Catching prompt metabolite dynamics in Escherichia coli with the BioScope at oxygen rich conditions.

The design and application of a BioScope, a mini plug-flow reactor for carrying out pulse response experiments, specifically designed for Escherichia coli is presented. Main differences with the previous design are an increased volume-specific membrane surface for oxygen transfer and significantly decreased sampling intervals. The characteristics of the new device (pressure drop, residence time distribution, plug-flow behavior and O2 mass transfer) were determined and evaluated. Subsequently, 2.8 mM glucose perturbation experiments on glucose-limited aerobic E. coli chemostat cultures were carried out directly in the chemostat as well as in the BioScope (for two time frames: 8 and 40 s). It was ensured that fully aerobic conditions were maintained during the perturbation experiments. To avoid metabolite leakage during quenching, metabolite quantification (glycolytic and TCA-cycle intermediates and nucleotides) was carried out with a differential method, whereby the amounts measured in the filtrate were subtracted from the amounts measured in total broth. The dynamic metabolite profiles obtained from the BioScope perturbations were very comparable with the profiles obtained from the chemostat perturbation. This agreement demonstrates that the BioScope is a promising device for studying in vivo kinetics in E. coli that shows much faster response (< 10 s) in comparison with eukaryotes.

[1]  J. Heijnen,et al.  Quantitative analysis of the microbial metabolome by isotope dilution mass spectrometry using uniformly 13C-labeled cell extracts as internal standards. , 2005, Analytical biochemistry.

[2]  W. A. Scheffers,et al.  Effect of benzoic acid on metabolic fluxes in yeasts: A continuous‐culture study on the regulation of respiration and alcoholic fermentation , 1992, Yeast.

[3]  Masaru Tomita,et al.  Dynamic responses of the intracellular metabolite concentrations of the wild type and pykA mutant Escherichia coli against pulse addition of glucose or NH3 under those limiting continuous cultures , 2005 .

[4]  J J Heijnen,et al.  Generating short-term kinetic responses of primary metabolism of Penicillium chrysogenum through glucose perturbation in the bioscope mini reactor. , 2006, Metabolic engineering.

[5]  Christina D. Smolke,et al.  The metabolic pathway engineering handbook , 2010 .

[6]  K. Dam,et al.  A method for the determination of changes of glycolytic metabolites in yeast on a subsecond time scale using extraction at neutral pH. , 1992 .

[7]  I. E. Nikerel,et al.  Model reduction and a priori kinetic parameter identifiability analysis using metabolome time series for metabolic reaction networks with linlog kinetics. , 2009, Metabolic engineering.

[8]  H. Lange,et al.  Analysis of glycolytic intermediates in Saccharomyces cerevisiae using anion exchange chromatography and electrospray ionization with tandem mass spectrometric detection , 2002 .

[9]  J. Heijnen,et al.  Short-Term Metabolome Dynamics and Carbon, Electron, and ATP Balances in Chemostat-Grown Saccharomyces cerevisiae CEN.PK 113-7D following a Glucose Pulse , 2006, Applied and Environmental Microbiology.

[10]  J. Heijnen,et al.  In vivo kinetics with rapid perturbation experiments in Saccharomyces cerevisiae using a second-generation BioScope. , 2006, Metabolic engineering.

[11]  G. Stephanopoulos.  Metabolic fluxes and metabolic engineering. , 1999, Metabolic engineering.

[12]  Matthias Reuss,et al.  New bioreactor-coupled rapid stopped-flow sampling technique for measurements of metabolite dynamics on a subsecond time scale. , 2002, Biotechnology and bioengineering.

[13]  Barbara M. Bakker,et al.  Can yeast glycolysis be understood in terms of in vitro kinetics of the constituent enzymes? Testing biochemistry. , 2000, European journal of biochemistry.

[14]  Matthias Reuss,et al.  In vivo dynamics of glycolysis in Escherichia coli shows need for growth‐rate dependent metabolome analysis , 2008, Biotechnology progress.

[15]  D Weuster-Botz,et al.  Automated sampling device for monitoring intracellular metabolite dynamics. , 1999, Analytical biochemistry.

[16]  C. Wittmann,et al.  Sampling for metabolome analysis of microorganisms. , 2007, Analytical chemistry.

[17]  J J Heijnen,et al.  Improved rapid sampling for in vivo kinetics of intracellular metabolites in Saccharomyces cerevisiae. , 2001, Biotechnology and bioengineering.

[18]  J. Heijnen,et al.  Metabolome dynamic responses of Saccharomyces cerevisiae to simultaneous rapid perturbations in external electron acceptor and electron donor. , 2007, FEMS yeast research.

[19]  Hilal Taymaz-Nikerel,et al.  Development and application of a differential method for reliable metabolome analysis in Escherichia coli. , 2009, Analytical biochemistry.

[20]  I. Chou,et al.  Recent developments in parameter estimation and structure identification of biochemical and genomic systems. , 2009, Mathematical biosciences.

[21]  R. Takors,et al.  Metabolomics: quantification of intracellular metabolite dynamics. , 2002, Biomolecular engineering.

[22]  Robert N. Goldberg,et al.  Thermodynamics of enzyme-catalyzed reactions - a database for quantitative biochemistry , 2004, Bioinform..

[23]  Don W. Green,et al.  Perry's Chemical Engineers' Handbook , 2007 .

[24]  J. Heijnen,et al.  Rapid sampling for analysis of in vivo kinetics using the BioScope: a system for continuous-pulse experiments. , 2002, Biotechnology and bioengineering.

[25]  Peter J.T. Verheijen,et al.  Data Reconciliation and Error Detection , 2009 .

[26]  Christoph Wittmann,et al.  Metabolic pathway analysis for rational design of L-methionine production by Escherichia coli and Corynebacterium glutamicum. , 2006, Metabolic engineering.

[27]  C. Chassagnole,et al.  Dynamic modeling of the central carbon metabolism of Escherichia coli. , 2002, Biotechnology and bioengineering.

[28]  Wolfgang Wiechert,et al.  Modeling and simulation: tools for metabolic engineering. , 2002, Journal of biotechnology.

[29]  C. Smolke.  The Metabolic Pathway Engineering Handbook : Fundamentals , 2009 .

[30]  J. Heijnen,et al.  Critical evaluation of sampling techniques for residual glucose determination in carbon‐limited chemostat culture of Saccharomyces cerevisiae , 2003, Biotechnology and bioengineering.

[31]  J. Heijnen,et al.  Quantitative physiological study of the fast dynamics in the intracellular pH of Saccharomyces cerevisiae in response to glucose and ethanol pulses. , 2008, Metabolic engineering.

[32]  J. Heijnen,et al.  Simultaneous quantification of free nucleotides in complex biological samples using ion pair reversed phase liquid chromatography isotope dilution tandem mass spectrometry. , 2009, Analytical biochemistry.

[33]  M. Reuss,et al.  In vivo analysis of glucose-induced fast changes in yeast adenine nucleotide pool applying a rapid sampling technique. , 1993, Analytical biochemistry.

[34]  H. S. Fogler,et al.  Elements of Chemical Reaction Engineering , 1986 .