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Metabolic profiling of Escherichia coli cultivations: evaluation of extraction and metabolite analysis procedures

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Abstract

The quantitative estimation of intracellular metabolite concentrations (metabolic profiling) is a prerequisite for a better understanding of biological processes and thus inevitable for the rational improvement of microbial production strains and process design. Since pool sizes of substrates regulate flux through different enzymes, the accurate determination of intracellular metabolite concentrations is necessary to understand in vivo reaction kinetics. Quantification of intracellular concentrations of glycolytic intermediates in Escherichia coli K12 was achieved by using a novel in situ rapid sampling and quenching procedure. A new extraction procedure using buffered hot water was established. By use of simultaneous multi-substrate feeding with various ratios of glucose, fructose and acetate during continuous cultivations several metabolic states were induced. Metabolic flux analysis and the newly developed metabolic profiling procedure were used to determine in vivo enzyme kinetics as exemplified for fructose 1,6-bisphosphate aldolase and citrate synthase.

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Acknowledgements

We would like to thank Elisabeth Eckers for her substantial laboratory help in establishing the extraction procedure.

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Author notes

  1. Ezequiel Franco-Lara

    Present address: Institut für Bioverfahrenstechnik, Technische Universität Braunschweig, Gaußstraße 17, 38106, Braunschweig, Germany

Authors and Affiliations

  1. Lehrstuhl für Bioverfahrenstechnik, Technische Universität München, Boltzmannstr. 15, 85748, Garching, Germany

    Julia Hiller, Ezequiel Franco-Lara & Dirk Weuster-Botz

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  1. Julia Hiller
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  2. Ezequiel Franco-Lara
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  3. Dirk Weuster-Botz
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Correspondence to Dirk Weuster-Botz.

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Hiller, J., Franco-Lara, E. & Weuster-Botz, D. Metabolic profiling of Escherichia coli cultivations: evaluation of extraction and metabolite analysis procedures. Biotechnol Lett 29, 1169–1178 (2007). https://doi.org/10.1007/s10529-007-9384-8

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  • DOI: https://doi.org/10.1007/s10529-007-9384-8

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