title: The impact of anti-apoptotic gene Bcl-2∆ expression on CHO central metabolism.
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. (13)C 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 (13)C 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.
authors.affiliation: Department of Chemical and Biomolecular Engineering, Vanderbilt University; PMB 351604, 2301 Vanderbilt Place, Nashville, TN 37235-1604, USA.
authors.forename: Neil
authors.initials: N
authors.lastname: Templeton
authors.affiliation: Department of Chemical and Biomolecular Engineering, Johns Hopkins University; 3400 North Charles Street, Baltimore, MD 21218, USA.
authors.forename: Abasha
authors.initials: A
authors.lastname: Lewis
authors.affiliation: Janssen Pharmaceutical J&J, Biologics Research, Biotechnology CoE; 1400 McKean Road, Spring House, PA, 19002 USA.
authors.forename: Haimanti
authors.initials: H
authors.lastname: Dorai
authors.affiliation: Department of Chemical and Biomolecular Engineering, Vanderbilt University; PMB 351604, 2301 Vanderbilt Place, Nashville, TN 37235-1604, USA.
authors.forename: Elaine A
authors.initials: EA
authors.lastname: Qian
authors.affiliation: Janssen Pharmaceutical J&J, Biologics Research, Biotechnology CoE; 1400 McKean Road, Spring House, PA, 19002 USA.
authors.forename: Marguerite P
authors.initials: MP
authors.lastname: Campbell
authors.affiliation: Janssen Pharmaceutical J&J, Biologics Research, Biotechnology CoE; 1400 McKean Road, Spring House, PA, 19002 USA.
authors.forename: Kevin D
authors.initials: KD
authors.lastname: Smith
authors.affiliation: Janssen Pharmaceutical J&J, Biologics Research, Biotechnology CoE; 1400 McKean Road, Spring House, PA, 19002 USA.
authors.forename: Steven E
authors.initials: SE
authors.lastname: Lang
authors.affiliation: Department of Chemical and Biomolecular Engineering, Johns Hopkins University; 3400 North Charles Street, Baltimore, MD 21218, USA.
authors.forename: Michael J
authors.initials: MJ
authors.lastname: Betenbaugh
authors.affiliation: Department of Chemical and Biomolecular Engineering, Vanderbilt University; PMB 351604, 2301 Vanderbilt Place, Nashville, TN 37235-1604, USA; Department of Molecular Physiology and Biophysics, Vanderbilt University; PMB 351604, 2301 Vanderbilt Place, Nashville, TN 37235-1604, USA. Electronic address: j.d.young@vanderbilt.edu.
authors.forename: Jamey D
authors.initials: JD
authors.lastname: Young
mesh_terms: D000818:Animals; D051017:Apoptosis Regulatory Proteins / Q000235:genetics / Q000378:metabolism*; D016466:CHO Cells; D006224:Cricetinae; D003412:Cricetulus; D019344:Lactic Acid / Q000378:metabolism*; D064688:Metabolic Flux Analysis / Q000379:methods*; D024101:Mitochondrial Proteins / Q000235:genetics / Q000378:metabolism*; D019253:Proto-Oncogene Proteins c-bcl-2 / Q000235:genetics / Q000378:metabolism*; D019289:Pyruvic Acid / Q000378:metabolism*; D015398:Signal Transduction / Q000502:physiology*
chemical_list: D051017:Apoptosis Regulatory Proteins; D024101:Mitochondrial Proteins; D019253:Proto-Oncogene Proteins c-bcl-2; D019344:Lactic Acid; D019289:Pyruvic Acid
keywords: Apoptosis; Bcl-2; Central metabolism; Chinese hamster ovary (CHO); Lactate; Metabolic flux analysis (MFA); Mitochondria