for improving homogeneity of a human-type N-glycan structure in Saccharomyces cerevisiae. Glycoconj J 33:189–199. CrossRefPubMedGoogle Scholar
  7. 7.
    Helenius A, Aebi M (2004) Roles of N-linked glycans in the endoplasmic reticulum. Annu Rev Biochem 73:1019–1049. CrossRefPubMedGoogle Scholar
  8. 8.
    Gemmill TR, Trimble RB (1999) Overview of N- and O-linked oligosaccharide structures found in various yeast species. Biochim Biophys Acta 1426:227–237CrossRefGoogle Scholar
  9. 9.
    Munro S (2001) What can yeast tell us about N -linked glycosylation in the Golgi apparatus? FEBS Lett 498:223–227CrossRefGoogle Scholar
  10. 10.
    Moremen KW, Tiemeyer M, Nairn AV (2012) Vertebrate protein glycosylation: diversity, synthesis and function. Nat Rev Mol Cell Biol 13:448–462. CrossRefPubMedPubMedCentralGoogle Scholar
  11. 11.
    Helenius J, Ng DT, Marolda CL et al (2002) Translocation of lipid-linked oligosaccharides across the ER membrane requires Rft1 protein. Nature 415:447–450. CrossRefPubMedGoogle Scholar
  12. 12.
    Abe H, Takaoka Y, Chiba Y, et al (2009) Development of a valuable yeast strain using a novel mutagenesis technique for the effective production of therapeutic glycoproteins. Glycobiology 19:428–436. CrossRefGoogle Scholar
  13. 13.
    Xu S, Zhang G-Y, Zhang H et al (2016) Effects of Rho1, a small GTPase on the production of recombinant glycoproteins in Saccharomyces cerevisiae. Microb Cell Factories 15:179. CrossRefGoogle Scholar
  14. 14.
    Kelleher DJ, Banerjee S, Cura AJ et al (2007) Dolichol-linked oligosaccharide selection by the oligosaccharyltransferase in protist and fungal organisms. J Cell Biol 177:29–37. CrossRefPubMedPubMedCentralGoogle Scholar
  15. 15.
    Samuelson J, Banerjee S, Magnelli P et al (2005) The diversity of dolichol-linked precursors to Asn-linked glycans likely results from secondary loss of sets of glycosyltransferases. Proc Natl Acad Sci U S A 102:1548–1553. CrossRefPubMedPubMedCentralGoogle Scholar
  16. 16.
    Nett JH, Stadheim TA, Li H et al (2011) A combinatorial genetic library approach to target heterologous glycosylation enzymes to the endoplasmic reticulum or the Golgi apparatus of Pichia pastoris. Yeast 28:237–252. CrossRefPubMedGoogle Scholar
  17. 17.
    Abeijon C, Robbins PW, Hirschberg CB (1996) Molecular cloning of the Golgi apparatus uridine diphosphate-N-acetylglucosamine transporter from Kluyveromyces lactis. Proc Natl Acad Sci U S A 93:5963–5968CrossRefGoogle Scholar
  18. 18.
    Frey AD, Aebi M (2015) An enzyme-based screening system for the rapid assessment of protein N-glycosylation efficiency in yeast. Glycobiology 25:252–257. CrossRefPubMedGoogle Scholar
  19. 19.
    de Ruijter JC, Frey AD (2015) Analysis of antibody production in Saccharomyces cerevisiae: effects of ER protein quality control disruption. Appl Microbiol Biotechnol 99:9061–9071. CrossRefPubMedGoogle Scholar
  20. 20.
    Bigge JC, Patel TP, Bruce JA et al (1995) Nonselective and efficient fluorescent labeling of glycans using 2-amino benzamide and anthranilic acid. Anal Biochem 230:229–238. CrossRefPubMedGoogle Scholar
  21. 21.
    Ciucanu I (2006) Per-O-methylation reaction for structural analysis of carbohydrates by mass spectrometry. Anal Chim Acta 576:147–155. CrossRefPubMedGoogle Scholar
  22. 22.
    Yu SY, Wu SW, Khoo KH (2006) Distinctive characteristics of MALDI-Q/TOF and TOF/TOF tandem mass spectrometry for sequencing of permethylated complex type N-glycans. Glycoconj J 23:355–369. CrossRefPubMedGoogle Scholar
  23. 23.
    Mumberg D, Muller R, Funk M (1994) Regulatable promoters of Saccharomyces cerevisiae: comparison of transcriptional activity and their use for heterologous expression. Nucleic Acids Res 22:5767–5768CrossRefGoogle Scholar
  24. 24.
    Taxis C, Knop M (2006) System of centromeric, episomal, and integrative vectors based on drug resistance markers for Saccharomyces cerevisiae. BioTechniques 40:73–78CrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2019

Authors and Affiliations

  • Mari A. Piirainen
    • 1
  • Heidi Salminen
    • 1
  • Alexander D. Frey
    • 1
    Email author
  1. 1.Department of Bioproducts and Biosystems, School of Chemical EngineeringAalto UniversityEspooFinland

Personalised recommendations