Long-term adaptation of Saccharomyces cerevisiae to the burden of recombinant insulin production

Authors

  • Ali Kazemi Seresht,

    Corresponding author
    1. Protein Expression, Novo Nordisk A/S, Måløv, Denmark
    • Industrial Biotechnology, Department of Chemical and Biological Engineering, Chalmers University of Technology, Gothenburg, Sweden
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  • Ana Luisa Cruz,

    1. Department of Biotechnology, Delft University of Technology and Kluyver Centre for Genomics of Industrial Fermentation, BC Delft, The Netherlands
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  • Erik de Hulster,

    1. Department of Biotechnology, Delft University of Technology and Kluyver Centre for Genomics of Industrial Fermentation, BC Delft, The Netherlands
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  • Marit Hebly,

    1. Department of Biotechnology, Delft University of Technology and Kluyver Centre for Genomics of Industrial Fermentation, BC Delft, The Netherlands
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  • Eva Akke Palmqvist,

    1. Protein Expression, Novo Nordisk A/S, Måløv, Denmark
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  • Walter van Gulik,

    1. Department of Biotechnology, Delft University of Technology and Kluyver Centre for Genomics of Industrial Fermentation, BC Delft, The Netherlands
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  • Jean-Marc Daran,

    1. Department of Biotechnology, Delft University of Technology and Kluyver Centre for Genomics of Industrial Fermentation, BC Delft, The Netherlands
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  • Jack Pronk,

    1. Department of Biotechnology, Delft University of Technology and Kluyver Centre for Genomics of Industrial Fermentation, BC Delft, The Netherlands
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  • Lisbeth Olsson

    1. Industrial Biotechnology, Department of Chemical and Biological Engineering, Chalmers University of Technology, Gothenburg, Sweden
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Correspondence to: Lisbeth Olsson

e-mail lisbeth.olsson@chalmers.se

+46(0)31 772 3805

+46(0)31 772 3801

ABSTRACT

High-level production of heterologous proteins is likely to impose a metabolic burden on the host cell and can thus affect various aspects of cellular physiology. A data-driven approach was applied to study the secretory production of a human insulin analog precursor (IAP) in Saccharomyces cerevisiae during prolonged cultivation (80 generations) in glucose-limited aerobic chemostat cultures. Physiological characterization of the recombinant cells involved a comparison with cultures of a congenic reference strain that did not produce IAP, and time-course analysis of both strains aimed at identifying the metabolic adaptation of the cells towards the burden of IAP production. All cultures were examined at high cell density conditions (30 g/L dry weight) to increase the industrial relevance of the results. The burden of heterologous protein production in the recombinant strain was explored by global transcriptome analysis and targeted metabolome analysis, including the analysis of intracellular amino acid pools, glycolytic metabolites, and TCA intermediates. The cellular re-arrangements towards IAP production were categorized in direct responses, for example, enhanced metabolism of amino acids as precursors for the formation of IAP, as well as indirect responses, for example, changes in the central carbon metabolism. As part of the long-term adaptation, a metabolic re-modeling of the IAP-expressing strain was observed, indicating an augmented negative selection pressure on glycolytic overcapacity, and the emergence of mitochondrial dysfunction. The evoked metabolic re-modeling of the cells led to less optimal conditions with respect to the expression and processing of the target protein and thus decreased the cellular expression capacity for the secretory production of IAP during prolonged cultivation. Biotechnol. Bioeng. 2013;110: 2749–2763. © 2013 Wiley Periodicals, Inc.

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