Patterns of indirect protein interactions suggest a spatial organization to metabolism

It has long been believed that cells organize their cytoplasm so as to efficiently channel metabolites between sequential enzymes. This metabolic channeling has the potential to yield higher metabolic fluxes as well as better regulatory control over metabolism. One mechanism for achieving such channeling is to ensure that sequential enzymes in a pathway are physically close to each other in the cell. We present evidence that indirect protein interactions between related enzymes represent a global mechanism for achieving metabolic channeling; the intuition being that protein interactions between enzymes and non-enzymatic mediator proteins are a powerful means of physically associating enzymes in a modular fashion. By analyzing the metabolic and protein-protein interactions networks of Escherichia coli, yeast and humans, we are able to show that all three species have many more indirect protein interactions linking enzymes that share metabolites than would be expected by chance. Moreover, these interactions are distributed non-randomly in the metabolic network. Our analyses in yeast and E. coli show that reactions possessing such interactions also show higher flux than do those lacking them. On the basis of these observations, we suggest that an important role of protein interactions with mediator proteins is to contribute to the spatial organization of the cell. This hypothesis is supported by the fact that these mediator proteins are also enriched with annotations related to signal transduction, a system where scaffolding proteins are known to limit cross-talk by controlling spatial localization.     

Topology of the global regulatory network of carbon limitation in Escherichia coli

One fundamental shortcoming of biotechnological processes operating under carbon-limiting conditions is the high-energy demand (maintenance) of the cells. Although the function of the central carbon metabolism in supplying precursors and energy for biosynthesis has been thoroughly characterized, its regulation and dynamic behaviour during carbon-limited growth has not yet been revealed. The current work demonstrates a time series of metabolic flux distributions during fed-batch cultivation of Escherichia coli K-12 W3110 applying a constant feed rate. The fluxes in glycolysis, pentose phosphate pathway and biosynthesis fell significantly, whereas TCA cycle fluxes remained constant. The flux redistribution resulted in an enhanced energy generation in the TCA cycle and consequently, in a 20% lower biomass yield. The intracellular alarmones ppGpp and cAMP accumulated in large quantities after the onset of nutrient limitation, subsequently declining to basal levels. The network topology of the regulation of the central metabolic pathways was identified so that the observed metabolic and regulatory behaviour can be described. This provides novel aspects of global regulation of the metabolism by the cra, crp and relA/spoT modulons. The work constitutes an important step towards dynamic mathematical modelling of regulation and metabolism, which is needed for the rational optimization of biotechnological processes.     

Integrating cybernetic modeling with pathway analysis provides a dynamic, systems-level description of metabolic control

Cybernetic modeling strives to uncover the inbuilt regulatory programs of biological systems and leverage them toward computational prediction of metabolic dynamics. Because of its focus on incorporating the global aims of metabolism, cybernetic modeling provides a systems-oriented approach for describing regulatory inputs and inferring the impact of regulation within biochemical networks. Combining cybernetic control laws with concepts from metabolic pathway analysis has culminated in a systematic strategy for constructing cybernetic models, which was previously lacking. The newly devised framework relies upon the simultaneous application of local controls that maximize the net flux through each elementary flux mode and global controls that modulate the activities of these modes to optimize the overall nutritional state of the cell. The modeling concepts are illustrated using a simple linear pathway and a larger network representing anaerobic E. coli central metabolism. The E. coli model successfully describes the metabolic shift that occurs upon deleting the pta-ackA operon that is responsible for fermentative acetate production. The model also furnishes predictions that are consistent with experimental results obtained from additional knockout strains as well as strains expressing heterologous genes. Because of the stabilizing influence of the included control variables, the resulting cybernetic models are more robust and reliable than their predecessors in simulating the network response to imposed genetic and environmental perturbations.     

Dynamic modeling of the central carbon metabolism of Escherichia coli

Application of metabolic engineering principles to the rational design of microbial production processes crucially depends on the ability to describe quantitatively the systemic behavior of the central carbon metabolism to redirect carbon fluxes to the product-forming pathways. Despite the importance for several production processes, development of an essential dynamic model for central carbon metabolism of Escherichia coli has been severely hampered by the current lack of kinetic information on the dynamics of the metabolic reactions. Here we present the design and experimental validation of such a dynamic model, which, for the first time, links the sugar transport system (i.e., phosphotransferase system [PTS]) with the reactions of glycolysis and the pentose-phosphate pathway. Experimental observations of intracellular concentrations of metabolites and cometabolites at transient conditions are used to validate the structure of the model and to estimate the kinetic parameters. Further analysis of the detailed characteristics of the system offers the possibility of studying important questions regarding the stability and control of metabolic fluxes.     

Effects of aeration on metabolic profiles of <Emphasis Type="Italic">Mortierella alpina</Emphasis> during the production of arachidonic acid

To investigate the metabolic regulation against oxygen supply, comparative metabolomics was performed to explore the metabolic responses of Mortierella alpina in the process of arachidonic acid (ARA) production. More than 110 metabolites involved in Embden–Meyerhof–Parnas pathway, pentose phosphate pathway, tricarboxylic acid cycle, inositol phosphate metabolism, fatty acid biosynthesis, and amino acid metabolism were identified by gas chromatography–mass spectrometry. Samples at different aeration rates were clearly distinguished by principal components analysis and partial least squares analysis, indicating that oxygen supply had a profound effect on the metabolism of M. alpina. Eleven major metabolites were identified as potential biomarkers to be primarily responsible for the difference of metabolism. Further study of metabolic changes with the relevant pathways demonstrated that the levels of several intermediate metabolites in relation to central carbon metabolism changed remarkably via both processes and citrate and malate was supposed to play vital roles in polyunsaturated acid (PUFA) synthesis. Increase of myo-inositol and sorbitol were probably for osmo-regulation and redox balance, while enhanced phosphoric acid and pyroglutamic acid were supposed to have function in the activation of signal transduction pathway for stress resistance. The present study provides a novel insight into the metabolic responses of M. alpina to aeration rates and the metabolic characteristics during the ARA fermentation.     

Analysis of NADPH supply during xylitol production by engineered Escherichia coli

Escherichia coli strain PC09 (DeltaxylB, cAMP-independent CRP (crp*) mutant) expressing an NADPH-dependent xylose reductase from Candida boidinii (CbXR) was previously reported to produce xylitol from xylose while metabolizing glucose [Cirino et al. (2006) Biotechnol Bioeng 95(6): 1167-1176]. This study aims to understand the role of NADPH supply in xylitol yield and the contribution of key central carbon metabolism enzymes toward xylitol production. Studies in which the expression of CbXR or a xylose transporter was increased suggest that enzyme activity and xylose transport are not limiting xylitol production in PC09. A constraints-based stoichiometric metabolic network model was used to understand the roles of central carbon metabolism reactions and xylose transport energetics on the theoretical maximum molar xylitol yield (xylitol produced per glucose consumed), and xylitol yields (Y(RPG)) were measured from resting cell biotransformations with various PC09 derivative strains. For the case of xylose-proton symport, omitting the Zwf (glucose-6-phosphate dehydrogenase) or PntAB (membrane-bound transhydrogenase) reactions or TCA cycle activity from the model reduces the theoretical maximum yield from 9.2 to 8.8, 3.6, and 8.0 mol xylitol (mol glucose)(-1), respectively. Experimentally, deleting pgi (encoding phosphoglucose isomerase) from strain PC09 improves the yield from 3.4 to 4.0 mol xylitol (mol glucose)(-1), while deleting either or both E. coli transhydrogenases (sthA and pntA) has no significant effect on the measured yield. Deleting either zwf or sucC (TCA cycle) significantly reduces the yield from 3.4 to 2.0 and 2.3 mol xylitol (mol glucose)(-1), respectively. Expression of a xylose reductase with relaxed cofactor specificity increases the yield to 4.0. The large discrepancy between theoretical maximum and experimentally determined yield values suggests that biocatalysis is compromised by pathways competing for reducing equivalents and dissipating energy. The metabolic role of transhydrogenases during E. coli biocatalysis has remained largely unspecified. Our results demonstrate the importance of direct NADPH supply by NADP+-utilizing enzymes in central metabolism for driving heterologous NADPH-dependent reactions, and suggest that the pool of reduced cofactors available for biotransformation is not readily interchangeable via transhydrogenase.     

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

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 [1-13C] and [U-13C]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 Entner-Doudoroff 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.     

Stress eating and tuning out: Cancer cells re-wire metabolism to counter stress

Abstract

Cancer cells reprogram metabolism to maintain rapid proliferation under often stressful conditions. Glycolysis and glutaminolysis are two central pathways that fuel cancer metabolism. Allosteric regulation and metabolite driven post-translational modifications of key metabolic enzymes allow cancer cells glycolysis and glutaminolysis to respond to changes in nutrient availability and the tumor microenvironment. While increased aerobic glycolysis (the Warburg effect) has been a noted part of cancer metabolism for over 80 years, recent work has shown that the elevated levels of glycolytic intermediates are critical to cancer growth and metabolism due to their ability to feed into the anabolic pathways branching off glycolysis such as the pentose phosphate pathway and serine biosynthesis pathway. The key glycolytic enzymes phosphofructokinase-1 (PFK1), pyruvate kinase (PKM2) and phosphoglycerate mutase 1 (PGAM1) are regulated by upstream and downstream metabolites to balance glycolytic flux with flux through anabolic pathways. Glutamine regulation is tightly controlled by metabolic intermediates that allosterically inhibit and activate glutamate dehydrogenase, which fuels the tricarboxylic acid cycle by converting glutamine derived glutamate to α-ketoglutarate. The elucidation of these key allosteric regulatory hubs in cancer metabolism will be essential for understanding and predicting how cancer cells will respond to drugs that target metabolism. Additionally, identification of the structures involved in allosteric regulation will inform the design of anti-metabolism drugs which bypass the off-target effects of substrate mimics. Hence, this review aims to provide an overview of allosteric control of glycolysis and glutaminolysis.     

Metabolic Flux Ratio Analysis of Genetic and Environmental Modulations of Escherichia coli Central Carbon Metabolism

The response of Escherichia coli central carbon metabolism to genetic and environmental manipulation has been studied by use of a recently developed methodology for metabolic flux ratio (METAFoR) analysis; this methodology can also directly reveal active metabolic pathways. Generation of fluxome data arrays by use of the METAFoR approach is based on two-dimensional (13)C-(1)H correlation nuclear magnetic resonance spectroscopy with fractionally labeled biomass and, in contrast to metabolic flux analysis, does not require measurements of extracellular substrate and metabolite concentrations. METAFoR analyses of E. coli strains that moderately overexpress phosphofructokinase, pyruvate kinase, pyruvate decarboxylase, or alcohol dehydrogenase revealed that only a few flux ratios change in concert with the overexpression of these enzymes. Disruption of both pyruvate kinase isoenzymes resulted in altered flux ratios for reactions connecting the phosphoenolpyruvate (PEP) and pyruvate pools but did not significantly alter central metabolism. These data indicate remarkable robustness and rigidity in central carbon metabolism in the presence of genetic variation. More significant physiological changes and flux ratio differences were seen in response to altered environmental conditions. For example, in ammonia-limited chemostat cultures, compared to glucose-limited chemostat cultures, a reduced fraction of PEP molecules was derived through at least one transketolase reaction, and there was a higher relative contribution of anaplerotic PEP carboxylation than of the tricarboxylic acid (TCA) cycle for oxaloacetate synthesis. These two parameters also showed significant variation between aerobic and anaerobic batch cultures. Finally, two reactions catalyzed by PEP carboxykinase and malic enzyme were identified by METAFoR analysis; these had previously been considered absent in E. coli cells grown in glucose-containing media. Backward flux from the TCA cycle to glycolysis, as indicated by significant activity of PEP carboxykinase, was found only in glucose-limited chemostat culture, demonstrating that control of this futile cycle activity is relaxed under severe glucose limitation.     

Proteomic studies of diauxic lag in the differentiating prokaryote Streptomyces coelicolor reveal a regulatory network of stress-induced proteins and central metabolic enzymes

Bacteria typically undergo intermittent periods of starvation and adaptation, emulated as diauxic growth in the laboratory. In association with growth arrest elicited by metabolic stress, the differentiating eubacterium Streptomyces coelicolor not only adapts its primary metabolism, but can also activate developmental programmes leading to morphogenesis and antibiotic biosynthesis. Here, we report combined proteomic and metabolomic data of S. coelicolor used to analyse global changes in gene expression during diauxic growth in a defined liquid medium. Cultures initially grew on glutamate, providing the nitrogen source and feeding carbon (as 2-oxoglutarate) into the TCA cycle, followed by a diauxic delay allowing reorientation of metabolism and a second round of growth supported by NH4+, formed during prediauxic phase, and maltose, a glycolytic substrate. Cultures finally entered stationary phase as a result of nitrogen starvation. These four physiological states had previously been defined statistically by their distinct patterns of protein synthesis and heat shock responses. Together, these data demonstrated that the rates of synthesis of heat shock proteins are determined not only by temperature increase but also by the patterns and rates of metabolic flux in certain pathways. Synthesis profiles for metabolic- and stress-induced proteins can now be interpreted by the identification of 204 spots (SWICZ database presented at http://proteom.biomed.cas.cz). Cluster analysis showed that the activity of central metabolic enzymes involved in glycolysis, the TCA cycle, starvation or proteolysis each displayed identifiable patterns of synthesis that logically underlie the metabolic state of the culture. Diauxic lag was accompanied by a structured regulatory programme involving the sequential activation of heat-, salt-, cold- and bacteriostatic antibiotic (pristinamycin I, PI)-induced stimulons. Although stress stimulons presumably provide protection during environmental- or starvation-induced stress, their identities did not reveal any coherent adaptive or developmental functions. These studies revealed interactive regulation of metabolic and stress response systems including some proteins known to support developmental programmes in S. coelicolor.     

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.     

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.     

Thermodynamically based profiling of drug metabolism and drug-drug metabolic interactions: a case study of acetaminophen and ethanol toxic interaction

Drug-drug metabolic interactions can result in unwanted side effects, including reduced drug efficacy and formation of toxic metabolic intermediates. In this work, thermodynamic constraints on non-equilibrium metabolite concentrations are used to reveal the biochemical interactions between the metabolic pathways of ethanol and acetaminophen (N-acetyl-p-aminophenol), two drugs known to interact unfavorably. It is known that many reactions of these pathways are coupled to the central energy metabolic reactions through a number of metabolites and the cellular redox potential. Based on these observations, a metabolic network model has been constructed and a database of thermodynamic properties for all participating metabolites and reactions has been compiled. Constraint-based computational analysis of the feasible metabolite concentrations reveals that the non-toxic pathways for APAP metabolism and the pathway for detoxifying N-acetyl-p-benzoquinoneimine (NAPQI) are inhibited by network interactions with ethanol metabolism. These results point to the potential utility of thermodynamically based profiling of metabolic network interactions in screening of drug candidates and analysis of potential toxicity.     

Structural analysis of metabolic networks based on flux centrality

Metabolic reactions are fundamental to living organisms, and a large number of reactions simultaneously occur at a given time in living cells transforming diverse metabolites into each other. There has been an ongoing debate on how to classify metabolites with respect to their importance for metabolic performance, usually based on the analysis of topological properties of genome scale metabolic networks. However, none of these studies have accounted quantitatively for flux in metabolic networks, thus lacking an important component of a cell’s biochemistry.We therefore analyzed a genome scale metabolic network of Escherichia coli by comparing growth under 19 different growth conditions, using flux balance analysis and weighted network centrality investigation. With this novel concept of flux centrality we generated metabolite rankings for each particular growth condition. In contrast to the results of conventional analysis of genome scale metabolic networks, different metabolites were top-ranking dependent on the growth condition. At the same time, several metabolites were consistently among the high ranking ones. Those are associated with pathways that have been described by biochemists as the most central part of metabolism, such as glycolysis, tricarboxylic acid cycle and pentose phosphate pathway. The values for the average path length of the analyzed metabolite networks were between 10.5 and 12.6, supporting recent findings that the metabolic network of E. coli is not a small-world network.     

Resolving the TCA cycle and pentose-phosphate pathway of Clostridium acetobutylicum ATCC 824: Isotopomer analysis, in vitro activities and expression analysis

Solventogenic clostridia are an important class of microorganisms that can produce various biofuels. One of the bottlenecks in engineering clostridia stems from the fact that central metabolic pathways remain poorly understood. Here, we utilized the power of (13) C-based isotopomer analysis to re-examine central metabolic pathways of Clostridium acetobutylicum ATCC 824. We demonstrate using [1,2-(13) C]glucose, MS analysis of intracellular metabolites, and enzymatic assays that C. acetobutylicum has a split TCA cycle where only Re-citrate synthase (CS) contributes to the production of α-ketoglutarate via citrate. Furthermore, we show that there is no carbon exchange between α-ketoglutarate and fumarate and that the oxidative pentose-phosphate pathway (oxPPP) is inactive. Dynamic gene expression analysis of the putative Re-CS gene (CAC0970), its operon, and all glycolysis, pentose-phosphate pathway, and TCA cycle genes identify genes and their degree of involvement in these core pathways that support the powerful primary metabolism of this industrial organism.