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Manuscript title Generating short-term kinetic responses of primary metabolism of Penicillium chrysogenum through glucose perturbation in the bioscope mini reactor.
PubMed ID 16807032
Journal Metabolic Engineering
Year 2006
Authors U. Nasution, W.M. Van Gulik, A. Proell, W.A. van Winden, J.J. Heijnen
Affiliations Department of Biotechnology, Delft University of Technology, Julianalaan 67, 2628 BC Delft, Netherlands
Keywords glucose pulse, in vivo kinetics, metabolome, Penicillium chrysogenum
Full text article Downloadarticle Nasution_2006.pdf
Project name not specified

Experiment Description info

Organism Penicillium chrysogenum
Strain DS12975
Data type time-series data of metabolites
Data units (µmol/gDW)
Execution date not specified

Experimental Details info

Temperature (0C) 25.0
pH 6.5
Carbon source glucose,
Culture mode chemostat
Process condition aerobic
Dilution rate (h-1) 0.05
Working volume (L) 4.0
Biomass concentration (g/L) 6.21 ± 0.16
Medium composition

16.5 g/L glucose·H2O, 1 g/L KH2PO4, 5 g/L (NH4)2SO4, 0.5 g/L MgSO4·7H2O, and 10 ml/L of trace element solution. The trace element solution contained 15 g/L Na2-EDTA·2H2O, 0.5 g/L CuSO4·5H2O, 2 g/L ZnSO4·7H2O, 2 g/L MnSO4·H2O, 4 g/L FeSO4·7H2O and 0.5 g/L CaCl2·2H2O. For penicillin-G production the side chain precursor phenylacetic acid (PAA) was added at a concentration of 0.94 g/L.

General protocol information Sampling method: rapid sampling of broth for measurement of intracellular metabolites was carried out at 5, 10, 15, 20, 25, 35, 50, 70 and 90 s after injection of the glucose solution.

Quenching procedure: 60 w/w % analytical grade methanol (ARCOS, Geel, Belgium) and water purified in a milli-QUFplus system (Millipore, Bedford, MA), buffered with 10 mM HEPES (Merck, Darmstadt, Germany) and adjusted to pH 7.5 with 3M KOA (Baker, Deventer, The Netherlands).

Extraction technique: hot ethanol

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

Methods description - Notes

Chemostat pulse experiments - for the pulse response experiment carried out in the chemostat 16 ml of a 125 g/L glucose solution was injected into the reactor within 1 s. This increased the residual glucose concentration to approximately 0.5 g/L. Subsequently, rapid sampling of broth for measurement of intracellular metabolites was carried out at 5, 10, 15, 20, 25, 35, 50, 70 and 90 s after injection of the glucose solution. Rapid sampling and quenching was carried out as described previously [1]. Sampling times were chosen similar to the sampling times applied for the BioScope experiments. Rapid sampling for determination of extracelluar metabolite concentrations was carried out according to Mashego et al. [2].
Metabolite extraction - metabolite extraction was carried out with the hot ethanol method [3], which was adapted for the extraction of P. chrysogenum cells. The adapted methods for cold centrifugation and washing of the cells, metabolite extraction and sample preparation have been described elsewhere [4].
Analytical procedures - The concentrations of the glycolytic and TCA cycle intermediates in the cell extracts were analyzed with isotope dilution mass spectrometry (IDMS) as described in Wu et al. [5]. Intracellular nucleotides were analyzed using LC-ESI-MS/MS. The nucleotides were separated by an ion pairing reversed phase HPLC method, using a XTerra MS C18 column (100mm x 1 mm) equipped with guard column (10mm x 2.1 mm) (both from Waters, Milford, USA) as described previously [5].
Concentrations of PAA and PenG in the culture supernatant were measured with high performance liquid chromatography (HPLC) as described in Christensen et al. [6]. The glucose concentration in the culture supernatant was analyzed spectrophotometrically (Agilent 8453 UV-Visible Spectroscopy System, Waldbronn, Germany) using a Boehringer Mannheim enzymatic bioanalysis kit according to the manufacturer’s instructions. The concentrations of organic acids (citrate, pyruvate, succinate, lactate and acetate) in the culture supernatant were determined by HPLC analysis with Aminex HPC-87H Column (Biorad, Hercules, USA) at 60 ºC with 5mM H2SO4 as the mobile phase.

[1] Lange, H.C., Heijnen, J.J., 2001. Improved rapid sampling for in vivo kinetics of intracellular metabolites in S. cerevisiae. Biotechnol. Bioeng. 75, 406–415.
[2] Mashego, M.R., van Gulik, W.M., Vinke, J.L., Heijnen, J.J., 2003. Critical evaluation of sampling techniques for residual glucose determination in carbon-limited chemostat culture of Saccharomyces cerevisiae. Biotechnol. Bioeng. 83, 395–399.
[3] Gonzalez, B., Francois, J., Renaud,M., 1997. A rapid and reliable method for metabolite extraction in yeast using boiling buffered ethanol. Yeast 13, 1347–1355.
[4] Nasution, U., van Gulik, W., Kleijin, R., Heijnen, J.J., 2006. Measurement of intracellular metabolites of primary metabolism and adenine nucleotides in chemostat cultivated P. chrysogenum. Biotechnol. Bioeng. 94, 159–166.
[5] Wu, L., Mashego, M.R., Van Dam, J.C., Proell, A.M., Vinke, J.L., Ras, C., van Winden, W.A., van Gulik, W.M., Heijnen, J.J., 2005. Quantitative analysis of the microbial metabolome by isotope dilution mass spectrometry using uniformly C-13-labeled cell extracts as internal standards. Anal. Biochem. 336, 164–171.
[6] Christensen, L.H., Mandrup, G., Nielsen, J., Villadsen, J., 1994. A robust liquid-chromatographic method for measurement of medium components during penicillin fermentations. Anal. Chim. Acta 296, 51–62.

Data file
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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 2013-06-21 17:03:42 UTC

Updated 2014-06-13 15:57:46 UTC

Version 0

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

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