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Metabolic Flux Rewiring in Mammalian Cell Cultures Young 1109 Available online at www.sciencedirect.com Metabolic flux rewiring in mammalian cell cultures 1,2 Jamey D Young Continuous cell lines (CCLs) engage in ‘wasteful’ glucose and by this substrate is also ‘wasted’ due to elevated production glutamine metabolism that leads to accumulation of inhibitory of ammonium and alanine [3]. These observations imply byproducts, primarily lactate and ammonium. Advances in that the metabolic phenotypes of CCLs are not pro- techniques for mapping intracellular carbon fluxes and profiling grammed to economize their use of carbon, nitrogen, or global changes in enzyme expression have led to a deeper energetic resources, but instead tend to increase their understanding of the molecular drivers underlying these nutrient uptake beyond what is required for growth [4]. metabolic alterations. However, recent studies have revealed This leads to accumulation of excreted byproducts, prim- that CCLs are not necessarily entrenched in a glycolytic or arily lactate and ammonium, which reduce cell viability glutaminolytic phenotype, but instead can shift their and recombinant protein yields and introduce unwanted metabolism toward increased oxidative metabolism as variability into cell culture bioprocesses [2]. nutrients become depleted and/or growth rate slows. Progress to understand dynamic flux regulation in CCLs has enabled the Minimizing wasteful byproduct accumulation has been a development of novel strategies to force cultures into desirable goal of the mammalian biotechnology industry for over 25 metabolic phenotypes, by combining fed-batch feeding years, but it still remains a poorly understood and often ill- strategies with direct metabolic engineering of host cells. controlled problem [5 ]. Furthermore, many production Addresses cultures can exhibit dramatic shifts in metabolic pheno- 1 Department of Chemical and Biomolecular Engineering, PMB 351604, type during the course of a typical bioprocess run, yet the Vanderbilt University, Nashville, TN 37235-1604, USA molecular mechanisms responsible for dynamic nutrient 2 Department of Molecular Physiology and Biophysics, PMB 351604, sensing and metabolic response to changing environmen- Vanderbilt University, Nashville, TN 37235-1604, USA tal conditions are only now beginning to emerge [6 ]. Corresponding author: Young, Jamey D ([email protected]) This review aims to present recent progress in under- standing the causes and consequences of metabolic repro- gramming in mammalian cell cultures, as well as Current Opinion in Biotechnology 2013, 24:1108–1115 engineering strategies that have been applied to suppress This review comes from a themed issue on Pharmaceutical undesirable metabolic phenotypes in industrial biopro- biotechnology cesses. Much of this progress has been enabled by Edited by Ajikumar Parayil and Federico Gago advances in techniques for mapping intracellular carbon For a complete overview see the Issue and the Editorial fluxes using isotope tracing and metabolic flux analysis (MFA), combined with approaches for profiling global Available online 28th May 2013 changes in expression and posttranslational modification 0958-1669/$ – see front matter, # 2013 Elsevier Ltd. All rights (PTM) of metabolic enzymes and regulatory proteins. reserved. http://dx.doi.org/10.1016/j.copbio.2013.04.016 Metabolic physiology of mammalian cell cultures Why do CCLs rely on aerobic glycolysis for proliferation? Introduction Although hypotheses abound as to the adaptive Continuous cell lines (CCLs) require constant availability advantage provided by aerobic glycolysis, little consensus of carbon, nitrogen, energy (ATP), and reductant has been achieved in the 85 years since this paradoxical (NADPH) to sustain their anabolic functions. Most CCLs, metabolic shift was first reported by the German bio- such as those used in industrial bioprocesses, rely heavily chemist Otto Warburg through his studies of rat tumor upon aerobic glycolysis to supply the energetic demands of tissues [7]. It is important to note that this effect is not cell growth, which involves rapid conversion of glucose to restricted to mammalian cells, but is in fact analogous to lactate even in the presence of abundant oxygen [1] the well-known Crabtree effect whereby yeast shift to (Figure 1a). However, glycolysis provides only 2 moles aerobic ethanol production during rapid growth on glu- of ATP per mole of glucose consumed, whereas mitochon- cose [8] or in response to cell-cycle dysregulation [9]. One drial oxidative phosphorylation (OXPHOS) can provide up explanation for this metabolic flux rewiring is that, while to 36 moles of ATP from the same amount of glucose. As a quiescent cells utilize mitochondria chiefly as a catabolic result, aerobic glycolysis is considered ‘wasteful’ from a engine to produce ATP, proliferating cells must repur- bioenergetic and biosynthetic standpoint because it does pose their mitochondria to supply biosynthetic intermedi- not make efficient use of glucose to supply either ATP or ates: citrate is exported to supply carbon for lipid carbon to the cell [2]. Increased consumption of glutamine biosynthesis, oxaloacetate and alpha-ketoglutarate may is also exhibited by many CCLs, but the nitrogen provided be withdrawn for amino acid or nucleotide biosynthesis, Current Opinion in Biotechnology 2013, 24:1108–1115 www.sciencedirect.com Metabolic flux rewiring in mammalian cell cultures Young 1109 Figure 1 (a) Glucose (b) Glucose PPP G6PG66P PPP G6PGG6 NADN + NAD+ ATP NADHN ATP NADH PyrPyyr Lactate PyrPy Lactate + + NH4 NH4 TCA TCA ATP Glutamine ATP Glutamine cycle cycle Exponential phase Stationary phase Current Opinion in Biotechnology Typical metabolic phenotypes of proliferating and non-proliferating CCLs. (a) Exponentially growing cultures exhibit aerobic glycolysis and rely on elevated glutamine consumption to fuel mitochondrial metabolism. This results in increased lactate and ammonium production as cells rewire their metabolism to maintain carbon, nitrogen, and redox balance. (b) Stationary phase cultures metabolize glucose mainly by oxidation in the TCA cycle, which provides much higher ATP yields and reduced byproduct accumulation. An increased proportion of incoming glucose is diverted into the oxidative pentose phosphate pathway to maintain NADPH levels. and flux rerouting via malic enzyme (ME) and isocitrate rate-limiting enzyme of glycolysis in many industrial cell dehydrogenase (IDH) reactions can be used to supply lines [16,17]. anabolic reducing power in the form of NADPH [10] 13 (Figure 2). Indeed, C MFA studies have recently shown Expression of both GLUT1 and HK2 is controlled by the that flux leaving the TCA cycle as citrate can at times transcription factors HIF1 and c-Myc [18] and the sig- exceed the flux entering it from pyruvate, with the naling protein Akt [19], which are often dysregulated in additional citrate supplied by reductive carboxylation immortalized cells. These same proteins also control of glutamine via IDH operating in the retrograde direc- expression of several other key glycolytic enzymes that tion [11,12 ] (Figure 3b). With their mitochondria redir- are commonly upregulated in proliferating CCLs ected toward anabolic processes that utilize glutamine as (Figure 2): phosphofructokinase 1 (PFK1), the bifunc- a major carbon substrate, proliferating cultures shift tional enzyme phosphofructokinase 2/fructose-2,6- toward aerobic glycolysis to satisfy their cellular demands bisphosphatase (PFK2/FBPase), lactate dehydrogenase for ATP production. A (LDHA), and the M2 isoform of pyruvate kinase (PK) [13]. The connection between increased expression of glycolytic enzymes and cell immortalization is further Metabolic flux rewiring is associated with altered strengthened by the finding that either spontaneously expression and activity of metabolic enzymes immortalized mouse embryonic fibroblasts (MEFs) or Although the shift to aerobic glycolysis has been a scien- oncogene-induced human fibroblasts increase their gly- tific paradox—and a stumbling block of the mammalian colytic rate, and inhibition of any one of many different biotech industry — for some time, the underlying mol- glycolytic enzymes can induce MEF senescence [20,21]. ecular alterations associated with metabolic flux rewiring in CCLs have been largely undefined. However, recent Upregulation of glycolysis in CCLs is typically accom- studies have identified transcriptional and proteomic panied by downregulation of enzymes that facilitate signatures associated with increased aerobic glycolysis mitochondrial translocation and oxidation of glucose- that are conserved across several different cell and tissue derived pyruvate. For example, Neermann and Wagner types [13]. Overexpression of the high-affinity glucose [16] measured no detectable level of pyruvate dehydro- transporter GLUT1 and the initial glycolytic enzyme genase (PDH) or pyruvate carboxylase (PC) activity in a hexokinase 2 (HK2) is frequently observed in cancer cells wide range of CCL cultures, and similarly Fitzpatrick and transformed cell lines [14,15] (Figure 2). Studies in et al. [17] reported no detectable PDH activity in an hybridoma, Chinese hamster ovary (CHO), and baby antibody-secreting murine hybridoma cell line hamster kidney (BHK) cells found that hexokinase (Figure 2). In contrast, activities of both PDH and PC activity was consistently lowest among all glycolytic were detectable in insect cell lines and primary liver cells enzymes examined, suggesting that it may be the [16]. Low activity of PDH in CCLs may be attributable
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