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Intracellular Metabolite Concentrations (intracellular + metabolite_concentration)

Distribution by Scientific Domains

Life Sciences	100%

Selected Abstracts

Respiratory oscillations in yeast: mitochondrial reactive oxygen species, apoptosis and time; a hypothesis

FEMS YEAST RESEARCH, Issue 4 2003
David Lloyd
Abstract Oscillatory metabolic activities occur more widely than is generally realised; detectability requires observation over extended times of single yeast cells or synchrony of individuals to provide a coherent population. Where oscillations in intracellular metabolite concentrations are observed, the phenomenon has been ascribed to sloppy control, energetic optimisation, signalling, temporal compartmentation of incompatible reactions, or timekeeping functions. Here we emphasise the consequences of respiratory oscillations as a source of mitochondrially generated reactive O2 metabolites. Temporal co-ordination of intracellular activities necessitates a time base. This is provided by an ultradian clock, and one result of its long-term operation is cyclic energisation of mitochondria, and thereby the generation of deleterious free radical species. Our hypothesis is that unrepaired cellular constituents and components (especially mitochondria) eventually lead to cellular senescence and apoptosis when a finite number of respiratory cycles has occurred. [source]

Analysis of Escherichia coli anaplerotic metabolism and its regulation mechanisms from the metabolic responses to altered dilution rates and phosphoenolpyruvate carboxykinase knockout

BIOTECHNOLOGY & BIOENGINEERING, Issue 2 2003
Chen Yang
Abstract The gluconeogenic phosphoenolpyruvate (PEP) carboxykinase is active in Escherichia coli during its growth on glucose. The present study investigated the influence of growth rates and PEP carboxykinase knockout on the anaplerotic fluxes in E. coli. The intracellular fluxes were determined using the complementary methods of flux ratio analysis and metabolic flux analysis based on [U- 13C6]glucose labeling experiments and 2D nuclear magnetic resonance (NMR) spectroscopy of cellular amino acids and glycerol. Significant activity of PEP carboxykinase was identified in wild-type E. coli, and the ATP dissipation for the futile cycling via this reaction accounted for up to 8.2% of the total energy flux. Flux analysis of pck deletion mutant revealed that abolishment of PEP carboxykinase activity resulted in a remarkably reduced flux through the anaplerotic PEP carboxylase and the activation of the glyoxylate shunt, with 23% of isocitrate found being channeled in the glyoxylate shunt. The changes in intracellular metabolite concentrations and specific enzyme activities associated with different growth rates and pck deletion, were also determined. Combining the measurement data of in vivo fluxes, metabolite concentrations and enzyme activities, the in vivo regulations of PEP carboxykinase flux, PEP carboxylation, and glyoxylate shunt in E. coli are discussed. © 2003 Wiley Periodicals, Inc. Biotechnol Bioeng 84: 129,144, 2003. [source]

The identification of enzyme targets for the optimization of a valine producing Corynebacterium glutamicum strain using a kinetic model

BIOTECHNOLOGY PROGRESS, Issue 3 2009
Jørgen Barsett Magnus
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. © 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2009 [source]

Integrated Sampling Procedure for Metabolome Analysis

BIOTECHNOLOGY PROGRESS, Issue 5 2006
Jochen Schaub
Metabolome analysis, the analysis of large sets of intracellular metabolites, has become an important systems analysis method in biotechnological and pharmaceutical research. In metabolic engineering, the integration of metabolome data with fluxome and proteome data into large-scale mathematical models promises to foster rational strategies for strain and cell line improvement. However, the development of reproducible sampling procedures for quantitative analysis of intracellular metabolite concentrations represents a major challenge, accomplishing (i) fast transfer of sample, (ii) efficient quenching of metabolism, (iii) quantitative metabolite extraction, and (iv) optimum sample conditioning for subsequent quantitative analysis. In addressing these requirements, we propose an integrated sampling procedure. Simultaneous quenching and quantitative extraction of intracellular metabolites were realized by short-time exposure of cells to temperatures ,95 °C, where intracellular metabolites are released quantitatively. Based on these findings, we combined principles of heat transfer with knowledge on physiology, for example, turnover rates of energy metabolites, to develop an optimized sampling procedure based on a coiled single tube heat exchanger. As a result, this sampling procedure enables reliable and reproducible measurements through (i) the integration of three unit operations into a one unit operation, (ii) the avoidance of any alteration of the sample due to chemical reagents in quenching and extraction, and (iii) automation. A sampling frequency of 5 s,1 and an overall individual sample processing time faster than 30 s allow observing responses of intracellular metabolite concentrations to extracellular stimuli on a subsecond time scale. Recovery and reliability of the unit operations were analyzed. Impact of sample conditioning on subsequent IC-MS analysis of metabolites was examined as well. The integrated sampling procedure was validated through consistent results from steady-state metabolite analysis of Escherichia coli cultivated in a chemostat at D = 0.1 h,1. [source]