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General Information info

Manuscript title Changes in substrate availability in Escherichia coli lead to rapid metabolite, flux and growth rate responses.
PubMed ID 23370343
Journal Metabolic Engineering
Year 2013
Authors Hilal Taymaz-Nikerel, Marjan De Mey, Gino Baart, Jo Maertens, Joseph J. Heijnen, Walter M. Van Gulik
Affiliations Department of Biotechnoloy, Delft University of Technology, Kluyver Center for Genomics of Industrial Fermentation Julianalaan 67, 2628 BC Delft, The Netherlands
Keywords Escherichia coli, intracellular metabolites, stimulus response
Full text article Downloadarticle Nikerel_2013.pdf
Project name not specified

Experiment Description info

Organism Escherichia coli
Strain K-12 MG1655
Data type time-series data of metabolites
Data units (µmol/gDW)
Execution date not specified

Experimental Details info

Temperature (0C) 37.0
pH 7.0
Carbon source glucose,
Culture mode chemostat
Process condition aerobic
Dilution rate (h-1) 0.1
Working volume (L) 4.0
Biomass concentration (g/L) 8.36 (Experiment 1) and 8.02 (Experiment 2)
Medium composition

The composition of the low Cl- minimal medium was, per liter: 1.25g(NH4)2SO4, 1.15g KH2PO4, 0.5g MgSO4.7H2O, 0.5g NaCl, 30g glucose.1H2O, 0.001 gthiamine–HCl, 2ml of trace elements solution and 0.2 ml silicone-based antifoaming agent (BDH,Poole,UK). The composition of the trace elements solution was described in Verduyn et al. [1].

[1] Verduyn, C., Postma, E., Scheffers, W.A., van Dijken, J.P., 1992. Effect of benzoic acid on metabolic fluxes in yeasts: a continuous-culture study on the regulation of respiration and alcoholic fermentation. Yeast 8 (7), 501–517

General protocol information Sampling method: differential method

Quenching procedure: not specified

Extraction technique: boiling ethanol

Sample analyzing method: enzymatic, GC-MS, LC-MS, HPLC-UV/RI

Methods description - Notes

Rapid substrate pulse experiments - Short-term rapid pulse experiments in the bioreactor and in the BioScope were carried out as described previously [1,2].
Rapid sampling for extra-/intracellular metabolites - the differential method [3] was applied to obtain the amounts of intracellular metabolites collected during the steady-state as well as during the transient states for both the bioreactor and the BioScope.The required broth and filtrate sampling was carried out as described by Taymaz-Nikerel et al. [3]. The steady-state preceding each pulse was sampled twice and each sample was analyzed in duplicate. For intracellular metabolite determination during the pulse experiment, only sampling of broth was performed [1,2] at each time point and analyzed in duplicate.
For measurements of glucose and possible secreted by-products (organic acids and alcohols) fast filtration sampling using cold stainless steel beads was applied (both for the bioreactor and the BioScope), as described previously [1,2].
Extraction of metabolites - metabolites were extracted in 75% boiling ethanol (3min, 90 ºC) as described in [3]. Before extraction, 100 mL of 100% U-13C-labeled cell extract was added to every sample as internal standard for isotope dilution mass spectrometry (IDMS)-based metabolite quantification [4]. The preparation of the 100% U-13C-labeled yeast cell extract can be found in [4].
Analytical techniques - measurement of cell dry weight, residual glucose, total organic carbon and intracellular metabolite concentrations (glycolysis, TCA cycle, PPP, adenine nucleotides and free amino acids) were carried out as described previously [1,3]. The filtrate samples for extracellular pyruvate determination during the pyruvate pulse were kept in the fridge (about 4 ºC) about a few days until analysis. Pyruvate concentration was measured with HPLC (Aminex HPX-87H ion exclusion column, Bio-Rad,CA,USA) with a refractive index detector (Waters 2414) and UV detector at 210 nm. The column was eluted with phosphoric acid (15 mM) at a column temperature of 59 ºC and a flow rate of 0.6 mL/min. The extracellular succinate concentration during the succinate pulse was quantified enzymatically (Boehringer Mannheim/R-Biopharm,Roche). The supernatant samples obtained after the pulses were extensively analyzed for the presence of possible by-products. HPLC (AminexHPX-87H ion exclusion column, Bio-Rad,CA, USA; the column was eluted with 1.5 mM phosphoric acid except for pyruvate quantification) and LC-MS/MS [5] analysis were performed to check the presence of ethanol, acetaldehyde, glyoxylate, acetate, formate, lactate, fumarate, pyruvate, oxaloacetate, malate, succinate, citrate and α-ketoglutarate.

[1] De Mey,M., Taymaz-Nikerel,H., Baart,G., Waegeman,H., Maertens,J., Heijnen,J.J., van Gulik,W.M., 2010. Catching prompt metabolite dynamics in Escherichia coli with the BioScope at oxygen rich conditions. Metab. Eng. 12 (5), 477–487.
[2] Taymaz-Nikerel, H.,vanGulik,W.M.,Heijnen,J.J., 2011. Escherichia coli responds with a rapid and large change in growth rate upon a shift from glucose-limited to glucose-excess conditions. Metab. Eng.13 (3), 307–318.
[3] Taymaz-Nikerel, H., de Mey,M., Ras,C., tenPierick,A., Seifar,R.M., vanDam,J.C., Heijnen, J.J., vanGulik,W.M., 2009. Development and application of a differential method for reliable metabolome analysis in Escherichia coli. Anal. Biochem. 386 (1), 9–19.
[4] Wu,L., Mashego,M.R., vanDam,J.C., Proell,A.M., Vinke,J.L., Ras,C., vanWinden,W.A., vanGulik,W.M., Heijnen,J.J., 2005. Quantitative analysis of the microbial metabolome by isotope dilution mass spectrometry using uniformly 13C labeled cell extracts as internal standards. Anal. Biochem. 336 (2), 164–171.
[5] van Dam,J.C., Eman,M.R., Frank,J., Lange,H.C., vanDedem,G.W.K., Heijnen,S.J., 2002. Analysis of glycolytic intermediates in Saccharomyces cerevisiae using anion exchange chromatography and electrospray ionization with tandem mass spectrometric detection. Anal. Chim. Acta 460 (2), 209–218.

Data file
Downloadmetabolites KIMODATAID75_v1.xlsx
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Submission and curation info

Entered by Administrator KiMoSysFirst name: Administrator
Affiliation: INESC-ID/IST
Interests: mathematical modeling, accessible data, use of data

Created 2013-06-25 15:06:47 UTC

Updated 2018-07-25 20:40:25 UTC

Version 1

Status (reviewed) 2013-12-06 17:17:38 UTC

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