Quantitative analysis of metabolic fluxes in Escherichia coli, using two-dimensional NMR spectroscopy and complete isotopomer models

Schmidt, Karsten; Nielsen, Jens

doi:10.1016/s0168-1656(99)00021-8

Cited by 155 publications

(139 citation statements)

References 17 publications

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“…The NADPH balance forces the PP pathway to operate only to satisfy the biosynthetic demands for NADPH and pentose precursors. Flux measurements in E. coli using 13 C-labelled glucose which are not dependent on NADPH stoichiometries are in close agreement with the above analysis: a PP pathway flux of 53 % mol mol 21 and a TCA cycle flux of 45 % mol mol 21 have been reported (Schmidt et al, 1999). Thus, the PP pathway utilization is intensified at the cost of the TCA cycle activity when growth rates increase in glucose-limited chemostat cultures.…”

Section: Growth Energetics and Metabolic Flux Distributions: A Linearsupporting

confidence: 85%

Metabolic flux analysis of Escherichia coli in glucose-limited continuous culture. I. Growth-rate-dependent metabolic efficiency at steady state

Kayser¹,

Weber²,

Hecht³

et al. 2005

Microbiology

166

170

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The Escherichia coli K-12 strain TG1 was grown at 28 6C in aerobic glucose-limited continuous cultures at dilution rates ranging from 0?044 to 0?415 h "1 . The rates of biomass formation, the specific rates of glucose, ammonium and oxygen uptake and the specific carbon dioxide evolution rate increased linearly with the dilution rate up to 0?3 h "1 . At dilution rates between 0?3 h "1 and 0?4 h "1 , a strong deviation from the linear increase to lower specific oxygen uptake and carbon dioxide evolution rates occurred. The biomass formation rate and the specific glucose and ammonium uptake rates did not deviate that strongly from the linear increase up to dilution rates of 0?4 h "1 . An increasing percentage of glucose carbon flow towards biomass determined by a reactor mass balance and a decreasing specific ATP production rate concomitant with a decreasing adenylate energy charge indicated higher energetic efficiency of carbon substrate utilization at higher dilution rates. Estimation of metabolic fluxes by a stoichiometric model revealed an increasing activity of the pentose phosphate pathway and a decreasing tricarboxylic acid cycle activity with increasing dilution rates, indicative of the increased NADPH and precursor demand for anabolic purposes at the expense of ATP formation through catabolic activities. Thus, increasing growth rates first result in a more energy-efficient use of the carbon substrate for biomass production, i.e. a lower portion of the carbon substrate is channelled into the respiratory, energy-generating pathway. At dilution rates above 0?4 h "1 , close to the wash-out point, respiration rates dropped sharply and accumulation of glucose and acetic acid was observed. Energy generation through acetate formation yields less ATP compared with complete oxidation of the sugar carbon substrate, but is the result of maximized energy generation under conditions of restrictions in the tricarboxylic acid cycle or in respiratory NADH turnover. Thus, the data strongly support the conclusion that, in aerobic glucose-limited continuous cultures of E. coli TG1, two different carbon limitations occur: at low dilution rates, cell growth is limited by cell-carbon supply and, at high dilution rates, by energy-carbon supply.

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Section: Growth Energetics and Metabolic Flux Distributions: A Linearsupporting

confidence: 85%

Metabolic flux analysis of Escherichia coli in glucose-limited continuous culture. I. Growth-rate-dependent metabolic efficiency at steady state

Kayser¹,

Weber²,

Hecht³

et al. 2005

Microbiology

166

170

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“…With decreasing oxygen availability, most prominently, the fraction of OAA originating through the TCA cycle decreases from 44% to 5%. This reveals a branched, noncyclic operation of the TCA cycle to fulfill exclusively biosynthetic requirements, as was also shown earlier [7,16,26]. Although the oxidative PP pathway is still active under anaerobic conditions (serine through glycolysis), its relative contribution to glucose catabolism is decreased from 19% to 5% (Fig.…”

Section: Metabolic Flux Ratio Analysis Of E Coli Under Different Envsupporting

confidence: 83%

Metabolic flux profiling of Escherichia coli mutants in central carbon metabolism using GC‐MS

Fischer

Sauer

2003

European Journal of Biochemistry

348

418

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We describe here a novel methodology for rapid diagnosis of metabolic changes, which is based on probabilistic equations that relate GC-MS-derived mass distributions in proteinogenic amino acids to in vivo enzyme activities. This metabolic flux ratio analysis by GC-MS provides a comprehensive perspective on central metabolism by quantifying 14 ratios of fluxes through converging pathways and reactions from [1-13 C] and [U-13 C]glucose experiments. Reliability and accuracy of this method were experimentally verified by successfully capturing expected flux responses of Escherichia coli to environmental modifications and seven knockout mutations in all major pathways of central metabolism. Furthermore, several mutants exhibited additional, unexpected flux responses that provide new insights into the behavior of the metabolic network in its entirety. Most prominently, the low in vivo activity of the EntnerDoudoroff pathway in wild-type E. coli increased up to a contribution of 30% to glucose catabolism in mutants of glycolysis and TCA cycle. Moreover, glucose 6-phosphate dehydrogenase mutants catabolized glucose not exclusively via glycolysis, suggesting a yet unidentified bypass of this reaction. Although strongly affected by environmental conditions, a stable balance between anaplerotic and TCA cycle flux was maintained by all mutants in the upper part of metabolism. Overall, our results provide quantitative insight into flux changes that bring about the resilience of metabolic networks to disruption.Keywords: Entner-Doudoroff pathway; flux analysis; fluxome; METAFoR analysis; pentose phosphate pathway.Comprehensive and quantitative understanding of biochemical reaction networks requires not only knowledge about their constituting components, but also information about the behavior of the network in its entirety. Toward this end, systems-oriented methodologies were developed that simultaneously access the level of reaction intermediates [1] or rates of reactions [2][3][4][5], also referred to as the metabolome [6] and the fluxome [7], respectively. The most important property of biochemical networks are the per se nonmeasurable in vivo reaction rates, which may be estimated by so-called metabolic flux analysis that provides a holistic perspective on metabolism.In its simplest form, metabolic flux analysis relies on flux balancing of extracellular consumption and secretion rates within a stoichiometric reaction model [5]. To increase reliability and resolution of such flux balancing analyses, additional information may be derived from 13 C-labeling experiments. In this approach, 13 C-labeled substrates are administered and 13 C-labeled products of metabolism are analyzed by methods that distinguish between different isotope labeling patterns, in particular NMR and MS [2,3,8]. In the most advanced methodology, a comprehensive isotope isomer (isotopomer) model of metabolism is used to map metabolic fluxes in an iterative fitting procedure on the isotopomer pattern of network metabolites that are deduced from NMR or M...

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“…In general, decarboxylation reactions, such as the oxidative reactions in the PPP and the TCA cycle, are frequently irreversible (30). However, the model predicted extremely high reversibility (exch ϭ 0.99) in the reaction that converts oxoglutarate to succinate compared to those in other microorganisms (41).…”

Section: Resultsmentioning

confidence: 92%

Flux Analysis of Central Metabolic Pathways in Geobacter metallireducens during Reduction of Soluble Fe(III)-Nitrilotriacetic Acid

Tang

Chakraborty

Martín

et al. 2007

Appl Environ Microbiol

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We analyzed the carbon fluxes in the central metabolism of Geobacter metallireducens strain GS-15 using 13 C isotopomer modeling. Acetate labeled in the first or second position was the sole carbon source, and Fenitrilotriacetic acid was the sole terminal electron acceptor. The measured labeled acetate uptake rate was 21 mmol/g (dry weight)/h in the exponential growth phase. The resulting isotope labeling pattern of amino acids allowed an accurate determination of the in vivo global metabolic reaction rates (fluxes) through the central metabolic pathways using a computational isotopomer model. The tracer experiments showed that G. metallireducens contained complete biosynthesis pathways for essential metabolism, and this strain might also have an unusual isoleucine biosynthesis route (using acetyl coenzyme A and pyruvate as the precursors). The model indicated that over 90% of the acetate was completely oxidized to CO 2 via a complete tricarboxylic acid cycle while reducing iron. Pyruvate carboxylase and phosphoenolpyruvate (PEP) carboxykinase were present under these conditions, but enzymes in the glyoxylate shunt and malic enzyme were absent. Gluconeogenesis and the pentose phosphate pathway were mainly employed for biosynthesis and accounted for less than 3% of total carbon consumption. The model also indicated surprisingly high reversibility in the reaction between oxoglutarate and succinate. This step operates close to the thermodynamic equilibrium, possibly because succinate is synthesized via a transferase reaction, and the conversion of oxoglutarate to succinate is a rate-limiting step for carbon metabolism. These findings enable a better understanding of the relationship between genome annotation and extant metabolic pathways in G. metallireducens.Geobacter species are known to be one of the dominant groups of microorganisms mediating iron reduction in the environment (21). They have been found to be ubiquitous in a myriad of subsurface environments. Detailed studies of their metabolism have revealed them to be capable of bioremediation of several heavy metals, including uranium, plutonium, technetium, and vanadium, as well as biodegradation of several organic contaminants, including monoaromatic hydrocarbons (16,17,26). More recently, Geobacter species have been used to generate electricity from waste organic matter (2, 15, 19). These unique metabolisms make Geobacter species important players in the contaminated subsurface environment (18). Geobacter metallireducens was the first iron-reducing organism isolated that coupled complete oxidation of organic acids with reduction of iron oxides (20,21). It completely oxidizes organic carbons such as fatty acids, alcohols, and monoaromatic compounds via the tricarboxylic acid (TCA) cycle (3, 20) coupled with the reduction of iron. The genomes of several Geobacter species have been sequenced, and proteome data are also available (5, 24). While the genome sequence and proteome are important for understanding Geobacter, they are not necessarily accurate represe...

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Quantitative analysis of metabolic fluxes in Escherichia coli, using two-dimensional NMR spectroscopy and complete isotopomer models

Cited by 155 publications

References 17 publications

Metabolic flux analysis of Escherichia coli in glucose-limited continuous culture. I. Growth-rate-dependent metabolic efficiency at steady state

Metabolic flux analysis of Escherichia coli in glucose-limited continuous culture. I. Growth-rate-dependent metabolic efficiency at steady state

Metabolic flux profiling of Escherichia coli mutants in central carbon metabolism using GC‐MS

Flux Analysis of Central Metabolic Pathways in Geobacter metallireducens during Reduction of Soluble Fe(III)-Nitrilotriacetic Acid

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