Size exclusion chromatography: an improved method to harvest Corynebacterium glutamicum cells for the analysis of cytosolic metabolites.
The efficient separation of Corynebacterium glutamicum cells from culture medium by size exclusion chromatography (SEC) is presented. Residue analysis demonstrated that this method effectively depletes extracellular compounds. For evaluation, SEC was compared with the common methods cold methanol treatment, fast centrifugation and fast filtration. For this purpose, samples of C. glutamicum cells from fermenter cultures were harvested and subjected to a metabolome analysis. In particular, the wild type strain C. glutamicum ATCC13032 and the lysine production strain C. glutamicum DM1730 were grown in a minimal or in a complex medium. Comparison of metabolite pool sizes after harvesting C. glutamicum cells by the methods mentioned above by gas chromatography coupled to mass spectrometry (GC-MS) revealed that SEC is the most suitable method when intracellular metabolite pools are to be measured during growth in complex media or in the presence of significant amounts of secreted metabolites. In contrast to the other methods tested, the SEC method turned out to be fast and able to remove extracellular compounds almost completely.
Persicke
Marcus
M
Institut für Genomforschung und Systembiologie, Centrum für Biotechnologie (CeBiTec), Universität Bielefeld, Universitätsstrasse 27, 33615 Bielefeld, Germany.
Plassmeier
Jens
J
Neuweger
Heiko
H
Rückert
Christian
C
Pühler
Alfred
A
Kalinowski
Jörn
J
D002498:Centrifugation / Q000379:methods; D002850:Chromatography, Gel / Q000379:methods*; D048230:Corynebacterium glutamicum / Q000254:growth & development / Q000302:isolation & purification*; D003470:Culture Media; D003600:Cytosol / Q000737:chemistry; D005285:Fermentation; D005374:Filtration / Q000379:methods; D055442:Metabolome
D003470:Culture Media