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Recent Advances in the Expression of Foreign Genes in Pichia pastoris

Bio/Technology volume 11, pages 905–910 (1993)Cite this article

Abstract

The Pichia pastoris heterologous gene expression system has been utilized to produce attractive levels of a variety of intracellular and extracellular proteins of interest. Recent advances in our understanding and application of the system have improved its utility even further. These advances include: (1) methods for the construction of P. pastoris strains with multiple copies of A0X1-promoter-driven expression cassettes; (2) mixed-feed culture strategies for high foreign protein volumetric productivity rates; (3) methods to reduce proteolysis of some products in high cell-density culture media; (4) tested procedures for purification of secreted products; and (5) detailed information on the structures of N-linked oligosaccharides on P. pastoris secreted proteins. In this review, these advances along with basic features of the P. pastoris system are described and discussed.

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References

  1. Hitzeman, R.A., Hagie, F.E., Levine, H.L., Goeddel, D.V., Ammerer, G. and Hall, B.D. 1981. Expression of human gene for interferon in yeast. Nature 293: 717–222.

    Article  CAS  Google Scholar 

  2. Buckholz, R.G. and Gleeson, M.A.G. 1991. Yeast systems for the commercial production of heterologous proteins. Bio/Technology 9: 1067–1072.

    Article  CAS  Google Scholar 

  3. Romanos, M.A., Scorer, C.A. and Clare, J.J. 1992. Foreign gene expression in yeast: a review. Yeast 8: 423–488.

    Article  CAS  Google Scholar 

  4. Wegner, E.H. 1983. Biochemical conversions by yeast fermentation at high-cell densities. U. S. Patent 44114329.

  5. Veenhuis, M., van Dijken, J.P. and Harder, W. 1983. The significance of peroxisomes in the metabolism of one-carbon compounds in yeasts. Adv. Microb. Physiol. 24: 1–82.

    Article  CAS  Google Scholar 

  6. Couderc, R. and Baratti, J. 1980. Oxidation of methanol by the yeast Pichia pastoris: purification and properties of alcohol oxidase. Agric. Biol. Chem. 44: 2279–2289.

    CAS  Google Scholar 

  7. Cregg, J.M., Tschopp, J.F., Digan, M.E., Siegel, R., Craig, W., Velicelebi, G, and Thill, G 1988. High-level expression and secretion of heterologous proteins from the methylotrophic yeast, Pichia pastoris, p. 48–49. In: Advances in Gene Technology: Protein Engineering and Production. Brew, K., Ahmad, F., Bialy, H., Black, S., Fenna, R. E., Puett, D., Scott, W. A., van Brunt, J., Voellmy, W., Whelan, W. J. and Woessner, J. F. (Eds.). IRLPress, Washington, DC.

    Google Scholar 

  8. Cregg, J.M. and Madden, K.R. 1988. Development of the methylotrophic yeast, Pichia pastoris, as a host system for the production of foreign proteins. Dev. Ind. Microbiol. 29: 33–41.

    CAS  Google Scholar 

  9. Digan, M.E., Tschopp, J., Grinna, L., Lair, S.V., Craig, W.S., Velicelebi, G., Siegel, R., Davis, G.R. and Thill, G.P. 1988. Secretion of heterologous proteins from the methylotrophic yeast, Pichia pastoris . Dev. Ind. Microbiol. 29: 59–65.

    CAS  Google Scholar 

  10. Cregg, J.M., Digan, M.E., Tschopp, J.F., Brierley, R.A., Craig, W.S., Velicelebi, G., Siegel, R.S. and Thill, G.P. 1989. Expression of foreign genes in Pichia pastoris, p. 343–352. In: Genetics and Molecular Biology of Industrial Microorganisms. Hershberger, C. L., Queener, S. W. andHegeman, G. (Eds.). American Society for Microbiology, Washington, DC.

    Google Scholar 

  11. Thill, G.P., Davis, G.R., Stillman, C., Holtz, G., Brierley, R., Engel, M., Buckholz, R., Kenney, J., Provow, S., Vedvick, T. and Siegel, R.S. 1990. Positive and negative effects of multi-copy integrated expression vectors on protein expression in Pichia pastoris, p. 477–490. In: Proceedings of the 6th International Symposium on Genetics of Microorganisms, Vol. II. HeslotH., Davies, J., Florent, I., Bobichon, L., Durand, G. and Penasse, L. (Eds.). Societe Francaise de Microbiologie, Paris.

    Google Scholar 

  12. Vedvick, T.S. Gene expression in yeast: Pichia pastoris . Curr. Opinion Biotechnol. 2: 742–745 1991.

    Article  CAS  Google Scholar 

  13. Tschopp, J.F. and Cregg, J.M. 1991. Heterologous gene expression in methylotrophic yeast, p. 305–322. In: Biology of Methylotrophs. Goldberg, I. and Roken, I. S. (Eds.). Butterworth-Heinemann, Stoneham, MA.

    Chapter  Google Scholar 

  14. Ellis, S.B., Brust, P.F., Koutz, P.J., Waters, A.F., Harpold, M.M. and Gingeras, T.R. 1985. Isolation of alcohol oxidase and two other methanol regulatable genes from the yeast Pichia pastoris . Mol. Cell. Biol. 5: 1111–1121.

    Article  CAS  Google Scholar 

  15. Cregg, J.M., Madden, K.R., Barringer, K.J., Thill, G.P. and Stillman, C.A. 1989. Functional characterization of the two alcohol oxidase genes from the yeast Pichia pastoris . Mol. Cell. Biol. 9: 1316–1323.

    Article  CAS  Google Scholar 

  16. Koutz, P., Davis, G.R., Stillman, C., Barringer, K., Cregg, J. and Thill, G. 1989. Structural comparison of the Pichia pastoris alcohol oxidase genes. Yeast 5: 167–177.

    Article  CAS  Google Scholar 

  17. Tschopp, J.F., Brust, P.F., Cregg, J.M., Stillman, C.A. and Gingeras, T.R. 1987. Expression of the lacZ gene from two methanol-regulated promoters in Pichia pastoris . Nucleic Acids Res. 15: 3859–3876.

    Article  CAS  Google Scholar 

  18. Cregg, J.M. 1987. Genetics of methylotrophic yeasts, p. 158–167. In: Proceedings of the Fifth International Symposium on Microbial Growth on C1 Compounds. Duine, J. A. and van Verseveld, H. W. (Eds.). Martinus Nijhoff Publishers, Dordrecht, The Netherlands.

    Chapter  Google Scholar 

  19. Liu, H., Tan, X., Veenhuis, M., McCollum, D. and Cregg, J.M. 1992. An efficient screen for peroxisome-deficient mutants of Pichia pastoris. J. Bacteriol. 174: 4943–4951.

    Article  CAS  Google Scholar 

  20. Cregg, J.M., Barringer, K.J., Messier, A.Y. and Madden, K.R. 1985. Pichia pastoris as a host system for transformations. Mol. Cell. Biol. 5: 3376–3385.

    Article  CAS  Google Scholar 

  21. Cregg, J.M. and Madden, K.R. 1987. Development of yeast transformation systems and construction of methanol-utilization-defective mutants of Pichia pastoris by gene disruption, p. 1–18. In: Biological Research on Industrial Yeasts, Vol. II. StewartG. G., Russell, I., Klein, R. D. and Hiebsch, R. R. (Eds.). CRC Press, Boca Raton, FL.

    Google Scholar 

  22. Cregg, J.M. and Madden, K.R. 1989. Use of site-specific recombination to regenerate selectable markers. Mol. Gen. Genet. 219: 320–323.

    Article  CAS  Google Scholar 

  23. Tschopp, J.F., Sverlow, G., Kosson, R., Craig, W. and Grinna, L. 1987. High-level secretion of glycosylated invertase in the methylotrophic yeast, Pichia pastoris . Bio/Technology 5: 1305–1308.

    CAS  Google Scholar 

  24. Vedvick, T., Buckholz, R.G., Engel, M., Urcan, M., Kinney, J., Provow, S., Siegel, R.S. and Thill, G.P. 1991. High-level secretion of biologically active aprotinin from the yeast Pichiapastoris. J. Ind. Microbiol. 7: 197–201.

    Article  CAS  Google Scholar 

  25. Clare, J.J., Romanos, M.A., Rayment, F.B., Rowedder, J.E., Smith, M.A., Payne, M.M., Sreekrishna, K. and Henwood, C.A. 1991. Production of mouse epidermal growth factor in yeast: high-level secretion using Pichia pastoris strains containing multiple gene copies. Gene 105: 205–212.

    Article  CAS  Google Scholar 

  26. Cregg, J.M., Tschopp, J.F., Stillman, C., Siegel, R., Akong, M., Craig, W.S., Buckholz, R.G., Madden, K.R., Kellaris, P.A., Davis, G.R., Smiley, B.L., Cruze, J., Torregrossa, R., Velicelebi, G. and Thill, G.P. 1987. High-level expression and efficient assembly of hepatitis B surface antigen in the methylotrophic yeast, Pichia pastoris . Bio/Technology 5: 479–485.

    CAS  Google Scholar 

  27. Digan, M.E., Lair, S.V., Brierley, R.A., Siegel, R.S., Williams, M.E., Ellis, S.B., Kellaris, P.A., Provow, S.A., Craig, W.S., Velicelebi, G., Harpold, M.M. and Thill, G.P. 1989. Continuous production of a novel lysozyme via secretion from the yeast, Pichia pastoris . Bio/Technology 7: 160–164.

    CAS  Google Scholar 

  28. Sreekrishna, K., Nelles, L., Potenz, R., Cruze, J., Mazzaferro, P., Fish, W., Fuke, M., Holden, K., Phelps, D., Wood, P. and Parker, K. 1989. High-level expression, purification, and characterization of recombinant human tumor necrosis factor synthesized in the methylotrophic yeast Pichia pastoris. Biochemistry 28: 4117–4125.

    Article  CAS  Google Scholar 

  29. Clare, J.J., Rayment, F.B., Ballantine, S.P., Sreekrishna, K. and Romanos, M.A. 1991. High-level expression of tetanus toxin fragment C in Pichia pastoris strains containing multiple tandem integrations of the gene. Bio/Technology 9: 455–460.

    CAS  Google Scholar 

  30. Barr, K.A., Hopkins, S.A. and Sreekrishna, K. 1992. Protocol for efficient secretion of HSA developed from Pichia pastoris . Pharm. Eng. 12: 48–51.

    Google Scholar 

  31. Romanos, M.A., Clare, J.J., Beesley, K.M., Rayment, F.B., Ballantine, S.P., Makoff, A.J., Dougan, G., Fairweather, N.F. and Charles, I.G. 1991. Recombinant Bordetella pertussis pertactin (P69) from the yeast Pichia pastoris: high-level production and immunological properties. Vaccine 9: 901–906.

    Article  CAS  Google Scholar 

  32. Siegel, R.S. and Brierley, R.A. 1989. Methylotrophic yeast Pichia pastoris produced in high-cell-density fermentations with high cell yields as vehicle for recombinant protein production. Biotechnol. Bioeng. 34: 403–404.

    Article  CAS  Google Scholar 

  33. Brierley, R.A., Siegel, R.S., Bussineau, C.M., Craig, W.S., Holtz, G.C., Davis, G.R., Buckholz, R.G., Thill, G.P., Wondrack, L.M., Digan, M.E., Harpold, M.M., Lair, S.V., Ellis, S.B. and Williams, M.E. 1989. Mixed feed recombinant yeast fermentation. International Patent Application, Publication No. WO 90/03431.

  34. Brierley, R.A., Bussineau, C., Kosson, R., Melton, A. and Siegel, R.S. 1990. Development of recombinant Pichia pastoris expressing the heterologous gene bovine lysozyme. Biochem. Eng. VI 589: 350–362.

    Google Scholar 

  35. Siegel, R.S., Buckholz, R.G., Thill, G.P. and Wondrack, L.M. 1990. Production of epidermal growth factor in methylotrophic yeast cells. International Patent Application, Publication No. WO 90/10697.

  36. Hirsch, H.H., Rendueles, P.S. and Wolf, D.H. 1989. Yeast (Saccharomyces cerevisiae) proteinases: structure, characteristics and function, p. 134–200. In: Molecular and Cell Biology of Yeasts. Walton, E. F. and Yarranton, G. T. (Eds.). Blackie and van Nostrand Reinhold, New York.

    Google Scholar 

  37. Goochee, C.F., Gramer, M.J., Andersen, D.C., Bahr, J.B. and Rasmussen, J.R. 1991. The oligosaccharides of glycoproteins: bioprocess factors affecting oligosaccharide structure and their effect on glycoprotein properties. Bio/Technology 9: 1347–1355.

    Article  CAS  Google Scholar 

  38. Kukuruzinska, M.A., Bergh, M.L.E. and Jackson, B.J. 1987. Protein glycosylation in yeast. Ann. Rev. Biochem. 56: 915–944.

    Article  CAS  Google Scholar 

  39. Grinna, L.S. and Tschopp, J.F. 1989. Size distribution and general structural features of N-linked oligosaccharides from the methylotrophic yeast, Pichia pastoris . Yeast 5: 107–115.

    Article  CAS  Google Scholar 

  40. Byrd, J.C., Tarentino, A.L., Maley, F., Atkinson, P.H. and Trimble, R.B. 1982. Glycoprotein synthesis in yeast identificaiton of Man8 GlcNAc2 as an essential intermediate in oligosaccharide processing. J. Biol. Chem. 257: 14657–14666.

    CAS  Google Scholar 

  41. Trimble, R.B., Atkinson, P.H., Tschopp, J.F., Townsend, R.R. and Maley, F. 1991. Structure of oligosaccharides on Saccharomyces SUC2 invertase secreted by the methylotrophic yeast Pichia pastoris . J. Biol. Chem. 266: 22807–22817.

    CAS  PubMed  Google Scholar 

  42. Wagner, S.L., Siegel, R.S., Vedvick, T.S., Raschke, W.C. and van Nostrand, W.E. 1992 High-level expression, purification and characterization of the Kunitz-type protease inhibitor domain of protease nexin-2/amyloid β-protein precursor. Biochem. Biophys. Res. Commun. 186: 1138–1145.

    Article  CAS  Google Scholar 

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  1. James M. Cregg: Corresponding author.

Authors and Affiliations

  1. Department of Chemical and Biological Sciences, Oregon Graduate Institute of Science and Technology, Beaverton, OR, 97006-1999

    James M. Cregg

  2. The Salk Institute Biotechnology/Industrial Associates, Inc., La Jolla, CA, 92037-4641

    Thomas S. Vedvick & William C. Raschke

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  3. William C. Raschke
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Cregg, J., Vedvick, T. & Raschke, W. Recent Advances in the Expression of Foreign Genes in Pichia pastoris. Nat Biotechnol 11, 905–910 (1993). https://doi.org/10.1038/nbt0893-905

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