Applied Microbiology and Biotechnology

, Volume 97, Issue 9, pp 3939–3948 | Cite as

Modulating heterologous protein production in yeast: the applicability of truncated auxotrophic markers

  • Ali Kazemi Seresht
  • Per Nørgaard
  • Eva Akke Palmqvist
  • Asser Sloth Andersen
  • Lisbeth Olsson
Biotechnologically Relevant Enzymes and Proteins

Abstract

The use of auxotrophic Saccharomyces cerevisiae strains for improved production of a heterologous protein was examined. Two different marker genes were investigated, encoding key enzymes in the metabolic pathways for amino acid (LEU2) and pyrimidine (URA3) biosynthesis, respectively. Expression plasmids, carrying the partly defective selection markers LEU2d and URA3d, were constructed. Two CEN.PK-derived strains were chosen and insulin analogue precursor was selected as a model protein. Different truncations of the LEU2 and URA3 promoters were used as the mean to titrate the plasmid copy number and thus the recombinant gene dosage in order to improve insulin productivity. Experiments were initially carried out in batch mode to examine the stability of yeast transformants and to select high yielding mutants. Next, chemostat cultivations were run at high cell density to address industrial applicability and long-term expression stability of the transformants. We found that the choice of auxotrophic marker is crucial for developing a yeast expression system with stable heterologous protein production. The incremental truncation of the URA3 promoter led to higher plasmid copy numbers and IAP yields, whereas the truncation of the LEU2 promoter caused low plasmid stability. We show that the modification of the level of the recombinant gene dosage by varying the degree of promoter truncation can be a strong tool for optimization of productivity. The application of the URA3d-based expression systems showed a high potential for industrial protein production and for further academic studies.

Keywords

Promoter truncation URA3d LEU2d Human insulin Plasmid copy number High cell density cultivation 

Notes

Acknowledgments

This work was financially supported by Novo Nordisk A/S and the Danish Ministry of Science Technology and Innovation (VTU).

Conflict of interest

The authors declare no competing interests.

References

  1. Çakar ZP, Sauer U, Bailey JE (1999) Metabolic engineering of yeast: the perils of auxotrophic hosts. Biotechnol Lett 21:611–616CrossRefGoogle Scholar
  2. Christianson TW, Sikorski RS, Dante M, Shero JH, Hieter P (1992) Multifunctional yeast high-copy-number shuttle vectors. Gene 110:119–122CrossRefGoogle Scholar
  3. Delorme E (1989) Transformation of Saccharomyces cerevisiae by electroporation. Appl Env Microbiol 55:2242–2246Google Scholar
  4. Dever TE (1997) Using GCN4 as a reporter of eIF2α phosphorylation and translational regulation in yeast. Method Enzymol 11:403–417CrossRefGoogle Scholar
  5. Erhart E, Hollenberg CP (1983) The presence of a defective LEU2 gene on 2μ DNA recombinant plasmids of Saccharomyces cerevisiae is responsible for curing and high copy number. J Bacteriol 156:625–635Google Scholar
  6. Gasser B, Sauer M, Maurer M, Stadlmayr G, Mattanovich D (2007) Transcriptomics-based identification of novel factors enhancing heterologous protein secretion in yeasts. Appl Environ Microb 73:6499–6507CrossRefGoogle Scholar
  7. Geymonat M, Spanos A, Sedgwick SG (2007) A Saccharomyces cerevisiae autoselection system for optimised recombinant protein expression. Gene 399:120–128CrossRefGoogle Scholar
  8. Gietz RD, Woods RA (2001) Genetic transformation of yeast. Biotechniques 30:816–831Google Scholar
  9. Gupta J (2002) Stability studies of recombinant Saccharomyces cerevisiae in the presence of varying selection pressure. Biotechnol Bioeng 78:475–488CrossRefGoogle Scholar
  10. Görgens JF, Planas J, van Zyl WH, Knoetze JH, Hahn-Hägerdal B (2004) Comparison of three expression systems for heterologous xylanase production by S. cerevisiae in defined medium. Yeast 21:1205–1217CrossRefGoogle Scholar
  11. Hinnebusch AG (1992) General and pathway-specific regulatory mechanisms controlling the synthesis of amino acid biosynthetic enzymes in Saccharomyces cerevisiae. In: Jones WE, Pringle JR, Broach JR (eds) The molecular and cellular biology of the yeast Saccharomyces gene expression 2. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, pp 319–414Google Scholar
  12. Jimenez A, Davies J (1980) Expression of a transposable antibiotic resistance element in Saccharomyces. Nature 287:869–871CrossRefGoogle Scholar
  13. Kern L, De Montigny J, Jund R, Lacroute F (1990) The FUR1 gene of Saccharomyces cerevisiae: cloning, structure and expression of wild-type and mutant alleles. Gene 88:149–157CrossRefGoogle Scholar
  14. Kjeldsen T, Ludvigsen S, Diers I, Balschmidt P, Sorensen AR, Kaarsholm NC (2002) Engineering-enhanced protein secretory expression in yeast with application to insulin. J Biol Chem 277:18245–182408CrossRefGoogle Scholar
  15. Krogh AM, Beck V, Christensen LH, Henriksen CM, Møller K, Olsson L (2008) Adaptation of Saccharomyces cerevisiae expressing a heterologous protein. J Biotechnol 137:28–33CrossRefGoogle Scholar
  16. Lichten M, Goldman AS (1995) Meiotic recombination hotspots. Annu Rev Genet 29:423–444CrossRefGoogle Scholar
  17. Loison G, Vidal A, Findeli A, Roitsch C, Balloul JM, Lemoine Y (1989) High level of expression of a protective antigen of schistosomes in Saccharomyces cerevisiae. Yeast 5:497–507CrossRefGoogle Scholar
  18. Lopes TS, de Wijs IJ, Steenhauer SI, Verbakel J, Planta RJ (1996) Factors affecting the mitotic stability of high-copy-number integration into the ribosomal DNA of Saccharomyces cerevisiae. Yeast 12:467–477CrossRefGoogle Scholar
  19. Moo-Young M, Chisti Y, Zhang Z, Garrido F, Banerjee U, Vlach D (1996) Bioprocessing with genetically modified and other organisms: case studies in processing constraints. Ann NY Acad Sci 782:391–401CrossRefGoogle Scholar
  20. Napp SJ, Da Silva NA (1993) Enhancement of cloned gene product synthesis via autoselection in recombinant Saccharomyces cerevisiae. Biotechnol Bioeng 41:801–810CrossRefGoogle Scholar
  21. Nielsen T, Holmberg S, Petersen J (1990) Regulated overproduction and secretion of yeast carboxypeptidase Y. Appl Microbiol Biot 33:307–312CrossRefGoogle Scholar
  22. Nishikawa S, Brodsky JL, Nakatsukasa K (2005) Roles of molecular chaperones in endoplasmic reticulum (ER) quality control and ER-associated degradation (ERAD). J Biochem 137:551–555CrossRefGoogle Scholar
  23. Okkels JS (1996) A URA3-promoter deletion in a pYES vector increases the expression level of a fungal lipase in Saccharomyces cerevisiae. Ann NY Acad Sci 782:202–207CrossRefGoogle Scholar
  24. Payne T, Finnis C, Evans L, Mead D, Avery S, Archer D, Sleep D (2008) Modulation of chaperone gene expression in mutagenized Saccharomyces cerevisiae strains developed for recombinant human albumin production results in increased production of multiple heterologous proteins. Appl Env Microbiol 74:7759–7766CrossRefGoogle Scholar
  25. Primrose S, Ehrlich S (1981) Isolation of plasmid deletion and study of their instability. Plasmid 6:193–201CrossRefGoogle Scholar
  26. Pronk JT (2002) Auxotrophic yeast strains in fundamental and applied research. Appl Env Microbiol 68:2095–2100CrossRefGoogle Scholar
  27. Ro D-K, Ouellet M, Paradise EM, Burd H, Eng D, Paddon CJ, Newman JD, Keasling JD (2008) Induction of multiple pleiotropic drug resistance genes in yeast engineered to produce an increased level of anti-malarial drug precursor, artemisinic acid. BMC Biotechnol 8:83CrossRefGoogle Scholar
  28. Robinson AS, Hines V, Wittrup KD (1994) Protein disulfide isomerase overexpression increases secretion of foreign proteins in Saccharomyces cerevisiae. Nature Biotechnol 12:381–384CrossRefGoogle Scholar
  29. Smith JD, Tang BC, Robinson AS (2004) Protein disulfide isomerase, but not binding protein, overexpression enhances secretion of a non-disulfide-bonded protein in yeast. Biotechnol Bioeng 85:340–350CrossRefGoogle Scholar
  30. Symington L, Brown A, Oliver SG, Greenwell P, Petes TD (1991) Genetic analysis of a meiotic recombination hotspot on chromosome III of Saccharomyces cerevisiae. Genetics 128:717–727Google Scholar
  31. Teixeira MC, Monteiro P, Jain P, Tenreiro S, Fernandes AR, Mira NP, Alenquer M, Freitas AT, Oliveira AL, Sa-Correia I (2006) The YEASTRACT database: a tool for the analysis of transcription regulatory associations in Saccharomyces cerevisiae. Nucleic Acids Res 34:D446–D451CrossRefGoogle Scholar
  32. Ugolini S, Tosato V, Bruschi CV (2002) Selective fitness of four episomal shuttle-vectors carrying HIS3, LEU2, TRP1, and URA3 selectable markers in Saccharomyces cerevisiae. Plasmid 47:94–107CrossRefGoogle Scholar
  33. Verduyn C, Postma E, Scheffers WA, Van Dijken JP (1992) Effect of benzoic acid on metabolic fluxes in yeasts: a continuous-culture study on the regulation of respiration and alcoholic fermentation. Yeast 8:501–517CrossRefGoogle Scholar
  34. Walsh G (2005) Therapeutic insulins and their large-scale manufacture. Appl Microbiol Biot 67:151–159CrossRefGoogle Scholar
  35. Wentz AE, Shusta EV (2007) A novel high-throughput screen reveals yeast genes that increase secretion of heterologous proteins. Appl Env Microbiol 73:1189–1198CrossRefGoogle Scholar
  36. Wu T, Lichten M (1994) Meiosis-induced double-strand break sites determined by yeast chromatin structure. Science 263:515–518CrossRefGoogle Scholar
  37. Zhang Z, Moo-Young M, Chisti Y (1996) Plasmid stability in recombinant Saccharomyces cerevisiae. Biotechnol Adv 14:401–435CrossRefGoogle Scholar

Copyright information

© Springer-Verlag 2012

Authors and Affiliations

  • Ali Kazemi Seresht
    • 1
    • 2
  • Per Nørgaard
    • 1
  • Eva Akke Palmqvist
    • 1
  • Asser Sloth Andersen
    • 1
  • Lisbeth Olsson
    • 2
  1. 1.Protein Expression, Novo Nordisk A/SMåløvDenmark
  2. 2.Industrial Biotechnology, Department of Chemical and Biological EngineeringChalmers University of TechnologyGothenburgSweden

Personalised recommendations