DataEntryID 55
    General information
      Manuscript title: Glucose-methanol co-utilization in Pichia pastoris studied by metabolomics and instationary 13C flux analysis.
      PubMed ID: http://www.ncbi.nlm.nih.gov/pubmed/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: https://www.kimosys.org/rails/active_storage/blobs/eyJfcmFpbHMiOnsibWVzc2FnZSI6IkJBaHBBblFFIiwiZXhwIjpudWxsLCJwdXIiOiJibG9iX2lkIn19--493b985a096556b80694399204bf8e52d456c714/Jorda_2013.pdf
      Project name: not specified

    Experiment description
      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
      Temperature (°C): 25.0
      pH: 5.0
      Carbon source: glucose
      Culture mode: chemostat
      Process condition: aerobic
      Dilution rate (h⁻¹): 0.09
      Working volume: 1.0 L
      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switch to the labelled feed using a dedicated rapid-sampling setup.; Quenching: Approximately 1 g of broth were rapidly withdrawn and immediately mixed with 5 ml of precooled quenching solution  at -40°C. ; Extraction list: boiling ethanol; Analysis list: GC-MS, LC-MS, HPLC-UV/RI;
      Methods description: 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.

------------References--------------
[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. http://doi.org/dsg2kz
[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. http://doi.org/bxd49p
[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. http://doi.org/cdphfq 
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    Submission and curation
      Entered by: Administrator KiMoSys
      Created: 2013-05-30 19:09:20 UTC
      Updated: 2020-04-24 16:10:32 UTC
      Version: 0
      Status: (reviewed) 2013-12-06 17:17:38 UTC
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