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


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.


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



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.


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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

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