The impact of anti-apoptotic gene Bcl-2∆ expression on CHO central metabolism |
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Institution: | 1. Department of Chemical and Biomolecular Engineering, Vanderbilt University; PMB 351604, 2301 Vanderbilt Place, Nashville, TN 37235-1604, USA;2. Department of Chemical and Biomolecular Engineering, Johns Hopkins University; 3400 North Charles Street, Baltimore, MD 21218, USA;3. Janssen Pharmaceutical J&J, Biologics Research, Biotechnology CoE; 1400 McKean Road, Spring House, PA, 19002 USA;4. Department of Molecular Physiology and Biophysics, Vanderbilt University; PMB 351604, 2301 Vanderbilt Place, Nashville, TN 37235-1604, USA;1. Department of Chemical and Biomolecular Engineering, Vanderbilt University, PMB 351604, Nashville, TN 37235-1604, USA;2. Department of Molecular Physiology and Biophysics, Vanderbilt University, PMB 351604, Nashville, TN 37235-1604, USA;1. Cell Technology Group, Department of Industrial Biotechnology/Bioprocess Design, School of Chemistry, Biotechnology and Health, KTH Royal Institute of Technology, Stockholm, Sweden;2. AdBIOPRO, VINNOVA Competence Centre for Advanced Bioproduction by Continuous Processing, Sweden;3. WCPR, Wallenberg Centre for Protein Research, Sweden;4. Department of Mathematics, Division of Optimization and Systems Theory, KTH Royal Institute of Technology, Stockholm, Sweden;1. Manchester Institute of Biotechnology, Faculty of Science and Engineering, University of Manchester, Manchester, UK;2. School of Biochemical Engineering, Pontificia Universidad Católica de Valparaíso, Valparaíso, Chile;3. Regional Center for Healthy Food Studies (CREAS) R17A10001, CONICYT REGIONAL, GORE Valparaiso, Chile;1. Institute for Chemical and Bioengineering, Department of Chemistry and Applied Biosciences, ETH Zurich, 8093 Zurich, Switzerland;2. Swiss Institute of Bioinformatics, 1015 Lausanne, Switzerland;3. Biotech Process Sciences, Merck Biopharma, 1804 Corsier-sur-Vevey, Switzerland;4. Department of Chemical Engineering, University of Chemistry and Technology, 166 28 Prague, Czech Republic;1. Department of Bioengineering, University of California, San Diego, La Jolla, CA 92093, USA;2. Novo Nordisk Foundation Center for Biosustainability at the School of Medicine, University of California, San Diego, La Jolla, CA 92093, USA;3. Department of Chemical and Biomolecular Engineering, National University of Singapore, 4 Engineering Drive 4, Singapore 117585, Singapore;5. Department of Biotechnology, University of Natural Resources and Life Sciences, Vienna, 1190 Vienna, Austria;6. Austrian Centre of Industrial Biotechnology, 1190 Vienna, Austria;7. Australian Institute for Bioengineering and Nanotechnology (AIBN), The University of Queensland, Corner College and Cooper Roads (Building 75), Brisbane, QLD 4072, Australia;8. Department of Chemical and Biomolecular Engineering, Johns Hopkins University, Baltimore, MD 21218, USA;9. Bioinformatics and Systems Biology Program, University of California, San Diego, La Jolla 92093, CA, USA;10. Department of Systems Biology, Technical University of Denmark, 2800 Kgs. Lyngby, Denmark;11. Novo Nordisk Foundation Center for Biosustainability, Technical University of Denmark, 2800 Kgs. Lyngby, Denmark;12. Centre for Biotechnology and Bioengineering, Department of Chemical Engineering and Biotechnology, University of Chile, Santiago 8370456, Chile;13. MATHomics, Center for Mathematical Modeling; Center for Genome Regulation (Fondap 15090007), University of Chile, Santiago 8370456, Chile;14. Center for Systems Biology, University of Iceland, 101 Reykjavik, Iceland;15. Computational Bioscience Research Centre, King Abdullah University of Science and Technology, Thuwal 23955, Saudi Arabia;16. Department of Pediatrics, University of California, San Diego, La Jolla, CA 92093, USA |
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Abstract: | Anti-apoptosis engineering is an established technique to prolong the viability of mammalian cell cultures used for industrial production of recombinant proteins. However, the effect of overexpressing anti-apoptotic proteins on central carbon metabolism has not been systematically studied. We transfected CHO-S cells to express Bcl-2∆, an engineered anti-apoptotic gene, and selected clones that differed in their Bcl-2∆ expression and caspase activity. 13C metabolic flux analysis (MFA) was then applied to elucidate the metabolic alterations induced by Bcl-2∆. Expression of Bcl-2Δ reduced lactate accumulation by redirecting the fate of intracellular pyruvate toward mitochondrial oxidation during the lactate-producing phase, and it significantly increased lactate re-uptake during the lactate-consuming phase. This flux redistribution was associated with significant increases in biomass yield, peak viable cell density (VCD), and integrated VCD. Additionally, Bcl-2∆ expression was associated with significant increases in isocitrate dehydrogenase and NADH oxidase activities, both rate-controlling mitochondrial enzymes. This is the first comprehensive 13C MFA study to demonstrate that expression of anti-apoptotic genes has a significant impact on intracellular metabolic fluxes, especially in controlling the fate of pyruvate carbon, which has important biotechnology applications for reducing lactate accumulation and enhancing productivity in mammalian cell cultures. |
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Keywords: | Apoptosis Central metabolism Mitochondria Lactate Metabolic flux analysis (MFA) Chinese hamster ovary (CHO) Bcl-2 |
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