Metabolic adaptation of Escherichia coli during temperature-induced recombinant protein production: 2. Redirection of metabolic fluxes

The impact of temperature-induced synthesis of human basic fibroblast growth factor (hFGF-2) in high-cell-density cultures of recombinant Escherichia coli was studied by estimating metabolic flux variations. Metabolic flux distributions in E. coli were calculated by means of a stoichiometric network and linear programming. After the temperature upshift, a substantially elevated energy demand for synthesis of hFGF-2 and heat shock proteins resulted in a redirection of metabolic fluxes. Catabolic pathways like the Embden-Meyerhof-Parnas pathway and the tricarboxylic acid (TCA) cycle showed significantly enhanced activities, leading to reduced flux to growth-associated pathways like the pentose phosphate pathway and other anabolic pathways. Upon temperature upshift, an excess of NADPH was produced in the TCA cycle by isocitrate dehydrogenase. The metabolic model predicted the involvement of a transhydrogenase generating additional NADH from NADPH, thereby increasing ATP regeneration in the respiratory chain. The influence of the temperature upshift on the host's metabolism was investigated by means of a control strain harboring the "empty" parental expression vector. The metabolic fluxes after the temperature upshift were redirected similarly to the production strain; the effects, however, were observed to a lesser extent and with different time profiles.     

Changes of in vivo fluxes through central metabolic pathways during the production of nystatin by Streptomyces noursei in batch culture

The central carbon metabolism of the nystatin-producing strain Streptomyces noursei ATCC 11455 was evaluated by 13C-labelling experiments. A batch fermentation was examined during the idiophase by GC-MS measurements of the labelling patterns of amino acids in the biomass. The labelling patterns of the amino acids and calculated fluxes of the central metabolism showed that changes in the primary and secondary metabolisms occurred simultaneously. Changes in the profiles for the integrated fluxes showed a decreased flux through the pentose phosphate pathway and an increased flux in the tricarboxylic acid cycle relative to the glucose uptake rate when the culture entered a phase with reduced specific growth rate and enhanced nystatin yield. The flux through the pentose phosphate pathway seemed to be adjusted according to the NADPH requirement during the different phases of the batch fermentation.     

Metabolic flux analysis of CHO cell metabolism in the late non‐growth phase

Chinese hamster ovary (CHO) cell cultures are commonly used for production of recombinant human therapeutic proteins. Often the goal of such a process is to separate the growth phase of the cells, from the non‐growth phase where ideally the cells are diverting resources to produce the protein of interest. Characterizing the way that the cells use nutrients in terms of metabolic fluxes as a function of culture conditions can provide a deeper understanding of the cell biology offering guidance for process improvements. To evaluate the fluxes, metabolic flux analysis of the CHO cell culture in the non‐growth phase was performed by a combination of steady‐state isotopomer balancing and stoichiometric modeling. Analysis of the glycolytic pathway and pentose phosphate pathway (PPP) indicated that almost all of the consumed glucose is diverted towards PPP with a high NADPH production; with even recycle from PPP to G6P in some cases. Almost all of the pyruvate produced from glycolysis entered the TCA cycle with little or no lactate production. Comparison of the non‐growth phase against previously reported fluxes from growth phase cultures indicated marked differences in the fluxes, in terms of the split between glycolysis and PPP, and also around the pyruvate node. Possible reasons for the high NADPH production are also discussed. Evaluation of the fluxes indicated that the medium strength, carbon dioxide level, and temperature with dissolved oxygen have statistically significant impacts on different nodes of the flux network. Biotechnol. Bioeng. 2011; 108:82–92. © 2010 Wiley Periodicals, Inc.     

Metabolic flux analysis of CHO cells at growth and non-growth phases using isotopic tracers and mass spectrometry

Chinese hamster ovary (CHO) cells are the main platform for production of biotherapeutics in the biopharmaceutical industry. However, relatively little is known about the metabolism of CHO cells in cell culture. In this work, metabolism of CHO cells was studied at the growth phase and early stationary phase using isotopic tracers and mass spectrometry. CHO cells were grown in fed-batch culture over a period of six days. On days 2 and 4, [1,2-¹³C] glucose was introduced and the labeling of intracellular metabolites was measured by gas chromatography-mass spectrometry (GC–MS) at 6, 12 and 24 h following the introduction of tracer. Intracellular metabolic fluxes were quantified from measured extracellular rates and ¹³C-labeling dynamics of intracellular metabolites using non-stationary ¹³C-metabolic flux analysis (¹³C-MFA). The flux results revealed significant rewiring of intracellular metabolic fluxes in the transition from growth to non-growth, including changes in energy metabolism, redox metabolism, oxidative pentose phosphate pathway and anaplerosis. At the exponential phase, CHO cell metabolism was characterized by a high flux of glycolysis from glucose to lactate, anaplerosis from pyruvate to oxaloacetate and from glutamate to α-ketoglutarate, and cataplerosis though malic enzyme. At the stationary phase, the flux map was characterized by a reduced flux of glycolysis, net lactate uptake, oxidative pentose phosphate pathway flux, and reduced rate of anaplerosis. The fluxes of pyruvate dehydrogenase and TCA cycle were similar at the exponential and stationary phase. The results presented here provide a solid foundation for future studies of CHO cell metabolism for applications such as cell line development and medium optimization for high-titer production of recombinant proteins.     

Robustness analysis of the Escherichia coli metabolic network

Genomic, biochemical, and strain-specific data can be assembled to define an in silico representation of the metabolic network for a select group of single cellular organisms. Flux-balance analysis and phenotypic phase planes derived therefrom have been developed and applied to analyze the metabolic capabilities and characteristics of Escherichia coli K-12. These analyses have shown the existence of seven essential reactions in the central metabolic pathways (glycolysis, pentose phosphate pathway, tricarboxylic acid cycle) for the growth in glucose minimal media. The corresponding seven gene products can be grouped into three categories: (1) pentose phosphate pathway genes, (2) three-carbon glycolytic genes, and (3) tricarboxylic acid cycle genes. Here we develop a procedure that calculates the sensitivity of optimal cellular growth to altered flux levels of these essential gene products. The results indicate that the E. coli metabolic network is robust with respect to the flux levels of these enzymes. The metabolic flux in the transketolase and the tricarboxylic acid cycle reactions can be reduced to 15% and 19%, respectively, of the optimal value without significantly influencing the optimal growth flux. The metabolic network also exhibited robustness with respect to the ribose-5-phosphate isomerase, and the ribose-5-phosephate isomerase flux was reduced to 28% of the optimal value without significantly effecting the optimal growth flux. The metabolic network exhibited limited robustness to the three-carbon glycolytic fluxes both increased and decreased. The development presented another dimension to the use of FBA to study the capabilities of metabolic networks.     

Comparative study on central metabolic fluxes of <Emphasis Type="Italic">Bacillus megaterium</Emphasis> strains in continuous culture using <Superscript>13</Superscript>C labelled substrates

Fluxes of central carbon metabolism [glycolysis, pentose phosphate pathway (PPP), tricarboxylic acid cycle (TCA cycle), biomass formation] were determined for several Bacillus megaterium strains (DSM319, WH320, WH323, MS941) in C- and N-limited chemostat cultures by ¹³C labelling experiments. The labelling patterns of proteinogenic amino acids were analysed by GC/MS and therefrom flux ratios at important nodes within the metabolic network could be calculated. On the basis of a stoichiometric metabolic model flux distributions were estimated for the different B. megaterium strains used at various cultivation conditions. Generally all strains exhibited similar metabolic flux distributions, however, several significant changes were found in (1) the glucose flux entering the PPP via the oxidative branch, (2) the reversibilities within the PPP, (3) the relative fluxes of pyruvate and acetyl-CoA fed to the TCA cycle, (4) the fluxes around the pyruvate node involving a futile cycle.     

Effect of different carbon sources on central metabolic fluxes and the recombinant production of a hydrolase from Thermobifida fusca in Bacillus megaterium

The recombinant Bacillus megaterium strain WH323 was employed for the inducible production and secretion of recombinant Thermobifida fusca hydrolase (TFH). Continuous cultivations were carried out in a chemostat using either glucose or pyruvate as sole carbon source. A remarkable increase of produced TFH was detected for the pyruvate-dependent cultivation compared to glucose-dependent growth. Estimation of intracellular carbon fluxes through the central metabolism for both growth conditions using (13)C-labelled substrates revealed noticeable changes of the fluxes through the tricarboxylic acid cycle, the pentose phosphate pathway and around the pyruvate node when protein production was induced. With pyruvate as sole carbon source the observed alterations of the fluxes yielded an increased production of ATP and NADPH both required for the anabolism. Additionally, the analysis of the corresponding secretome revealed significantly reduced amounts of extracellular proteases in the medium compared to glucose-grown cultivations. Thus, pyruvate-dependent chemostat cultivation was identified as a favourable condition for production and secretion of recombinant TFH using B. megaterium as production host.     

Aerobic glucose metabolism of Saccharomyces kluyveri: growth, metabolite production, and quantification of metabolic fluxes.

The growth and product formation of Saccharomyces kluyveri was characterized in aerobic batch cultivation on glucose. At these conditions it was found that ethyl acetate was a major overflow metabolite in S. kluyveri. During the exponential-growth phase on glucose ethyl acetate was produced at a constant specific rate of 0.12 g ethyl acetate per g dry weight per hour. The aerobic glucose metabolism in S. kluyveri was found to be less fermentative than in S. cerevisiae, as illustrated by the comparably low yield of ethanol on glucose (0.08 +/- 0.02 g/g), and high yield of biomass on glucose (0.29 +/- 0.01 g/g). The glucose metabolism of S. kluyveri was further characterized by the new and powerful techniques of metabolic network analysis. Flux distributions in the central carbon metabolism were estimated for respiro-fermentative growth in aerobic batch cultivation on glucose and respiratory growth in aerobic glucose-limited continuous cultivation. It was found that in S. kluyveri the flux into the pentose phosphate pathway was 18.8 mmole per 100 mmole glucose consumed during respiratory growth in aerobic glucose-limited continuous cultivation. Such a low flux into the pentose phosphate pathway cannot provide the cell with enough NADPH for biomass formation which is why the remaining NADPH will have to be provided by another pathway. During batch cultivation of S. kluyveri the tricarboxylic acid cycle was working as a cycle with a considerable flux, that is in sharp contrast to what has previously been observed in S. cerevisiae at the same growth conditions, where the tricarboxylic acid cycle operates as two branches. This indicates that the respiratory system was not significantly repressed in S. kluyveri during batch cultivation on glucose.     

Overcoming the metabolic burden of protein secretion in Schizosaccharomyces pombe – A quantitative approach using 13C-based metabolic flux analysis

Protein secretion in yeast is generally associated with a burden to cellular metabolism. To investigate this metabolic burden in Schizosaccharomyces pombe, we constructed a set of strains secreting the model protein maltase in different amounts. We quantified the influence of protein secretion on the metabolism applying ¹³C-based metabolic flux analysis in chemostat cultures. Analysis of the macromolecular biomass composition revealed an increase in cellular lipid content at elevated levels of protein secretion and we observed altered metabolic fluxes in the pentose phosphate pathway, the TCA cycle, and around the pyruvate node including mitochondrial NADPH supply. Supplementing acetate to glucose or glycerol minimal media was found to improve protein secretion, accompanied by an increased cellular lipid content and carbon flux through the TCA cycle as well as increased mitochondrial NADPH production. Thus, systematic metabolic analyses can assist in identifying factors limiting protein secretion and in deriving strategies to overcome these limitations.     

Metabolic flux distribution for γ-linolenic acid synthetic pathways in<Emphasis Type="Italic">Spirulina platensis</Emphasis>

Spirulina produces γ-linolenic acid (GLA), an important pharmaceutical substance, in a relatively low level compared with fungi and plants, prompting more research to improve its GLA yield. In this study, metabolic flux analysis was applied to determine the cellular metabolic flux distributions in the GLA synthetic pathways of twoSpirulina strains, wild type BP and a high-GLA producing mutant Z19/2. Simplified pathways involving the GLA synthesis ofS. platensis formulated comprise of photosynthesis, gluconeogenesis, the pentose phosphate pathway, the anaplerotic pathway, the tricarboxylic cycle, the GLA synthesis pathway, and the biomass synthesis pathway. A stoichiometric model reflecting these pathways contains 17 intermediates and 22 reactions. Three fluxes—the bicarbonate (C-source) uptake rate, the specific growth rate, and the GLA synthesis rate—were measured and the remaining fluxes were calculated using linear optimization. The calculation showed that the flux through the reaction converting acetyl-CoA into malonyl-CoA in the mutant strain was nearly three times higher than that in the wild-type strain. This finding implies that this reaction is rate controlling. This suggestion was supported by experiments, in which the stimulating factors for this reaction (NADPH and MgCl₂) were added into the culture medium, resulting in an increased GLA-synthesis rate in the wild type strain.     

一种中间代谢途径代谢通量的定量分析方法

以¹³C标记的碳源,用二维核磁共振技术(¹H-¹³C,HMQC)测定代谢中产生的氨基酸标记模式,研究对中间代谢途径胞内代谢通量进行定量分析的方法.通过开发软件包,改进同位素分布的数学模型,提出了反应映射矩阵(RMM)等概念.由简化算法,提高程序的执行效率,建立了定量分析胞内代谢通量的平台.代谢模型涉及了糖酵解途径、磷酸戊糖途径、三羧酸循环、几种回补反应、发酵途径和氨基酸合成途径.     

Profiling of central metabolism in human cancer cells by two-dimensional NMR, GC-MS analysis, and isotopomer modeling

Tracking metabolic profiles has the potential to reveal crucial enzymatic steps that could be targeted in the drug discovery process. It is of special importance for various types of cancer known to be associated with substantial rewiring of metabolic networks. Here we introduce an integrated approach for the analysis of metabolome that allows us to simultaneously assess pathway activities (fluxes) and concentrations of a large number of the key components involved in central metabolism of human cells. This is accomplished by in vivo labeling with [U-¹³C]glucose followed by two-dimensional nuclear magnetic resonance (NMR) spectroscopy and gas chromatography-mass spectrometry (GC-MS) analysis. A comprehensive isotopomer model was developed, which enabled us to compare fluxes through the key central metabolic pathways including glycolysis, pentose phosphate pathway, tricarboxylic acid cycle, anaplerotic reactions, and biosynthetic pathways of fatty acids and amino acids. The validity and strength of this approach is illustrated by its application to a number of perturbations to breast cancer cells, including exposure to hypoxia, drug treatment, and tumor progression. We observed significant differences in the activities of specific metabolic pathways resulting from these perturbations and providing new mechanistic insights. Based on these findings we conclude that the developed metabolomic approach constitutes a promising analytical tool for revealing specific metabolic phenotypes in a variety of cell types and pathological conditions. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users. Chen Yang and Adam D. Richardson contributed equally to this work.     

Metabolic network analysis on Phaffia rhodozyma yeast using C–labeled glucose and gas chromatography-mass spectrometry

Carotenoid production by microorganisms, as opposed to chemical synthesis, could fulfill an ever-increasing demand for ‘all natural’ products. The yeast Phaffia rhodozyma has received considerable attention because it produces the red pigment astaxanthin, commonly used as an animal feed supplement. In order to have a better understanding of its metabolism, labeling experiments with [1-¹³C]glucose were conducted with the wildtype strain (CBS5905 T) and a hyper-producing carotenoid strain (J4-3) in order to determine their metabolic network structure and estimate intracellular fluxes.Amino acid labeling patterns, as determined by GC–MS, were in accordance with a metabolic network consisting of the Embden–Meyerhof–Parnas pathway, the pentose phosphate pathway, and the TCA cycle. Glucose was mainly consumed along the pentose phosphate pathway (65% for wildtype strain), which reflected high NADPH requirements for lipid biosynthesis. Although common to other oleaginous yeast, there was no, or very little, malic enzyme activity for carbon-limited growth. In addition, there was no evidence of phosphoketolase activity. The central carbon metabolism of the mutant strain was similar to that of the wildtype strain, though the relative pentose phosphate flux was lower and the TCA cycle flux in accordance with the biomass yield being lower.     

Chemostat culture of Bacillus caldotenax at 65°C: Activity and regulation of the main metabolic pathways

Bacillus caldotenax was cultivated in chemostat experiments at 65°C with a chemically defined minimal medium. Glycolysis, tricarboxylic acid cycle, pentose phosphate pathway and the respiratory chain were active as demonstrated by measuring the corresponding enzymes. No enzyme activity of the Entner-Doudoroff pathway could be detected. The specific activities of the citrate cycle enzymes were up to 10 times higher as compared to the enzymes of glycolysis. At dilution rates between 0.3 and 2.2 h^-1 none of the main metabolic pathways was regulated. In contrast the isocitrate lyase was regulated (drop of activity with increasing growth rates). As a result of a batch culture with glucose and acetate as carbon sources a regulation model was proposed: glucose, or a metabolite of glucose, represses the isocitrate lyase; in the absence of glucose acetate acts as an inducer.Abbreviations DCIP dichlorphenol indophenol - ED Entner-Doudoroff pathway - EMP Emden-Meyerhof-Parnas pathway - ICL isocitrate lyase - PP pentose phosphate pathway - TCC tricarbonic acid cycle     

Response of the central metabolism in Corynebacterium glutamicum to the use of an NADH-dependent glutamate dehydrogenase

The extensive use of 13C enrichments in precursor metabolites for flux quantification does not rely on NADPH stoichiometries and can therefore be used to quantify reducing power fluxes. As an application of this concept, the NADPH fluxes were quantified in an L-lysine producer of Corynebacterium glutamicum grown into metabolic and isotopic steady state with [1-13C]glucose. In this case, where the organism's NADPH-dependent glutamate dehydrogenase consumes reducing power, the NADPH flux generated is 210% (molar flux relative to glucose uptake rate) with its major part (72% of the total) generated via the pentose phosphate pathway activity. An isogenic strain in which the glutamate dehydrogenase of C. glutamicum was replaced by the NADH-dependent glutamate dehydrogenase of Peptostreptococcus asaccharolyticus was made and the metabolite fluxes were again estimated. The major response to this local perturbation is a drastically reduced NADPH generation of only 139%. Most of the NADPH (62% of the total) is now generated via the tricarboxylic acid cycle activity. This shows the extraordinary flexibility of the central metabolism and provides a picture of the global regulatory properties of the central metabolism. Furthermore, a detailed analysis of the fluxes and exchange fluxes within the anaplerotic reactions is given. It is hypothesized that these reactions might also serve to balance the total reducing power budget as well as the energy budget within the cell.     

Comparison of dynamic responses of cellular metabolites in <Emphasis Type="Italic">Escherichia coli</Emphasis> to pulse addition of substrates

We conducted an integrated study of cell growth parameters, product formation, and the dynamics of intracellular metabolite concentrations using Escherichia coli with genes knocked out in the glycolytic and oxidative pentose phosphate pathway (PPP) for glucose catabolism. We investigated the same characteristics in the wild-type strain, using acetate or pyruvate as the sole carbon source. Dramatic effects on growth parameters and extracellular and intracellular metabolite concentrations were observed after blocking either glycolytic breakdown of glucose by inactivation of phosphoglucose isomerase (disruption of pgi gene) or pentose phosphate breakdown of glucose by inactivation of glucose-6-phosphate dehydrogenase (disruption of zwf gene). Reducing power (NADPH) was mainly produced through PPP when the pgi gene was knocked out, while NADPH was produced through the tricarboxylic acid (TCA) cycle by isocitrate dehydrogenase or NADP-linked malic enzyme when the zwf gene was knocked out. As expected, when the pgi gene was knocked out, intracellular concentrations of PPP metabolites were high and glycolytic and concentrations of TCA cycle pathway metabolites were low. In the zwf gene knockout, concentrations of PPP metabolites were low and concentrations of intracellular glycolytic and TCA cycle metabolites were high.     

Nonlinear Dependency of Intracellular Fluxes on Growth Rate in Miniaturized Continuous Cultures of Escherichia coli

A novel mini-scale chemostat system was developed for the physiological characterization of 10-ml cultures. The parallel operation of eight such mini-scale chemostats was exploited for systematic ¹³C analysis of intracellular fluxes over a broad range of growth rates in glucose-limited Escherichia coli. As expected, physiological variables changed monotonously with the dilution rate, allowing for the assessment of maintenance metabolism. Despite the linear dependence of total cellular carbon influx on dilution rate, the distribution of almost all major fluxes varied nonlinearly with dilution rate. Most prominent were the distinct maximum of glyoxylate shunt activity and the concomitant minimum of tricarboxylic acid cycle activity at low to intermediate dilution rates of 0.05 to 0.2 h⁻¹. During growth on glucose, this glyoxylate shunt activity is best understood from a network perspective as the recently described phosphoenolpyruvate (PEP)-glyoxylate cycle that oxidizes PEP (or pyruvate) to CO₂. At higher or extremely low dilution rates, in vivo PEP-glyoxylate cycle activity was low or absent. The step increase in pentose phosphate pathway activity at around 0.2 h⁻¹ was not related to the cellular demand for the reduction equivalent NADPH, since NADPH formation was 20 to 50% in excess of the anabolic demand at all dilution rates. The results demonstrate that mini-scale continuous cultivation enables quantitative and parallel characterization of intra- and extracellular phenotypes in steady state, thereby greatly reducing workload and costs for stable-isotope experiments.     

l-Valine biosynthesis during batch and fed-batch cultivations of Corynebacterium glutamicum: Relationship between changes in bacterial growth rate and intracellular metabolism

A transition in the bacterial growth rate to below maximum was found to be an optimum parameter of cellular physiology to increase the activity of acetohydroxy acid synthase, a regulatory enzyme in l-valine synthesis, and amino acid overproduction by Corynebacterium glutamicum ATCC 13032 recombinants under batch and fed-batch cultivation conditions. An increase in l-valine synthesis under transient situations when cellular growth rate was downregulated was correlated to a decrease in the activity of aconitase, a key enzyme in the tricarboxylic acid cycle (TCA) of C. glutamicum, and, in contrast, to an increase in the activity of glucose-6-phosphate dehydrogenase, a key enzyme in the pentose phosphate pathway (PPP). The increase in amino acid synthesis was also directly related to a drastic increase in intracellular pyruvate concentration. Thus, an increase in intracellular pyruvate availability and NADPH₂ generation by PPP could be the metabolic origins of the increased l-valine overproduction by growth restrained C. glutamicum cell culture.     

Metabolic network analysis on Phaffia rhodozyma yeast using 13C-labeled glucose and gas chromatography-mass spectrometry

Carotenoid production by microorganisms, as opposed to chemical synthesis, could fulfill an ever-increasing demand for 'all natural' products. The yeast Phaffia rhodozyma has received considerable attention because it produces the red pigment astaxanthin, commonly used as an animal feed supplement. In order to have a better understanding of its metabolism, labeling experiments with [1-(13)C]glucose were conducted with the wildtype strain (CBS5905T) and a hyper-producing carotenoid strain (J4-3) in order to determine their metabolic network structure and estimate intracellular fluxes. Amino acid labeling patterns, as determined by GC-MS, were in accordance with a metabolic network consisting of the Embden-Meyerhof-Parnas pathway, the pentose phosphate pathway, and the TCA cycle. Glucose was mainly consumed along the pentose phosphate pathway ( approximately 65% for wildtype strain), which reflected high NADPH requirements for lipid biosynthesis. Although common to other oleaginous yeast, there was no, or very little, malic enzyme activity for carbon-limited growth. In addition, there was no evidence of phosphoketolase activity. The central carbon metabolism of the mutant strain was similar to that of the wildtype strain, though the relative pentose phosphate flux was lower and the TCA cycle flux in accordance with the biomass yield being lower.     

Estradiol stimulates the biosynthetic pathways of breast cancer cells: detection by metabolic flux analysis

Selective estrogen receptor (ER) modulators are highly successful breast cancer therapies, but they are not effective in patients with ER negative and selective estrogen receptor modulator (SERM)-resistant tumors. Understanding the mechanisms of estrogen-stimulated proliferation may provide a route to design estrogen-independent therapies that would be effective in these patients. In this study, metabolic flux analysis was used to determine the intracellular fluxes that are significantly affected by estradiol stimulation in MCF-7 breast cancer cells. Intracellular fluxes were calculated from nuclear magnetic resonance (NMR)-generated isotope enrichment data and extracellular metabolite fluxes, using a specific flux analysis algorithm. The metabolic pathway model used by the algorithm includes glycolysis, the tricarboxylic acid cycle (TCA cycle), the pentose phosphate pathway, glutamine catabolism, pyruvate carboxylase, and malic enzyme. The pathway model also incorporates mitochondrial compartmentalization and reversible trans-mitochondrial membrane reactions to more accurately describe the role of mitochondria in cancer cell proliferation. Flux results indicate that estradiol significantly increases carbon flow through the pentose phosphate pathway and increases glutamine consumption. In addition, intra-mitochondrial malic enzyme was found to be inactive and the malate-aspartate shuttle (MAS) was only minimally active. The inactivity of these enzymes indicates that glutamine is not oxidized within mitochondria, but is consumed primarily to provide biosynthetic precursors. The excretion of glutamine carbons from the mitochondria has the secondary effect of limiting nicotinamide adenine dinucleotide (NADH) recycle, resulting in NADH buildup in the cytosol and the excretion of lactate. The observed dependence of breast cancer cells on pentose phosphate pathway activity and glutamine consumption for estradiol-stimulated biosynthesis suggests that these pathways may be targets for estrogen-independent breast cancer therapies.