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Journal ArticleDOI

Network analysis of intermediary metabolism using linear optimization. I. Development of mathematical formalism.

21 Feb 1992-Journal of Theoretical Biology (J Theor Biol)-Vol. 154, Iss: 4, pp 421-454
TL;DR: Analysis of metabolic networks using linear optimization theory allows one to quantify and understand the limitations imposed on the cell by its metabolic stoichiometry, and to understand how the flux through each pathway influences the overall behavior of metabolism.
About: This article is published in Journal of Theoretical Biology.The article was published on 1992-02-21 and is currently open access. It has received 255 citations till now.
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Journal ArticleDOI
TL;DR: This protocol provides a helpful manual for all stages of the reconstruction process and presents a comprehensive protocol describing each step necessary to build a high-quality genome-scale metabolic reconstruction.
Abstract: Network reconstructions are a common denominator in systems biology. Bottom–up metabolic network reconstructions have been developed over the last 10 years. These reconstructions represent structured knowledge bases that abstract pertinent information on the biochemical transformations taking place within specific target organisms. The conversion of a reconstruction into a mathematical format facilitates a myriad of computational biological studies, including evaluation of network content, hypothesis testing and generation, analysis of phenotypic characteristics and metabolic engineering. To date, genome-scale metabolic reconstructions for more than 30 organisms have been published and this number is expected to increase rapidly. However, these reconstructions differ in quality and coverage that may minimize their predictive potential and use as knowledge bases. Here we present a comprehensive protocol describing each step necessary to build a high-quality genome-scale metabolic reconstruction, as well as the common trials and tribulations. Therefore, this protocol provides a helpful manual for all stages of the reconstruction process.

1,574 citations

Journal ArticleDOI
TL;DR: A predictive algorithm is formulated in order to apply the flux balance model to describe unsteady-state growth and by-product secretion in aerobic batch, fed-batch, and anaerobic batch cultures.
Abstract: Flux balance models of metabolism use stoichiometry of metabolic pathways, metabolic demands of growth, and optimality principles to predict metabolic flux distribution and cellular growth under specified environmental conditions. These models have provided a mechanistic interpretation of systemic metabolic physiology, and they are also useful as a quantitative tool for metabolic pathway design. Quantitative predictions of cell growth and metabolic by-product secretion that are experimentally testable can be obtained from these models. In the present report, we used independent measurements to determine the model parameters for the wild-type Escherichia coli strain W3110. We experimentally determined the maximum oxygen utilization rate (15 mmol of O2 per g [dry weight] per h), the maximum aerobic glucose utilization rate (10.5 mmol of Glc per g [dry weight] per h), the maximum anaerobic glucose utilization rate (18.5 mmol of Glc per g [dry weight] per h), the non-growth-associated maintenance requirements (7.6 mmol of ATP per g [dry weight] per h), and the growth-associated maintenance requirements (13 mmol of ATP per g of biomass). The flux balance model specified by these parameters was found to quantitatively predict glucose and oxygen uptake rates as well as acetate secretion rates observed in chemostat experiments. We have formulated a predictive algorithm in order to apply the flux balance model to describe unsteady-state growth and by-product secretion in aerobic batch, fed-batch, and anaerobic batch cultures. In aerobic experiments we observed acetate secretion, accumulation in the culture medium, and reutilization from the culture medium. In fed-batch cultures acetate is cometabolized with glucose during the later part of the culture period.(ABSTRACT TRUNCATED AT 250 WORDS)

1,128 citations


Cites background from "Network analysis of intermediary me..."

  • ...A metabolic steady state is assumed, in which the metabolic pathway flux leading to the formation of a metabolite and that leading to the degradation of a metabolite must balance, which generates the flux balance equation (3, 13):...

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Journal ArticleDOI
TL;DR: The flux balance methodology allows the quantitative interpretation of metabolic physiology, gives an interpretation of experimental data, provides a guide to metabolic engineering, enables optimal medium formulation, and provides a method for bioprocess optimization.
Abstract: Recently, there has been an increasing interest in stoichiometric analysis of metabolic flux distributions. Flux balance methods only require information about metabolic reaction stoichiometry, metabolic requirements for growth, and the measurement of a few strain–specific parameters. This information determines the domain of stoichiometrically allowable flux distributions that may be taken to define a strain's “metabolic genotype”. Within this domain a single flux distribution is sought based on assumed behavior, such as maximal growth rates. The optimal flux distributions are calculated using linear optimization and may be taken to represent the strain's “metabolic phenotype” under the particular conditions. This flux balance methodology allows the quantitative interpretation of metabolic physiology, gives an interpretation of experimental data, provides a guide to metabolic engineering, enables optimal medium formulation, and provides a method for bioprocess optimization. This spectrum of applications, and its ease of use, makes the metabolic flux balance model a potentially valuable approach for the design and optimization of bioprocesses.

1,006 citations

Journal ArticleDOI
TL;DR: A generic approach to combine numerical optimization methods with biochemical kinetic simulations is described, suitable for use in the rational design of improved metabolic pathways with industrial significance and for solving the inverse problem of metabolic pathways.
Abstract: MOTIVATION The simulation of biochemical kinetic systems is a powerful approach that can be used for: (i) checking the consistency of a postulated model with a set of experimental measurements, (ii) answering 'what if?' questions and (iii) exploring possible behaviours of a model. Here we describe a generic approach to combine numerical optimization methods with biochemical kinetic simulations, which is suitable for use in the rational design of improved metabolic pathways with industrial significance (metabolic engineering) and for solving the inverse problem of metabolic pathways, i.e. the estimation of parameters from measured variables. RESULTS We discuss the suitability of various optimization methods, focusing especially on their ability or otherwise to find global optima. We recommend that a suite of diverse optimization methods should be available in simulation software as no single one performs best for all problems. We describe how we have implemented such a simulation-optimization strategy in the biochemical kinetics simulator Gepasi and present examples of its application. AVAILABILITY The new version of Gepasi (3.20), incorporating the methodology described here, is available on the Internet at http://gepasi.dbs.aber.ac.uk/softw/Gepasi. html. CONTACT prm@aber.ac.uk

722 citations


Cites methods from "Network analysis of intermediary me..."

  • ...A few groups, notably that of Heinrich, have indeed applied analytical optimization methods (e.g. Heinrich et al., 1987, 1997; Schuster and Heinrich, 1987, 1991; Savinell and Palsson, 1992; Klipp and Heinrich, 1994) to several pathway schemes to investigate the conditions for maximal flux, minimal concentrations, and a series of other criteria....

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  • ...…notably that of Heinrich, have indeed applied analytical optimization methods (e.g. Heinrich et al., 1987, 1997; Schuster and Heinrich, 1987, 1991; Savinell and Palsson, 1992; Klipp and Heinrich, 1994) to several pathway schemes to investigate the conditions for maximal flux, minimal…...

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Journal ArticleDOI
TL;DR: Fundamental issues associated with its formulation and use are reviewed and use to compute optimal growth states are reviewed.

565 citations


Cites background or methods from "Network analysis of intermediary me..."

  • ...DOI 10.1016/j.mib.2010.03.003 Introduction Flux balance analysis (FBA) [1] is a widely used approach for studying biochemical networks, in particular the genome-scale metabolic network reconstructions that have been built in the past decade [2,3]....

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  • ...With metabolic models becoming available for a growing number of organisms [5] and high-throughput technologies enabling the construction of many more each year [6], FBA is an important tool for harnessing the knowledge encoded in these models....

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  • ...Available online at www.sciencedirect.com The biomass objective function Adam M Feist1 and Bernhard O Palsson2 Flux balance analysis (FBA) is a mathematical approach for analyzing the flow of metabolites through a metabolic network....

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  • ...of ATP production, (3) minimizing total nutrient uptake, and (4) minimize redox metabolism through minimizing NADH production Linear programming Hybridoma cell line central metabolism (83 reactions, 42 metabolites) [11] (1) Aerobic batch bioreactor with growth, uptake, secretion, and protein production rates [20] Optimization of biomass production can be used to examine growth characteristics and explain observed phenomena [13] 1997 Max....

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  • ...This issue was recognized in the very first paper on large-scale network analysis using FBA [11,12] where a series of selected objective functions were used to find which one fit the data the best....

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References
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Journal Article
TL;DR: The capacity of the various tumor lines for the reoxidation of cytoplasmic NADH via the malate-aspartate shuttle approaches 20% of the total respiratory rate of the cells and thus appears to be sufficient to account for the mitochondrial re Roxidation of that fraction of glycolytic NADH not reoxidized by pyruvate and lactate dehydrognenase in the cy toplasm.
Abstract: The activity of the malate-aspartate shuttle for the reoxidation of cytoplasmic reduced nicotinamide adenine dinucleotide (NADH) by mitochondria was assessed in six lines of rodent ascites tumor cells (two strains of Ehrlich ascites carcinoma, Krebs II carcinoma, Novikoff hepatoma, AS-30D hepatoma, and L1210 mouse leukemia). All the tumor cells examined showed mitochondrial reoxidation of cytoplasmic NADH, as evidenced by the accumulation of pyruvate when the cells were incubated aerobically with L-lactate. Reoxidation of cytoplasmic NADH thus generated was completely inhibited by the transaminase inhibitor aminooxyacetate. The involvement of the respiratory chain in the reoxidation of cytoplasmic NADH was demonstrated by the action of cyanide, rotenone, and antimycin A, which strongly inhibited the formation of pyruvate from added L-lactate. Compounds that inhibit the carrier-mediated entry of malate into mitochondria, such as butylmalonate, benzenetricarboxylate, and iodobenzylmalonate, also inhibited the accumulation of pyruvate from added L-lactate by the tumor cells. The maximal rate of the malate-aspartate shuttle was established by addtion of arsenite to inhibit the mitochondrial oxidation of the pyruvate formed from added lactate. The capacity of the various tumor lines for the reoxidation of cytoplasmic NADH via the malate-aspartate shuttle approaches 20% of the total respiratory rate of the cells and thus appears to be sufficient to account for the mitochondrial reoxidation of that fraction of glycolytic NADH not reoxidized by pyruvate and lactate dehydrognenase in the cytoplasm.

92 citations

Journal ArticleDOI
TL;DR: It is suggested that under physiological conditions the transamination pathway is a less favourable route for the oxidation of glutamate (produced by hydrolysis of glutamine) in Ehrlich ascites-tumour cells, and perhaps also kidney, than the glutamate dehydrogenase pathway, as the production of acetyl-CoA strongly inhibits the first mechanism.
Abstract: 1. Pyruvate strongly inhibited aspartate production by mitochondria isolated from Ehrlich ascites-tumour cells, and rat kidney and liver respiring in the presence of glutamine or glutamate; the production of14CO2 from l-[U-14C]glutamine was not inhibited though that from l-[U-14C]glutamate was inhibited by more than 50%. 2. Inhibition of aspartate production during glutamine oxidation by intact Ehrlich ascites-tumour cells in the presence of glucose was not accompanied by inhibition of CO2 production. 3. The addition of amino-oxyacetate, which almost completely suppressed aspartate production, did not inhibit the respiration of the mitochondria in the presence of glutamine, though the respiration in the presence of glutamate was inhibited. 4. Glutamate stimulated the respiration of kidney mitochondria in the presence of glutamine, but the production of aspartate was the same as that in the presence of glutamate alone. 5. The results suggest that the oxidation of glutamate produced by the activity of mitochondrial glutaminase can proceed almost completely through the glutamate dehydrogenase pathway if the transamination pathway is inhibited. This indicates that the oxidation of glutamate is not limited by a high [NADPH]/[NADP+] ratio. 6. It is suggested that under physiological conditions the transamination pathway is a less favourable route for the oxidation of glutamate (produced by hydrolysis of glutamine) in Ehrlich ascites-tumour cells, and perhaps also kidney, than the glutamate dehydrogenase pathway, as the production of acetyl-CoA strongly inhibits the first mechanism. The predominance of the transamination pathway in the oxidation of glutamate by isolated mitochondria can be explained by a restricted permeability of the inner mitochondrial membrane to glutamate and by a more favourable location of glutamate–oxaloacetate transaminase compared with that of glutamate dehydrogenase.

90 citations

Journal ArticleDOI
TL;DR: Data from batch growth curves of mouse LS cells cultivated at controlled dissolved oxygen partial pressures were used to calculate the weight of cells produced per mole of adenosine triphosphate generated (YArv), and a theoretical relationship was developed which allowed the biosynthetic and maintenance energy requirements to be estimated.
Abstract: SUMMARY Data from batch growth curves of mouse LS cells cultivated at controlled dissolved oxygen partial pressures were used to calculate the weight of cells produced per mole of adenosine triphosphate generated (YArv). These values agree well with those reported for bacteria. A theoretical relationship was developed which allowed the biosynthetic and maintenance energy requirements to be estimated. The biosynthesis of LS cells required i-6 x io" 11 moles of ATP/cell. The maintenance energy, which is a function of growth rate, was 2-9 x io"" 11 moles ATP/new cell when the mean generation time was 1-15 days. The proportion of the total energy used for maintenance under these conditions was 65 %. This corresponds to a value of less than 10 % for bacterial maintenance when the organisms are grown at near their maximum rate. A comparison of biosynthetic energy requirements indicates that bacteria and moulds require about 4 times as much energy as animal cells to generate the same weight of cell material. Possible explanations of this difference are discussed.

89 citations

Book ChapterDOI
01 Jan 1986
TL;DR: In addition to its specific roles in multiple tissues, glutamine is the primary amino group donor in synthesis of purines and pyrimidines, amino sugars, pyridine nucleotides, and asparagine in mammalian cells.
Abstract: Glutamine is the most abundant amino acid in plasma and most tissues (Van Slyke et al., 1943). Because of both empirical reasoning and cellular requirements determined experimentally, it is the most abundant amino acid in most cell culture media (Ham and McKeehan, 1979). Although other amino acids have metabolic functions in addition to protein and peptide synthesis, glutamine is the most versatile (Krebs, 1980). It is the major source of urinary nitrogen and a key factor in acid—base balance in mammals. The carbon skeleton of glutamine is an important precursor of glucose in kidney cortex and thus contributes to renal gluconeogenesis (Krebs, 1963; Goodman et al., 1966). Glutamine is a vehicle for transporting nitrogen among tissues. Skeletal muscle is the principal site of glutamine production. Release of glutamine from muscle is nearly four times that that can be accounted for by direct protein breakdown (Blackshear et al., 1975; Pardridge and Casenello-Ertl, 1979; Garber, 1980). The principal site of net glutamine metabolism appears to be the gut (Windmueller and Spaeth, 1974; Hanson and Parsons, 1977) followed by the liver (Blackshear et al., 1975). Glutamine is a key metabolite for elimination of toxic ammonia in nerve tissue and may be an important precursor of glutamate and a-aminobutyrate, a synaptic transmitter (Waelsch, 1960; Takagaki et al.,1961). In addition to its specific roles in multiple tissues, glutamine is the primary amino group donor in synthesis of purines and pyrimidines, amino sugars, pyridine nucleotides, and asparagine in mammalian cells. The reader is referred to the following books and reviews for an in-depth picture of the role of glutamine (and glutamate) in mammals: Meister (1956, 1965), Lund et al. (1970), Prusiner and Stadtman (1973), Shepartz (1973), Meister (1978), Munro (1978), Mora and Palacios (1980), Kovacevic and McGivan (1983).

89 citations

Journal ArticleDOI
TL;DR: The main route of glutamine and glutamate entrance into the citric acid cycle via 2-oxoglutarate in lymphocytes appears to be transamination by aspartate aminotransferase rather than oxidative deamination by glutamate dehydrogenase.
Abstract: Pathways of glutamine metabolism in resting and proliferating rat thymocytes and established human T- and B-lymphoblastoid cell lines were evaluated by in vitro incubations of freshly prepared or cultured cells for one to two hours with [U 14 C]glutamine. Complete recovery of glutamine carbons utilized in products allowed quantification of the pathways of glutamine metabolism under the experimental conditions. Partial oxidation of glutamine via 2-oxoglutarate in a truncated citric acid cycle to CO 2 and oxaloacetate, which then was converted to aspartate, accounted for 76% and 69%, respectively, of the glutamine metabolized beyond the stage of glutamate by resting and proliferating thymocytes. Similar results were obtained with the lymphoblastoid T- and B-cell lines. Complete oxidation to CO 2 in the citric acid cycle via 2-oxoglutarate dehydrogenase and isocitrate dehydrogenase accounted for only 25% and 7%, respectively. In proliferating cells a substantial amount of glutamine carbons was also recovered in pyruvate, alanine, and especially lactate. The main route of glutamine and glutamate entrance into the citric acid cycle via 2-oxoglutarate in lymphocytes appears to be transamination by aspartate aminotransferase rather than oxidative deamination by glutamate dehydrogenase. In the presence of glucose as a second substrate, glutamine utilization and aspartate formation markedly decreased, but complete oxidation of glutamine carbons to CO 2 increased to 37% and 23%, respectively, in resting and proliferating cells. The dipeptide, glycyl- l -glutamine, which is more stable than free glutamine, can substitute for glutamine in thymocyte cultures at higher concentrations.

80 citations

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Linear optimization theory is a mathematical formalism used to analyze metabolic networks and understand the limitations and behavior of metabolism.