title: Long-term adaptation of Saccharomyces cerevisiae to the burden of recombinant insulin production.
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.
authors.affiliation: Industrial Biotechnology, Department of Chemical and Biological Engineering, Chalmers University of Technology, Kemivaegen 10, 41296, Gothenburg, Sweden.
authors.forename: Ali
authors.initials: A
authors.lastname: Kazemi Seresht
authors.forename: Ana Luisa
authors.initials: AL
authors.lastname: Cruz
authors.forename: Erik
authors.initials: E
authors.lastname: de Hulster
authors.forename: Marit
authors.initials: M
authors.lastname: Hebly
authors.forename: Eva Akke
authors.initials: EA
authors.lastname: Palmqvist
authors.forename: Walter
authors.initials: W
authors.lastname: van Gulik
authors.forename: Jean-Marc
authors.initials: JM
authors.lastname: Daran
authors.forename: Jack
authors.initials: J
authors.lastname: Pronk
authors.forename: Lisbeth
authors.initials: L
authors.lastname: Olsson
mesh_terms: D000220:Adaptation, Biological / Q000502:physiology; D000596:Amino Acids / Q000378:metabolism; D050976:Basic-Leucine Zipper Transcription Factors / Q000032:analysis / Q000378:metabolism; D019149:Bioreactors / Q000382:microbiology*; D018929:Cell Culture Techniques / Q000379:methods; D016000:Cluster Analysis; D005822:Genetic Vectors; D006801:Humans; D061385:Insulins / Q000032:analysis / Q000235:genetics / Q000378:metabolism*; D053858:Metabolic Networks and Pathways / Q000502:physiology; D055442:Metabolome / Q000502:physiology; D011994:Recombinant Proteins / Q000032:analysis / Q000235:genetics / Q000378:metabolism*; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000378:metabolism / Q000502:physiology*; D029701:Saccharomyces cerevisiae Proteins / Q000032:analysis / Q000378:metabolism; D059467:Transcriptome / Q000502:physiology
chemical_list: D000596:Amino Acids; D050976:Basic-Leucine Zipper Transcription Factors; C501391:GCN4 protein, S cerevisiae; D061385:Insulins; D011994:Recombinant Proteins; D029701:Saccharomyces cerevisiae Proteins
keywords: heterologous protein; metabolome; mitochondrial dysfunction; prolonged chemostat; time-course analysis; transcriptome