Universität Stuttgart
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Item Open Access A systems biology approach to analyse leaf carbohydrate metabolism in Arabidopsis thaliana(2011) Henkel, Sebastian; Nägele, Thomas; Hörmiller, Imke; Sauter, Thomas; Sawodny, Oliver; Ederer, Michael; Heyer, Arnd G.Plant carbohydrate metabolism comprises numerous metabolite interconversions, some of which form cycles of metabolite degradation and re-synthesis and are thus referred to as futile cycles. In this study, we present a systems biology approach to analyse any possible regulatory principle that operates such futile cycles based on experimental data for sucrose (Scr) cycling in photosynthetically active leaves of the model plant Arabidopsis thaliana. Kinetic parameters of enzymatic steps in Scr cycling were identified by fitting model simulations to experimental data. A statistical analysis of the kinetic parameters and calculated flux rates allowed for estimation of the variability and supported the predictability of the model. A principal component analysis of the parameter results revealed the identifiability of the model parameters. We investigated the stability properties of Scr cycling and found that feedback inhibition of enzymes catalysing metabolite interconversions at different steps of the cycle have differential influence on stability. Applying this observation to futile cycling of Scr in leaf cells points to the enzyme hexokinase as an important regulator, while the step of Scr degradation by invertases appears subordinate.Item Open Access Basic regulatory principles of Escherichia coli's electron transport chain for varying oxygen conditions(2014) Henkel, Sebastian; Beek, Alexander ter; Steinsiek, Sonja; Stagge, Stefan; Bettenbrock, Katja; Teixeira de Mattos, M. Joost; Sauter, Thomas; Sawodny, Oliver; Ederer, MichaelFor adaptation between anaerobic, micro-aerobic and aerobic conditions Escherichia coli's metabolism and in particular its electron transport chain (ETC) is highly regulated. Although it is known that the global transcriptional regulators FNR and ArcA are involved in oxygen response it is unclear how they interplay in the regulation of ETC enzymes under micro-aerobic chemostat conditions. Also, there are diverse results which and how quinones (oxidised/reduced, ubiquinone/other quinones) are controlling the ArcBA two-component system. In the following a mathematical model of the E. coli ETC linked to basic modules for substrate uptake, fermentation product excretion and biomass formation is introduced. The kinetic modelling focusses on regulatory principles of the ETC for varying oxygen conditions in glucose-limited continuous cultures. The model is based on the balance of electron donation (glucose) and acceptance (oxygen or other acceptors). Also, it is able to account for different chemostat conditions due to changed substrate concentrations and dilution rates. The parameter identification process is divided into an estimation and a validation step based on previously published and new experimental data. The model shows that experimentally observed, qualitatively different behaviour of the ubiquinone redox state and the ArcA activity profile in the micro-aerobic range for different experimental conditions can emerge from a single network structure. The network structure features a strong feed-forward effect from the FNR regulatory system to the ArcBA regulatory system via a common control of the dehydrogenases of the ETC. The model supports the hypothesis that ubiquinone but not ubiquinol plays a key role in determining the activity of ArcBA in a glucose-limited chemostat at micro-aerobic conditions.Item Open Access Model-based analysis of an adaptive evolution experiment with Escherichia coli in a pyruvate limited continuous culture with glycerol(2012) Feuer, Ronny; Gottlieb, Katrin; Viertel, Gero; Klotz, Johannes; Schober, Steffen; Bossert, Martin; Sawodny, Oliver; Sprenger, Georg; Ederer, MichaelBacterial strains that were genetically blocked in important metabolic pathways and grown under selective conditions underwent a process of adaptive evolution: certain pathways may have been deregulated and therefore allowed for the circumvention of the given block. A block of endogenous pyruvate synthesis from glycerol was realized by a knockout of pyruvate kinase and phosphoenolpyruvate carboxylase in E. coli. The resulting mutant strain was able to grow on a medium containing glycerol and lactate, which served as an exogenous pyruvate source. Heterologous expression of a pyruvate carboxylase gene from Corynebacterium glutamicum was used for anaplerosis of the TCA cycle. Selective conditions were controlled in a continuous culture with limited lactate feed and an excess of glycerol feed. After 200–300 generations pyruvate-prototrophic mutants were isolated. The genomic analysis of an evolved strain revealed that the genotypic basis for the regained pyruvate-prototrophy was not obvious. A constraint-based model of the metabolism was employed to compute all possible detours around the given metabolic block by solving a hierarchy of linear programming problems. The regulatory network was expected to be responsible for the adaptation process. Hence, a Boolean model of the transcription factor network was connected to the metabolic model. Our model analysis only showed a marginal impact of transcriptional control on the biomass yield on substrate which is a key variable in the selection process. In our experiment, microarray analysis confirmed that transcriptional control probably played a minor role in the deregulation of the alternative pathways for the circumvention of the block.Item Open Access A rapid method for the extraction and analysis of carotenoids and other hydrophobic substances suitable for systems biology studies with photosynthetic bacteria(2013) Bóna-Lovász, Judit; Bóna, Aron; Ederer, Michael; Sawodny, Oliver; Ghosh, RobinA simple, rapid, and inexpensive extraction method for carotenoids and other non-polar compounds present in phototrophic bacteria has been developed. The method, which has been extensively tested on the phototrophic purple non-sulphur bacterium Rhodospirillum rubrum, is suitable for extracting large numbers of samples, which is common in systems biology studies, and yields material suitable for subsequent analysis using HPLC and mass spectroscopy. The procedure is particularly suitable for carotenoids and other terpenoids, including quinones, bacteriochlorophyll a and bacteriopheophytin a, and is also useful for the analysis of polar phospholipids. The extraction procedure requires only a single step extraction with a hexane/methanol/water mixture, followed by HPLC using a Spherisorb C18 column, with a mobile phase consisting of acetone-water and a non-linear gradient of 50%-100% acetone. The method was employed for examining the carotenoid composition observed during microaerophilic growth of R. rubrum strains, and was able to determine 18 carotenoids, 4 isoprenoid-quinones, bacteriochlorophyll a and bacteriopheophytin a as well as four different phosphatidylglycerol species of different acyl chain compositions. The analytical procedure was used to examine the dynamics of carotenoid biosynthesis in the major and minor pathways operating simultaneously in a carotenoid biosynthesis mutant of R. rubrum.Item Open Access Model-based characterization of inflammatory gene expression patterns of activated macrophages(2016) Rex, Julia; Albrecht, Ute; Ehlting, Christian; Thomas, Maria; Zanger, Ulrich M.; Sawodny, Oliver; Häussinger, Dieter; Ederer, Michael; Feuer, Ronny; Bode, Johannes G.Macrophages are cells with remarkable plasticity. They integrate signals from their microenvironment leading to context-dependent polarization into classically (M1) or alternatively (M2) activated macrophages, representing two extremes of a broad spectrum of divergent phenotypes. Thereby, macrophages deliver protective and pro-regenerative signals towards injured tissue but, depending on the eliciting damage, may also be responsible for the generation and aggravation of tissue injury. Although incompletely understood, there is emerging evidence that macrophage polarization is critical for these antagonistic roles. To identify activation-specific expression patterns of chemokines and cytokines that may confer these distinct effects a systems biology approach was applied. A comprehensive literature-based Boolean model was developed to describe the M1 (LPS-activated) and M2 (IL-4/13-activated) polarization types. The model was validated using high-throughput transcript expression data from murine bone marrow derived macrophages. By dynamic modeling of gene expression, the chronology of pathway activation and autocrine signaling was estimated. Our results provide a deepened understanding of the physiological balance leading to M1/M2 activation, indicating the relevance of co-regulatory signals at the level of Akt1 or Akt2 that may be important for directing macrophage polarization.Item Open Access Modeling time delay in the NFκB signaling pathway following low dose IL-1 stimulation(2011) Witt, Johannes; Barisic, Sandra; Sawodny, Oliver; Ederer, Michael; Kulms, Dagmar; Sauter, ThomasStimulation of human epithelial cells with IL-1 (10 ng/ml) + UVB radiation results in sustained NFκB activation caused by continuous IKKbeta phosphorylation. We have recently published a strictly reduced ordinary differential equation model elucidating the involved mechanisms. Here, we compare model extensions for low IL-1 doses (0.5 ng/ml), where delayed IKKbeta phosphorylation is observed. The extended model including a positive regulatory element, most likely auto-ubiquitination of TRAF6, reproduces the observed experimental data most convincingly. The extension is shown to be consistent with the original model and contains very sensitive processes which may serve as potential intervention targets.Item Open Access A mathematical model of metabolism and regulation provides a systems-level view of how Escherichia coli responds to oxygen(2014) Ederer, Michael; Steinsiek, Sonja; Stagge, Stefan; Rolfe, Matthew D.; Beek, Alexander tek; Knies, David; Teixeira de Mattos, M. Joost; Sauter, Thomas; Green, Jeffrey; Poole, Robert K.; Bettenbrock, Katja; Sawodny, OliverThe efficient redesign of bacteria for biotechnological purposes, such as biofuel production, waste disposal or specific biocatalytic functions, requires a quantitative systems-level understanding of energy supply, carbon and redox metabolism. The measurement of transcript levels, metabolite concentrations and metabolic fluxes per se gives an incomplete picture. An appreciation of the interdependencies between the different measurement values is essential for systems-level understanding. Mathematical modeling has the potential to provide a coherent and quantitative description of the interplay between gene expression, metabolite concentrations and metabolic fluxes. Escherichia coli undergoes major adaptations in central metabolism when the availability of oxygen changes. Thus, an integrated description of the oxygen response provides a benchmark of our understanding of carbon, energy and redox metabolism. We present the first comprehensive model of the central metabolism of E. coli that describes steady-state metabolism at different levels of oxygen availability. Variables of the model are metabolite concentrations, gene expression levels, transcription factor activities, metabolic fluxes and biomass concentration. We analyze the model with respect to the production capabilities of central metabolism of E. coli. In particular, we predict how precursor and biomass concentration are affected by product formation.Item Open Access Thermokinetic modeling and model reduction of reaction networks(2010) Ederer, Michael; Gilles, Ernst Dieter (Prof. Dr.-Ing. Dr. h.c. mult.)This work introduces the thermokinetic modeling formalism (TKM). TKM is a framework for thermodynamically consistent, kinetic modeling and model reduction of biochemical reaction networks. Kinetic models describe the dynamics of the concentrations and fluxes in a biochemical reaction network by means of the network stoichiometry and the kinetic rate equations. The laws of thermodynamics constrain the possible dynamics of reaction networks and thus constrain physically feasible kinetic models. Especially for large networks, as they are considered in computational systems biology, finding thermodynamically consistent parameters can be difficult. TKM is a convenient and user-friendly formalism to build thermodynamically consistent kinetic models. The TKM formalism is based on thermokinetic potentials of compounds and thermokinetic forces of reactions. These quantities are derived from chemical potentials and Gibbs reaction energies. In the case of ideal dilute solutions, thermokinetic potentials are proportional to the corresponding concentrations. The constant proportionality factors are the thermokinetic capacities of the compounds. In the case of mass-action kinetics, the thermokinetic forces and the reaction fluxes are proportional: The constant proportionality factors are the thermokinetic resistances of the reactions. Non-ideal solutions or complex kinetics lead to non-constant, state-dependent capacities and resistances. Each model described by capacities and resistances is thermodynamically consistent and structurally fulfills the Wegscheider conditions. In addition, each thermodynamically consistent, kinetic model can be expressed by capacities and resistances. Thus, the use of these quantities provides a simple and comprehensive way for thermodynamically consistent modeling. If a thermokinetic model fulfills certain conditions, the model size can be reduced by suited transformation and reduction steps. In particular, the model size can be reduced if the model contains conservation relations or stoichiometric cycles. Further, a reduction is possible if resistances or capacities have a value of zero. Capacities of zero correspond to quasi-stationary compounds and resistances of zero correspond to reactions in rapid equilibrium. Due to the formal structure of thermokinetic models, model reduction based on the rapid equilibrium assumption is particularly simple. It can be easily applied to reaction rules as they are used to describe protein-protein interaction networks with inherent combinatorial complexity. Thermokinetic models can be depicted in a diagram as a connection of basic network elements representing the compounds and reactions. Several model reduction methods can be formulated as graphical rules, which allow for a simple and intuitive reduction of the model size. The TKM formalism is used to model the oxygen response of the bacterium Escherichia coli, which is strongly determined by thermodynamic constraints. In order to restrict the model to the relevant parameters and dynamics, model reduction techniques are applied. The model is able to explain the measured metabolic fluxes and concentrations in the wild type and a regulatory mutant in dependence of the oxygen availability. This example also shows that TKM is useful for modeling large networks. TKM unifies thermodynamic and kinetic approaches for the modeling of biochemical reaction networks in a natural and formally appealing way. In particular, it introduces thermodynamic flow-force relationships into kinetic modeling. In this way, TKM guarantees the thermodynamic consistency of the model equations. In the conventional kinetic modeling approach, the kinetic parameters are formally attributed to reactions but not compounds. However, the equilibrium constants that, in the conventional modeling approach, are ratios of kinetic parameters are solely determined by the thermodynamic properties of the compounds. This finally may lead to kinetic models violating thermodynamic constraints unless the Wegscheider conditions are explicitly considered. TKM clearly distinguishes between the thermodynamic parameters, the capacities, and the kinetic parameters, the resistances. Thus, TKM provides a thermodynamically consistent parameterization of kinetic models. TKM also provides thermodynamically consistent and conveniently usable model reduction methods. Altogether, TKM strongly simplifies the mathematical modeling of complex biochemical networks.