Metabolic fluxes in riboflavin-producing Bacillus subtilis

Sauer, Uwe; Hatzimanikatis, Vassily; Bailey, James E.; Hochuli, Michel; Szyperski, Thomas; Wüthrich, Kurt

doi:10.1038/nbt0597-448

Cited by 270 publications

(338 citation statements)

References 27 publications

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“…The system of linear equations was solved uniquely with constraints obtained from six of the above calculated flux ratios (i.e. serine through glycolysis, pyruvate through Entner-Doudoroff pathway (ED pathway), oxaloacetate from phosphoenolpyruvate (PEP), PEP from oxaloacetate, pyruvate from malate, and PEP through PP pathway) that were formulated as linearly independent equations as described previously (23,39,40). Briefly, the sum of the weighed square residuals of the constraints from both metabolite balances and flux ratios was minimized by using the MATLAB function fmincon, and the residuals were weighed by dividing through the experimental error (39).…”

Section: Methodsmentioning

confidence: 99%

“…Indeed, organisms lacking transhydrogenases, such as the yeast Saccharomyces cerevisiae, cannot tolerate imbalances between catabolic NADPH production and anabolic NADPH consumption (18,19), unless a soluble isoform is expressed (19,20). Further means of NADPH reoxidation must exist, however, because isotopic tracer-based carbon flux analysis revealed that at least some bacteria without transhydrogenase homologues exhibit a similar uncoupling (21)(22)(23)(24)(25). In Escherichia coli, overexpression of the soluble transhydrogenase UdhA was shown to partially restore the growth phenotype of a phosphoglucose isomerase mutant with impaired NADPH metabolism (26), but its phys-iological role in wild-type E. coli remains obscure.…”

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confidence: 99%

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The Soluble and Membrane-bound Transhydrogenases UdhA and PntAB Have Divergent Functions in NADPH Metabolism of Escherichia coli

Sauer

Canonaco

Heri

et al. 2004

Journal of Biological Chemistry

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530

526

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Pentose phosphate pathway and isocitrate dehydrogenase are generally considered to be the major sources of the anabolic reductant NADPH. As one of very few microbes, Escherichia coli contains two transhydrogenase isoforms with unknown physiological function that could potentially transfer electrons directly from NADH to NADP ؉ and vice versa. Using defined mutants and metabolic flux analysis, we identified the proton-translocating transhydrogenase PntAB as a major source of NADPH in E. coli. During standard aerobic batch growth on glucose, 35-45% of the NADPH that is required for biosynthesis was produced via PntAB, whereas pentose phosphate pathway and isocitrate dehydrogenase contributed 35-45% and 20 -25%, respectively. The energy-independent transhydrogenase UdhA, in contrast, was essential for growth under metabolic conditions with excess NADPH formation, i.e. growth on acetate or in a phosphoglucose isomerase mutant that catabolized glucose through the pentose phosphate pathway. Thus, both isoforms have divergent physiological functions: energy-dependent reduction of NADP ؉ with NADH by PntAB and reoxidation of NADPH by UdhA. Expression appeared to be modulated by the redox state of cellular metabolism, because genetic and environmental manipulations that increased or decreased NADPH formation down-regulated pntA or udhA transcription, respectively. The two transhydrogenase isoforms provide E. coli primary metabolism with an extraordinary flexibility to cope with varying catabolic and anabolic demands, which raises two general questions: why do only a few bacteria contain both isoforms, and how do other organisms manage NADPH metabolism?About 1,000 anabolic reactions synthesize the macromolecular components that make up functional cells (1, 2), but only 11 intermediates of central carbon metabolism and the cofactors ATP, NADH, and NADPH constitute the core of this intricate biochemical network (3, 4). These intermediates and cofactors must be supplied through the catabolism of different substrates at appropriate rates and stoichiometries for balanced growth; hence, anabolism and catabolism are delicately balanced and regulated to enable growth under fluctuating environmental conditions. Although chemically very similar, the redox cofactors NADH and NADPH serve distinct biochemical functions and participate in more than 100 enzymatic reactions (5). The electrons of the main respiratory cofactor NADH are transferred primarily to oxygen, thereby driving oxidative phosphorylation of ADP to ATP (3,4,6). NADPH, in contrast, exclusively drives anabolic reduction reactions. Despite the important role in linking the fundamental processes of catabolism and anabolism, however, even a qualitative understanding of NADPH metabolism is still missing for most organisms.The primary NADPH-generating reactions are considered to be the oxidative pentose phosphate (PP) 1 pathway and the NADPH-dependent isocitrate dehydrogenase in the TCA cycle (Fig.

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Section: Methodsmentioning

confidence: 99%

mentioning

confidence: 99%

The Soluble and Membrane-bound Transhydrogenases UdhA and PntAB Have Divergent Functions in NADPH Metabolism of Escherichia coli

Sauer

Canonaco

Heri

et al. 2004

Journal of Biological Chemistry

Self Cite

530

526

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“…This requires assumptions on the energy metabolism [Y ATP , stoichiometry of oxidative phosphorylation (P/O) ratio, NADH balance, NADPH balance]. Stoichiometric parameters such as P/O ratios or ATP-yields, often originate from wild type strains and continuous cultures and may not hold true in cases of highly engineered strains [5]. Moreover, such parameters may not stay constant during batch cultivations with changes of the physiological status of the cells.…”

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confidence: 99%

Application of MALDI‐TOF MS to lysine‐producing Corynebacterium glutamicum

Wittmann

Heinzle

2001

European Journal of Biochemistry

113

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In the present work, a novel comprehensive approach of 13 C-tracer studies with labeling measurements by MALDI-TOF MS, and metabolite balancing was developed to elucidate key fluxes in the central metabolism of lysine producing Corynebacterium glutamicum during batch culture. MALDI-TOF MS methods established allow the direct quantification of labeling patterns of low molecular mass Corynebacterium products from 1 mL of diluted culture supernatant. A mathematical model of the central Corynebacterium metabolism was developed, that describes the carbon transfer through the network via matrix calculations in a generally applicable way and calculates steady state mass isotopomer distributions of the involved metabolites. The model was applied for both experimental planning of tracer experiments and parameter estimation. Metabolic fluxes were calculated from stoichiometric data and from selected mass intensity ratios of lysine, alanine, and trehalose measured by MALDI-TOF MS in tracer experiments either with 1-13 C glucose or with mixtures of 13 C 6 / 12 C 6 glucose. During the phase of maximum lysine production C. glutamicum ATCC 21253 exhibited high relative fluxes into the pentose phosphate pathway of 71%, a highly reversible glucose-6-phosphate isomerase, significant backfluxes from the tricarboxylic acid cycle to the pyruvate node consuming the lysine precursor oxaloacetate, 36% net flux of anaplerotic carboxylation and 63% contribution of the dehydrogenase branch in the lysine biosynthetic pathway. Due to the straightforward and simple measurements of selected labeling patterns by MALDI-TOF MS sensitively reflecting the flux parameters of interest, the presented approach has an excellent potential to extend metabolic flux analysis from single experiments with enormous experimental effort to a broadly applied technique.Keywords: MALDI-TOF mass spectrometry; metabolic flux analysis; Corynebacterium glutamicum; lysine; 13 C tracer.Production of amino acids by Corynebacterium glutamicum belongs to the major processes in industrial biotechnology. The world market for amino acids amounts to about 3 billion US dollars, whereby lysine with a worldwide production of about 250 000 tonnes per annum is one of the most important products [1]. Optimization of cultivation strategies and producer strains have led to increased yields and rates of amino-acid production by Corynebacteria. To a large extent, the strain improvement was based on random mutagenesis and subsequent selection [2,3]. In the last decade the powerful tools of genetic engineering allowed the targeted amplification and disruption of selected genes, which led to a number of mutants with increased capabilities of lysine secretion [4]. However, practically achieved yields are still far from theoretical optima, leaving a significant potential for further improvement. To achieve such improvements in a targeted and efficient way, detailed knowledge of intracellular carbon flux distributions and their regulation is needed. Data on genome, transcriptome and proteome ...

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“…Such analytically deduced flux ratios were also used successfully as constraints for metabolic flux analysis [13][14][15]. Based on probabilistic equations, a more general methodology was developed to simultaneously identify network topology and multiple flux partitioning ratios [16,17].…”

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

Fischer

Sauer

2003

European Journal of Biochemistry

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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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Metabolic fluxes in riboflavin-producing Bacillus subtilis

Cited by 270 publications

References 27 publications

The Soluble and Membrane-bound Transhydrogenases UdhA and PntAB Have Divergent Functions in NADPH Metabolism of Escherichia coli

The Soluble and Membrane-bound Transhydrogenases UdhA and PntAB Have Divergent Functions in NADPH Metabolism of Escherichia coli

Application of MALDI‐TOF MS to lysine‐producing Corynebacterium glutamicum

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

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