-
Johannes Bongaerts,
Simon Esser,
Volker Lorbach,
Lóay Al-Momani,
Michael A Müller,
Dirk Franke,
Christoph Grondal,
Anja Kurutsch,
Robert Bujnicki, Ralf Takors,
Leon Raeven,
Marcel Wubbolts,
Roel Bovenberg,
Martin Nieger,
Melanie Schürmann,
Natalie Trachtmann,
Stefan Kozak,
Georg A Sprenger,
Michael Müller
Angewandte Chemie International Edition 08/2011; 50(34):7781-6. · 13.45 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: The enzyme targets for the rational optimization of a Corynebacterium glutamicum strain constructed for valine production are identified by analyzing the control of flux in the valine/leucine pathway. The control analysis is based on measurements of the intracellular metabolite concentrations and on a kinetic model of the reactions in the investigated pathway. Data-driven and model-based methods are used and evaluated against each other. The approach taken gives a quantitative evaluation of the flux control and it is demonstrated how the understanding of flux control is used to reach specific recommendations for strain optimization. The flux control coefficients (FCCs) with respect to the valine excretion rate were calculated, and it was found that the control is distributed mainly between the acetohydroxyacid synthase enzyme (FCC = 0.32), the branched chain amino acid transaminase (FCC = 0.27), and the exporting translocase (FCC = 0.43). The availability of the precursor pyruvate has substantial influence on the valine flux, whereas the cometabolites are less important as demonstrated by the calculation of the respective response coefficients. The model is further used to make in-silico predictions of the change in valine flux following a change in enzyme level. A doubling of the enzyme level of valine translocase will result in an increase in valine flux of 31%. By optimizing the enzyme levels with respect to valine flux it was found that the valine flux can be increased by a factor 2.5 when the optimal enzyme levels are implemented.
Biotechnology Progress 05/2009; 25(3):754-62. · 2.34 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: A highly selective and sensitive method for identification and quantification of intracellular metabolites involved in central carbon metabolism (including glycolysis, pentose phosphate pathway and tricarboxylic acid cycle) by means of liquid chromatography-tandem quadrupole mass spectrometry (LC-MS/MS) was developed. The volatile ion pair modifier tributylammonium acetate (TBAA) was employed in the mobile phase for simultaneously separation of 29 negatively charged compounds including sugar phosphates, nucleotides, and carboxylic acids on a common C18 reversed-phase column. Method validation results displayed that limits of detection (LODs) calculated according to DIN (German Institute for Standardization) 32645 are mostly below 60 nM, only with the exception of pyruvate and malate. The calibration curves showed excellent linearity mainly over three orders of magnitude with correlation coefficients R(2)>0.9982. This LC-MS/MS method was successfully applied to determine these metabolites in cell extracts of Escherichia coli. Most of the intracellular metabolites were found within the detection range and the relative standard deviations of the measurements were smaller than 5.65% (n=5) for a cell extract sample.
Journal of Chromatography 05/2007; 1147(2):153-64. · 4.53 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: A novel approach to (13)C metabolic flux analysis (MFA) is presented using cytosolic metabolite pool sizes and their (13)C labeling data from an isotopically non-stationary (13)C labeling experiment (INST-CLE). The procedure is demonstrated with an E. coli wild type strain grown at fed batch conditions. The intra cellular labeling dynamics are excited by a sudden step increase of the (13)C portion in the substrate feed. Due to unchanged saturation of the substrate uptake system, the metabolic fluxes remain constant during the following sampling time period of only 16s, in which 20 samples are taken by an automated rapid sampling device immediately stopping metabolism by methanol quenching. Subsequent cell disruptive sample preparation and LC-MS/MS enabled simultaneous determination of pool sizes and mass isotopomers of intra cellular metabolites requiring detection limits in the nM range. Based on this data the new computational flux analysis tool 13CFLUX/INST is used to determine the intra cellular fluxes based on a complex carbon labeling network model. The measured data is in good agreement with the model predictions, thus proving the applicability of the new isotopically non-stationary (13)C metabolic flux analysis (INST-(13)C-MFA) concept. Moreover, it is shown that significant new information with respect to flux identifiability, non-measurable pool sizes, data consistency, or large storage pools can be taken from the novel kind of experimental data. This offers new insight into the biological operation of the metabolic network in vivo.
Journal of Biotechnology 05/2007; 129(2):249-67. · 3.05 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: Two new concepts, "Limitation Potential" and "Constraint Limitation Sensitivity" are introduced that use definitions derived from metabolic flux analysis (MFA) and metabolic network analysis (MNA). They are applied to interpret a measured flux distribution in the context of all possible flux distributions and thus combine MFA with MNA. The proposed measures are used to quantify and compare the influence of intracellular fluxes on the production yield. The methods are purely based on the stoichiometry of the network and constraints that are given from irreversible fluxes. In contrast to metabolic control analysis (MCA), within this approach no information about the kinetic mechanisms are needed. A limitation potential (LP) is defined as the reduction of the reachable (theoretical) maximum by a measured flux. Measured fluxes that strongly narrow the reachable maximum are assumed to be limiting as the network has no ability to counterbalance the restriction due to the observed flux. In a second step, the sensitivity of the reduced maximum is regarded. This measure provides information about the necessitated changes to reach higher yields. The methods are applied to interpret the capabilities of a network based on measured fluxes for a L-phenylalanine producer. The strain was examined by a series of experiments and three flux maps of the production phase are analyzed. It can be shown that the reachable yield is drastically reduced by the measured efflux into the TCA cycle, while the oxidative pentose-phosphate pathway only plays a secondary role on the reachable maximum.
Biotechnology and Bioengineering 07/2006; 94(2):263-72. · 3.95 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: A model discriminating experimental design approach for fed-batch processes has been developed and applied to the fermentative production of L-valine by a genetically modified Corynebacterium glutamicum strain possessing multiple auxotrophies as an example. Being faced with the typical situation of uncertain model information based on preliminary experiments, model discriminating design was successfully applied to improve discrimination between five competing models. Within the same modeling and experimental design framework, also the planning of an optimized production process with respect to the total volumetric productivity is shown. Simulation results were experimentally affirmed, yielding an increased total volumetric productivity of 6.2 mM L-valine per hour. However, also so far unknown metabolic mechanisms were observed in the optimized process, underlining the importance of process optimization during modeling to avoid problems of extreme extrapolation of model predictions during the final process optimization.
Biotechnology and Bioengineering 09/2005; 91(3):356-68. · 3.95 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: Supporting the evolutionary modeling process of dynamic biochemical networks based on sampled in vivo data requires more than just simulation. In the course of the modeling process, the modeler is typically concerned not only with a single model but also with sequences, alternatives and structural variants of models. Powerful automatic methods are then required to assist the modeler in the organization and the evaluation of alternative models. Moreover, the structure and peculiarities of the data require dedicated tool support.
To support all stages of an evolutionary modeling process, a new general formalism for the combinatorial specification of large model families is introduced. It allows for automatic navigation in the space of models and excludes biologically meaningless models on the basis of elementary flux mode analysis. An incremental usage of the measured data is supported by using splined data instead of state variables. With MMT2, a versatile tool has been developed as a computational engine intended to be built into a tool chain. Using automatic code generation, automatic differentiation for sensitivity analysis and grid computing technology, a high performance computing environment is achieved. MMT2 supplies XML model specification and several software interfaces. The performance of MMT2 is illustrated by several examples from ongoing research projects.
http://www.simtec.mb.uni-siegen.de/
wiechert@simtec.mb.uni-siegen.de.
Bioinformatics 05/2005; 21(8):1617-25. · 5.47 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: So far it is mainly transcriptome and proteome analysis that has been applied to elucidate the correlation between genotype and phenotype although thorough metabolome studies can provide substantial information on the control of the metabolism at the biochemical level. Stimulus-response experiments, i.e. the investigation of metabolism dynamics after a glucose pulse (pulse experiment), can be used to study the in vivo enzyme kinetics offering insight into underlying reaction mechanisms. Usually, this requires rapid cell quenching combined with cell inactivation to'freeze' the microbial metabolism response at a definite time-lag after pulse stimulation. To access the 'frozen' metabolic reply, adequate analytical methods are needed to measure intracellular metabolite concentrations in the cell extract. As shown in the introductory review part, stimulus-response experiments were usually applied to study central metabolism dynamics in wildtype strains. Our own results, presented in the second part of the contribution, indicate that stimulus-response experiments should also be applied to analyse pathway dynamics in anabolic routes. Using the example of the aromatic amino acid pathway, an LC-MS/MS technique is presented that allows the quantification of intracellular pools of central metabolism as well as of the aromatic amino acid pathway. Based on the analytical approach metabolic profiling is performed to monitor the metabolism dynamics after a glucose pulse experiment allowing the conclusion that pulse stimulation is transmitted to the anabolic pathway of interest.
Advances in biochemical engineering/biotechnology 02/2005; 92:173-96. · 1.64 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: Using the pyruvate production strain Escherichia coli YYC202 ldhA::Kan different process alternatives are studied with the aim of preventing potential product inhibition by appropriate product separation. This strain is completely blocked in its ability to convert pyruvate into acetyl-CoA or acetate, resulting in acetate auxotrophy during growth in glucose minimal medium. Continuous experiments with cell retention, repetitive fed-batch, and an in situ product recovery (ISPR) process with fully integrated electrodialysis were tested. Although the continuous approach achieved a high volumetric productivity (QP) of 110 g L(-1) d(-1), this approach was not pursued because of long-term production strain instabilities. The highest pyruvate/glucose molar yield of up to 1.78 mol mol(-1) together with high QP 145 g L(-1) d(-1) and high pyruvate titers was achieved by the repetitive fed-batch approach. To separate pyruvate from fermentation broth a fully integrated continuous process was developed. In this process electrodialysis was used as a separation unit. Under optimum conditions a (calculated) final pyruvate titer of >900 mmol L(-1) (79 g L(-1)) was achieved.
Biotechnology and Bioengineering 04/2004; 85(6):638-46. · 3.95 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: Cyclohexadiene-trans-5,6-diols such as (S,S)-2,3-dihydroxy-2,3-dihydrobenzoic acid (2,3-trans-CHD) have been shown to be of importance as chiral starting materials for the syntheses of bioactive substances, especially for the syntheses of carbasugars. By using methods of metabolic-pathway engineering, the Escherichia coli genes entB and entC, which encode isochorismatase and isochorismate synthase, were cloned and over-expressed in E. coli strains with a deficiency of entA, which encodes 2,3-dihydroxybenzoate synthase. A 30-fold increase in the corresponding EntB/EntC enzyme activities affects the accumulation of 2,3-trans-CHD in the cultivation medium. Although the strains did not contain deletions in chorismate-utilising pathways towards aromatic amino acids, neither chorismate nor any other metabolic intermediates were found as by-products. Fermentation of these strains in a 30 L pH-controlled stirred tank reactor showed that 2,3-trans-CHD could be obtained in concentrations of up to 4.6 g L(-1). This demonstrates that post-chorismate metabolites are accessible on a preparative scale by using techniques of metabolic-pathway engineering. Isolation and separation from fermentation salts could be performed economically in one step through anion-exchange chromatography or, alternatively, by reactive extraction. Starting from 2,3-trans-CHD as an example, we established short syntheses towards new carbasugar derivatives.
Chemistry 10/2003; 9(17):4188-96. · 5.93 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: The rational improvement of microbial strains for the production of primary and secondary metabolites ('metabolic engineering') requires a quantitative understanding of microbial metabolism. A process by which this information can be derived from dynamic fermentation experiments is presented. By applying a substrate pulse to a substrate-limited, steady state culture, cellular metabolism is shifted away from its metabolic steady state. With the aid of a rapid sampling and quenching routine it is possible to take 4-5 samples per second during this process, thus capturing the metabolic response to this stimulus. Over 30 metabolites, nucleotides and cofactors from Escherichia coli metabolism can be extracted and analysed using a range of different techniques, for example enzymatic assays, HPLC and LC-MS methods. Using different substrates as limiting and pulse-substrates (glucose, glycerol), different metabolic pathways and substrate uptake systems are investigated. The resulting plots of intracellular metabolite concentrations against time serve as a data basis for modelling microbial metabolic networks.
Biomolecular Engineering 07/2002; 19(1):5-15. · 3.17 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: The identification of metabolic regulation is a major concern in metabolic engineering. Metabolic regulation phenomena depend on intracellular compounds such as enzymes, metabolites and cofactors. A complete understanding of metabolic regulation requires quantitative information about these compounds under in vivo conditions. This quantitative knowledge in combination with the known network of metabolic pathways allows the construction of mathematical models that describe the dynamic changes in metabolite concentrations over time. Rapid sampling combined with pulse experiments is a useful tool for the identification of metabolic regulation owing to the transient data they provide. Enzymatic tests in combination with ESI-LC-MS (Electrospray Ionization Liquid Chromatographic Tandem Mass Spectrometry) and HPLC measurements have been used to identify up to 30 metabolites and nucleotides from rapid sampling experiments. A metabolic modeling tool (MMT) that is built on a relational database was developed specifically for analysis of rapid sampling experiments. The tool allows to construct complex pathway models with information stored in the relational database. Parameter fitting and simulation algorithms for the resulting system of Ordinary Differential Equations (ODEs) are part of MMT. Additionally explicit sensitivity functions are calculated. The integration of all necessary algorithms in one tool allows fast model analysis and comparison. Complex models have been developed to describe the central metabolic pathways of Escherichia coli during a glucose pulse experiment.
In silico biology 02/2002; 2(4):467-84.
-
01/2002
-
Computer science and biology: Proceedings of the German Conference on Bioinformatics, GCB 2001, October 7-10, 2001, Braunschweig, Germany; 01/2001
-
[show abstract]
[hide abstract]
ABSTRACT: The regioselective oxidation of terfenadine with the fungi Cunninghamella blakesleeana was studied as a biochemical alternative for the chemical synthesis of the antihistaminic drug fexofenadine. It was demonstrated that C. blakesleeana oxidises the tert-butyl group of terfenadine to the corresponding alcohol 1-[4-(1,1-dimethyl-2-hydroxyethyl)phenyl]-4-[4-(hydroxydiphenylmethyl)-1-piperidinyl]-1-butanol. A continuous process for regioselective oxidation of terfenadine was developed. Terfenadine was supplied micro-crystalline due to the low solubility in water. Optimum reaction conditions with respect to medium composition, temperature, pH, pO2, co-substrate and feeding rates were found by means of reaction engineering studies. A cross-flow microfiltration unit was operated in a by-pass of a lab-scale stirred tank reactor for retention of the biocatalysts and the micro-crystalline substrate. The alcohol was continuously removed with the filtrate to minimise product inhibition. Continuous biotransformation of micro-crystalline terfenadine with C. blakesleeana in the membrane reactor system with a dilution rate of 33 h at co-substrate concentrations of about 1 up to 3 g/l glycerol in the reactor resulted in a space–time yield of 145 mg of alcohol/l/day and an alcohol yield of 71%. The produced alcohol was easily isolated from the filtrate by adsorption on XAD-4 resin followed by eluation with methanol (concentration factor 7).
Journal of Molecular Catalysis B: Enzymatic.
-
[show abstract]
[hide abstract]
ABSTRACT: The quantitative comprehension of microbial metabolic networks is a prerequisite for an efficient rational strain improvement (“metabolic engineering”). It is therefore necessary to accurately determine the concentration of a large number of reactants (i.e., metabolites, nucleotides, cofactors) in order to understand “in vivo” reaction kinetics. Quantification of intracellular concentrations of glycolytic intermediates and nucleotides in Escherichia coli K12 using a perchloric acid extraction and an LC-ESI-MS method was achieved. Intracellular metabolites (e.g., glucose 6-phosphate, fructose 1,6-bisphosphate, 6-phospho gluconate, acetyl-CoA, adenine nucleotides) were quantified under defined (glucose-limited steady-state) growth conditions. The method was verified by comparing the intracellular metabolite concentrations measured via LC-ESI-MS with enzymatic determinations. It is thus possible to identify and quantify more than 15 intracellular metabolites in parallel with a minimal amount of sample volume.
Analytical Biochemistry.
-
[show abstract]
[hide abstract]
ABSTRACT: The intracellular concentrations of the valine and leucine pathway intermediates in a Corynebacterium glutamicum strain were measured during a transient state. The data were obtained by performing a glucose stimulus-response experiment with the use of a rapid sampling device and advanced mass spectrometry. The glucose stimulus resulted in a 3-fold increase in the intracellular pyruvate concentration within less than a second, demonstrating the very fast interactions in metabolic networks. The samples were taken at subsecond intervals for a time period of 25 s. The time courses of the metabolite concentrations formed the experimental basis of a mathematical model simulating the fluxes and concentrations in the valine/leucine pathway. The implementation of a model selection criterion based on the second law of thermodynamics is demonstrated to be essential for the identification of realistic and unique models. Large differences between the enzyme properties determined in vitro and those determined in vivo by the model were observed with the in vivo maximal rates being almost an order of magnitude larger than the in vitro maximal rates. The transamination of ketoisovalerate (KIV) to valine is carried out mainly by the transaminase B enzyme, with the transaminase C enzyme playing a minor role. The availability of the cofactors NADP and NADPH only has modest influence on the flux through the valine pathway, while the influence of NAD and NADH on the flux through the leucine pathway is negligible.
Biotechnology Progress 22(4):1071-83. · 2.34 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: With the aid of the recently developed Sensor reactor system, a series of three subsequent (13)C labeling experiments was performed mirroring the l-phenylalanine (l-Phe) production phase of a recombinant E. coli strain that was cultivated under industry-like conditions in a 300 L bioreactor. On the basis of the data from NMR labeling analysis, three subsequent flux patterns were successfully derived monitoring the l-Phe formation during an observation window from 14 to 23.3 h process time. Linear programming was performed to identify optimal flux patterns for l-Phe formation. Additionally, flux sensitivity analysis was used to identify the most promising metabolic engineering target. As a result, high rates of phosphoenolpyruvate (PEP) to pyruvate (PYR) conversion were identified as the most important reason for deterioration of the l-Phe/glucose yield from 20 to finally 11 mol %. Considering the characteristics of the enzyme kinetics involved, the working hypothesis was formulated that phosphoenolpyruvate synthase activity was increasingly hampered by rising oxaloacetate and 2-oxoglutarate concentrations, while at the same time pyruvate kinase activity arose due to activation by fructose 1,6-diphosphate. Hence, pps overexpression should be performed to optimize the existing production strain.
Biotechnology Progress 20(3):706-14. · 2.34 Impact Factor
