High-throughput metabolic flux analysis based on gas chromatography–mass spectrometry derived 13C constraints

Fischer, Eliane; Zamboni, Nicola; Sauer, Uwe

doi:10.1016/j.ab.2003.10.036

Cited by 270 publications

(279 citation statements)

References 24 publications

Supporting

Mentioning

265

Contrasting

Unclassified

Order By: Relevance

“…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%

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

514

526

View full text Add to dashboard Cite

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.

show abstract

Section: Methodsmentioning

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

514

526

View full text Add to dashboard Cite

show abstract

“…These two assumptions were considered reasonable because flux ratio variation at the glucose-6-phosphate node and introduction of the glyoxylate shunt as an alternative anaplerotic pathway did not significantly influence the net formation of redox equivalents. Error minimization was carried out as described by Fischer et al (21). The NADPH pool was balanced, allowing the estimation of a net NADH formation rate.…”

Section: Methodsmentioning

confidence: 99%

NADH Availability Limits Asymmetric Biocatalytic Epoxidation in a Growing Recombinant Escherichia coli Strain

Bühler

Park

Blank

et al. 2008

Appl Environ Microbiol

View full text Add to dashboard Cite

Styrene can efficiently be oxidized to (S)-styrene oxide by recombinant Escherichia coli expressing the styrene monooxygenase genes styAB from Pseudomonas sp. strain VLB120. Targeting microbial physiology during whole-cell redox biocatalysis, we investigated the interdependency of styrene epoxidation, growth, and carbon metabolism on the basis of mass balances obtained from continuous two-liquid-phase cultures. Full induction of styAB expression led to growth inhibition, which could be attenuated by reducing expression levels. Operation at subtoxic substrate and product concentrations and variation of the epoxidation rate via the styrene feed concentration allowed a detailed analysis of carbon metabolism and bioconversion kinetics. Fine-tuned styAB expression and increasing specific epoxidation rates resulted in decreasing biomass yields, increasing specific rates for glucose uptake and the tricarboxylic acid (TCA) cycle, and finally saturation of the TCA cycle and acetate formation. Interestingly, the biocatalysis-related NAD(P)H consumption was 3.2 to 3.7 times higher than expected from the epoxidation stoichiometry. Possible reasons include uncoupling of styrene epoxidation and NADH oxidation and increased maintenance requirements during redox biocatalysis. At epoxidation rates of above 21 mol per min per g cells (dry weight), the absence of limitations by O 2 and styrene and stagnating NAD(P)H regeneration rates indicated that NADH availability limited styrene epoxidation. During glucose-limited growth, oxygenase catalysis might induce regulatory stress responses, which attenuate excessive glucose catabolism and thus limit NADH regeneration. Optimizing metabolic and/or regulatory networks for efficient redox biocatalysis instead of growth (yield) is likely to be the key for maintaining high oxygenase activities in recombinant E. coli.Oxygenases catalyze regio-and enantioselective oxyfunctionalization reactions and have a considerable potential in the area of asymmetric organic synthesis (2, 35). These enzymes typically depend on coenzymes such as NAD(P)H and are unstable outside cells, and they often consist of multiple components. Thus, for synthetic applications, their use in whole cells is favored over the use of isolated enzymes (7,18).The productivity of oxygenase-based whole-cell biocatalysts is influenced by various factors, such as maximal enzyme activity, enzyme level, substrate mass transfer, substrate and/or product inhibition/toxicity, and cofactor regeneration (7,13,54,68). Enzyme synthesis and cofactor regeneration are related to host metabolism, which in turn may be affected by the toxicity of organic substrates and products. Such toxicity can efficiently be attenuated by regulated substrate feeding (1, 11) and in situ product removal (7,38,61,66,74). Yet, microbial cells, especially in a nongrowing state, often lose biooxidation activity over time, which may be due to oxygenase inactivation and/or the metabolic burden imposed by redox-coenzyme withdrawal and reactive oxygen species formed via...

show abstract

“…Cell pellets and supernatants were stored at −20°C. Sample preparation was performed as described previously (13,44,45). Briefly, cell pellets were hydrolyzed in 6 M HCl at 105°C for 24 h in sealed 2-mL tubes and subsequently dried at 60°C in a heating block under a stream of air.…”

mentioning

confidence: 99%

Functioning of a metabolic flux sensor in Escherichia coli

Kochanowski

Volkmer

Gerosa

et al. 2012

Proc. Natl. Acad. Sci. U.S.A.

179

183

View full text Add to dashboard Cite

Regulation of metabolic operation in response to extracellular cues is crucial for cells' survival. Next to the canonical nutrient sensors, which measure the concentration of nutrients, recently intracellular "metabolic flux" was proposed as a novel impetus for metabolic regulation. According to this concept, cells would have molecular systems ("flux sensors") in place that regulate metabolism as a function of the actually occurring metabolic fluxes. Although this resembles an appealing concept, we have not had any experimental evidence for the existence of flux sensors and also we have not known how these flux sensors would work in detail. Here, we show experimental evidence that supports the hypothesis that Escherichia coli is indeed able to measure its glycolytic flux and uses this signal for metabolic regulation. Combining experiment and theory, we show how this flux-sensing function could emerge from an aggregate of several molecular mechanisms: First, the system of reactions of lower glycolysis and the feedforward activation of fructose-1,6-bisphosphate on pyruvate kinase translate flux information into the concentration of the metabolite fructose-1,6-bisphosphate. The interaction of this "flux-signaling metabolite" with the transcription factor Cra then leads to flux-dependent regulation. By responding to glycolytic flux, rather than to the concentration of individual carbon sources, the cell may minimize sensing and regulatory expenses. R egulation of metabolic operation is crucial for cells' survival. The canonical view is that this regulation occurs in response to extracellular cues, where, for instance, nutrient-specific transmembrane or intracellular receptors sense the presence of a nutrient and transfer respective commands to the regulatory machinery (1-5).Recently, however, a novel impetus for metabolic regulation was proposed: cells could regulate their metabolism as a function of the actually occurring intracellular metabolic fluxes (6, 7). According to this concept, changes in extracellular nutrient abundances would first-in a rather passive manner-result in changes in intracellular metabolic fluxes. In a second instance, the metabolic fluxes would be sensed by molecular systems ("flux sensors"), which in turn would transmit the sensed "flux signal" to the regulatory machinery that consequently would adjust metabolic operation (6). On the basis of a detailed mathematical model of Escherichia coli's central metabolism and its regulation, Kotte et al. suggested that this organism would have such flux sensors in place that establish a correlation between a metabolic flux and the concentration of certain so-called flux-signaling metabolites, which in turn affect the activity of transcription factors and thus would allow for transcriptional regulation in a flux-dependent manner.Using intracellular flux to regulate metabolism is an appealing concept as it would omit the need for nutrient-specific sensors for many different nutrients, and would allow the integration of multiple nutrient inputs directl...

show abstract

High-throughput metabolic flux analysis based on gas chromatography–mass spectrometry derived 13C constraints

Cited by 270 publications

References 24 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

NADH Availability Limits Asymmetric Biocatalytic Epoxidation in a Growing Recombinant Escherichia coli Strain

Functioning of a metabolic flux sensor in Escherichia coli

Contact Info

Product

Resources

About