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Manuscript title Glucose-methanol co-utilization in Pichia pastoris studied by metabolomics and instationary 13C flux analysis.
PubMed ID 23448228
Journal BMC Systems Biology
Year 2013
Authors Joel Jordà, Camilo Suarez, Marc Carnicer, Angela ten Pierick, Joseph J Heijnen, Walter van Gulik, Pau Ferrer, Joan Albiol and Aljoscha Wahl
Affiliations Department of Chemical Engineering, Escola d’Enginyeria, Universitat Autònoma de Barcelona, Bellaterra, Cerdanyola del Vallès, Spain
Keywords Pichia pastoris, intracellular, extracellular, glucose, glucose:methanol
Full text article Downloadarticle Jorda_2013.pdf
Project name not specified

Experiment Description info

Organism Pichia pastoris
Strain X-33
Data type metabolites at steady-state
Data units (µmol/l) and (µmol/gDW)
Execution date not specified

Experimental Details info

Temperature (0C) 25.0
pH 5.0
Carbon source glucose,
Culture mode chemostat
Process condition aerobic
Dilution rate (h-1) 0.09
Working volume (L) 1.0
Biomass concentration (g/L) 4.0
Medium composition

7.04 g/l Glucose monohydrate, 1.6 g/l Methanol, 0.915 g/l citric acid, 2 g/l (NH4)2 HPO4, 0.3 g/l MgSO4.7H2O, 1.4 g/l KH2PO4, 0.01 g/l CaCl2 · 2H2O, 0.5 ml/l trace salt solution, 0.3 ml/l biotin (0.2 g/l)

General protocol information Sampling method: One hour before the switch to the labelled feed using a dedicated rapid-sampling setup.

Quenching procedure: Approximately 1 g of broth were rapidly withdrawn and immediately mixed with 5 ml of precooled quenching solution at -40°C.

Extraction technique: boiling ethanol

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

Methods description - Notes

Sampling and measurement of metabolite concentrations - For quenching and extraction, the protocol recently described for P. pastoris growing on glucose [1] was used.
The metabolite extraction was performed with 75% (v/v) aqueous ethanol at 95 °C. Concentrations were measured from two independent chemostat experiments in triplicate. The labelling enrichment was measured from one wash-in experiment by sampling at 20 time-points. The sampling times were selected based on the theoretical calculations by Nöh and co-workers [2], exponentially increasing intervals over a total of 6 hours to also achieve steady state in large intracellular pools like glutamate. The first sample is taken at 5 s after the switch to labelled material, which is the maximum velocity for manual sampling. All further sample processing steps were carried out as described previously [3].
Quantification of extracellular metabolites - Triplicate samples (5 ml) for extracellular metabolite analyses were centrifuged at 6,000 rpm for 3 min in a micro centrifuge (Minispin, Eppendorf) to remove the cells, and subsequently filtered through 0.45 mm-filters (Millipore type HAWP). Glucose, methanol, and other extracellular compounds were analyzed by HPLC analysis using an ionic exchange column, (ICSep ICE-COREGEL 87H3, Transgenomic). The mobile phase was 6 mM sulphuric acid.

[1] Carnicer M, Canelas AB, Pierick A, Zeng Z, Dam J, Albiol J, Ferrer P, Heijnen JJ, Gulik W: Development of quantitative metabolomics for Pichia pastoris. Metabolomics 2011, 8:284–298.
[2] Nöh K, Wahl A, Wiechert W: Computational tools for isotopically instationary 13C labeling experiments under metabolic steady state conditions. Metab Eng 2006, 8:554–577.
[3] Douma RD, de Jonge LP, Jonker CTH, Seifar RM, Heijnen JJ, van Gulik WM: Intracellular metabolite determination in the presence of extracellular abundance, Application to the penicillin biosynthesis pathway in Penicillium chrysogenum. Biotechnol Bioeng 2010, 107:105–115.

Data file
Downloadmetabolites KIMODATAID55_v0.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 2013-05-30 19:09:20 UTC

Updated 2014-06-12 22:27:39 UTC

Version 0

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

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