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Advances in the production of human therapeutic proteins in yeasts and filamentous fungi

Nature Biotechnology volume 22, pages 1409–1414 (2004)Cite this article

A Corrigendum to this article was published on 01 December 2004

Abstract

Yeast and fungal protein expression systems are used for the production of many industrially relevant enzymes, and are widely used by the research community to produce proteins that cannot be actively expressed in Escherichia coli or require glycosylation for proper folding and biological activity. However, for the production of therapeutic glycoproteins intended for use in humans, yeasts have been less useful because of their inability to modify proteins with human glycosylation structures. Yeast glycosylation is of the high-mannose type, which confers a short in vivo half-life to the protein and may render it less efficacious or even immunogenic. Several ways of humanizing yeast-derived glycoproteins have been tried, including enzymatically modifying proteins in vitro and modulating host glycosylation pathways in vivo. Recent advances in the glycoengineering of yeasts and the expression of therapeutic glycoproteins in humanized yeasts have shown significant promise, and are challenging the current dominance of therapeutic protein production based on mammalian cell culture.

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Figure 1: Protein structure of interferon-β illustrates the impact of glycosylation on hydrodynamic volume; glycosylated interferon-β (top) and glycosylated interferon-β with glycosylation highlighted in blue (bottom).
Figure 2: Glycosylation pathways in humans and yeast.
Figure 3: MALDI-TOF mass spectrometry of oligosaccharides released from purified kringle 3 protein produced in wild-type P. pastoris and glycoengineered strains of P. pastoris.

References

  1. Walsh, G. Biopharmaceutical Benchmarks—2003. Nat. Biotechnol. 21, 865–870 (2003).

    Article  CAS  PubMed  Google Scholar 

  2. Punt, P.J., van Biezen, N., Conesa, A., Albers, A., Mangnus, J. & van den Hondel, C. Filamentous fungi as cell factories for heterologous protein production. Trends Biotechnol. 20, 200–206 (2002)

    Article  CAS  PubMed  Google Scholar 

  3. Werten, M.W.T., van den Bosch, T.J., Wind, R.D., Mooibroek, H. & De Wolf, F.A. High-yield secretion of recombinant gelatins by Pichia pastoris. Yeast 15, 1087–1096 (1999).

    Article  CAS  PubMed  Google Scholar 

  4. Humphreys, A. & Boersig, C. Cholesterol drugs dominate. MedAdNews 22, 42–57 (2003).

    Google Scholar 

  5. Coloma, M.J., Clift, A., Wims, L. & Morrison, S.L. The role of carbohydrate in the assembly and function of polymeric IgG. Mol Immunol. 37, 1081–1090 (2000).

    Article  CAS  PubMed  Google Scholar 

  6. Mistry, P.K., Wraight, E.P. & Cox, T.M. Therapeutic delivery of proteins to macrophages: implications for treatment of Gaucher's disease. The Lancet, 348, 1555–1559 (1996).

    Article  CAS  Google Scholar 

  7. Grabowski, G.A. et al. Enzyme therapy in type 1 Gaucher disease: comparative efficacy of mannose-terminated glycocerebrosidase from natural and recombinant sources. Ann. Intern. Med. 122, 33–39 (1995).

    Article  CAS  PubMed  Google Scholar 

  8. Grabenhorst, E., Schlenke, P., Pohl, S., Nimtz, M. & Conradt, H.S. Genetic engineering of recombinant glycoproteins and the glycosylation pathway in mammalian host cells. Glyconj. J. 16, 81–97 (1999).

    Article  CAS  Google Scholar 

  9. Suzuki, T., Kitajima, K., Inoue, S. & Inoue, Y. N-glycosylation/deglycosylation as a mechanism for the post-translational modification/remodification of proteins. Glycoconj J. 12, 189–193 (1995).

    Article  Google Scholar 

  10. Takeuchi, M. et al. Relationship between sugar chain structure and biological activity of recombinant human erythropoietin produced in Chinese hamster ovary cells. Proc. Natl. Acad. Sci. USA. 86, 7819–7822 (1989).

    Article  CAS  PubMed  Google Scholar 

  11. Yuen, C.T. et al. Relationships between the N-glycan structures and biological activities of recombinant human erythropoietins produced using different culture conditions and purification procedures. Br. J. Haematol. 121, 511–526 (2003).

    Article  CAS  PubMed  Google Scholar 

  12. Erbayraktar, S. et al. Asialoerythropoietin is a nonerythropoietic cytokine with broad neuroprotective activity in vivo. Proc. Natl. Acad. Sci. USA 100, 6741–6746 (2003). Erratum in: Proc. Natl. Acad. Sci. USA 100, 9102 (2003).

    Article  CAS  PubMed  Google Scholar 

  13. Friedman, B. et al. A comparison of the pharmacological properties of carbohydrate remodeled recombinant and placental-derived beta-glucocerebrosidase: implications for clinical efficacy in treatment of Gaucher disease. Blood 93, 2807–2816 (1999).

    Article  CAS  PubMed  Google Scholar 

  14. Umana, P., Jean-Mairet, J., Moudry, R., Amstutz, H. & Bailey, J.E. Engineered glycoforms of an antineuroblastoma IgG1 with optimized antibody-dependent cellular cytotoxic activity. Nat. Biotechnol. 17, 176–180 (1999).

    Article  CAS  PubMed  Google Scholar 

  15. Warner, T.G. Enhancing therapeutic glycoprotein production in Chinese hamster ovary cells by metabolic engineering endogenous gene control with antisense DNA and gene targeting. Glycobiology 9, 841–850 (1999).

    Article  CAS  PubMed  Google Scholar 

  16. Warner, T.G. Metabolic engineering glycosylation: biotechnology's challenge to the glycobiologist in the next millennium. in Part IV Biology of Saccharides. (eds. Ernst, B., Hart, G. & Sinay, P.) 1043–1064 (VCH-Wiley, New York, 2001).

    Google Scholar 

  17. Cregg, J.M., Cereghino, J.L., Shi, J. & Higgins, D.R. Recombinant protein expression in Pichia pastoris. Mol. Biotechnol. 16, 23–52 (2000).

    Article  CAS  PubMed  Google Scholar 

  18. Mochizuki, S. et al. Expression and characterization of recombinant human antithrombin III in Pichia pastoris. Protein Expr. Purif. 23, 55–65 (2001).

    Article  CAS  PubMed  Google Scholar 

  19. Ward, M. et al. Characterization of humanized antibodies secreted by Aspergillus niger. Appl. Environ. Microbiol. 70, 2567–2576 (2004).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  20. Ogunjimi, A.A., Chandler, J.M., Gooding, C.M., Recinos, A. & Choudary, P.V. High-level secretory expression of immunologically active intact antibody from the yeast P. pastoris. Biotechnol. Letters, 21, 561–567 (1999).

    Article  CAS  Google Scholar 

  21. Maras, M. & Contreras, R. Methods for modifying carbohydrate moieties. US patent 5,834,251 (Dec. 30, 1994).

  22. Maras, M. et al. In vitro conversion of the carbohydrate moiety of fungal glycoproteins to mammalian-type oligosaccharides. Eur. J. Biochem. 249, 701–707 (1997).

    Article  CAS  PubMed  Google Scholar 

  23. Kornfeld, R. & Kornfeld, S. Assembly of asparagine-linked oligosaccharides. Annu. Rev. Biochem. 54, 631–664 (1985).

    Article  CAS  PubMed  Google Scholar 

  24. Brockhausen, I., Carver, J.P. & Schachter, H. Control of glycoprotein synthesis. The use of oligosaccharide substrates and HPLC to study the sequential pathway for N-acetylglucosaminyltransferases I, II, III, IV, V and VI in the biosynthesis of highly branched N-glycans by hen oviduct membranes. Biochem. Cell Biol. 66, 1134–1151 (1988).

    Article  CAS  PubMed  Google Scholar 

  25. Silberstein, S. & Gilmore, R. Biochemistry, molecular biology and genetics of oligosaccharyltransferase. FASEB J. 10, 849–858 (1996).

    Article  CAS  PubMed  Google Scholar 

  26. Gemmill, T.R. & Trimble, R.B. Overview of N- and O-linked oligosaccharide structures found in various yeast species. Biochim. Biophys. Acta 1426, 227–237 (1999).

    Article  CAS  PubMed  Google Scholar 

  27. Tulsiani, D.R., Hubbard, S.C., Robbins, P.W. & Touster, O. Alpha-D-mannosidases of rat liver Golgi membranes. Mannosidase II is the GlcNAcMan5-cleaving enzyme in glycoprotein biosynthesis and mannosidases IA and IB are the enzymes converting Man9 precursors to Man5 intermediates. J. Biol. Chem. 257, 3660–3668 (1982).

    PubMed  CAS  Google Scholar 

  28. Kalsner, I., Hintz, W., Reid, L.S. & Schachter, H. Insertion into Aspergillus nidulans of functional UDP-GlcNAc: α3-D-mannoside β-1,2-N-acetyl-glucosaminyltransferase I, the enzyme catalyzing the first committed step from oligomannose to hybrid and complex N-glycans. Glycoconj. J. 12, 360–370 (1995).

    Article  CAS  PubMed  Google Scholar 

  29. Martinet, W., Maras, M., Saelens, X., Jou, W.M. & Contreras, R. Modification of the protein glycosylation pathway in the methylotrophic yeast, Pichia pastoris. Biotechnol. Letters 20, 1171–1177 (1998).

    Article  CAS  Google Scholar 

  30. Chiba, Y. et al. Production of human compatible high mannose-type (Man5GlcNAc2) sugar chains in Saccharomyces cerevisiae. J. Biol. Chem. 273, 26298–26304 (1998).

    Article  CAS  PubMed  Google Scholar 

  31. Choi, B.K. et al. Use of combinatorial genetic libraries to humanize N-linked glycosylation in the yeast Pichia pastoris. Proc. Natl. Acad. Sci. USA 100, 5022–5027 (2003).

    Article  CAS  PubMed  Google Scholar 

  32. Stadheim, T.A., Bobrowicz, P. & Wildt, S. Methods for eliminating mannosylphosphorylation of glycans in the production of glycoproteins. US Patent Appl. No. 60/532,461 (December 23, 2003).

  33. Vervecken, W. et al. In vivo synthesis of mammalian-like, hybrid-type N-glycans in Pichia pastoris. Appl Environ Microbiol. 70, 2639–2646 (2004).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  34. Hamilton, S.R. et al. Production of complex human glycoproteins in yeast. Science, 301, 1244–1246 (2003).

    Article  CAS  PubMed  Google Scholar 

  35. Bobrowicz, P. et al. Engineering of an artificial glycosylation pathway blocked in core oligosaccharide assembly in the yeast Pichia pastoris: production of complex humanized glycoproteins with terminal galactose. Glycobiology 14, 757–766 (2004).

    Article  CAS  PubMed  Google Scholar 

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Authors and Affiliations

  1. the Department of Biological Sciences and the Department of Chemistry, Thayer School of Engineering, Dartmouth College, Hanover, 03755, New Hampshire, USA

    Tillman U Gerngross

  2. GlycoFi Inc., 21 Lafayette Street, Lebanon, 03766, New Hampshire, USA

    Tillman U Gerngross

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Correspondence to Tillman U Gerngross.

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T.U.G. has equity exceeding 5% in a company (GlycoFi) that develops yeast-based protein expression systems.

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Gerngross, T. Advances in the production of human therapeutic proteins in yeasts and filamentous fungi. Nat Biotechnol 22, 1409–1414 (2004). https://doi.org/10.1038/nbt1028

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