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Approximately 1 g of broth were rapidly withdrawn and immediately mixed with 5 ml of precooled quenching solution at -40°C.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 m
... easured 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
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: LC/MS, GC-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.
------------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.
[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.
KiMoSys (https://kimosys.org). Data EntryID 55 (Pichia pastoris). [online], [Accessed 12 December 2024]. Available from: https://doi.org/10.34619/5zhw-s051