Metabolic quenching of Corynebacterium glutamicum: efficiency of methods and impact of cold shock
- 322 Downloads
Representative and valid cytoplasmic concentrations are essential for ensuring the significance of results in the field of metabolome analysis. One of the most crucial points in this respect is the sampling itself. A rapid and sudden stopping of the metabolism on a timescale that is much faster than the conversion rates of investigated metabolites is worthwhile. This can be achieved by applying of cold methanol quenching combined with reproducible, fast, and automated sampling. Unfortunately, quenching the metabolism by a sharp temperature shift leads to what is known as cold shock or the cell-leakage effect. In the present work, we applied a microstructure heat exchanger to analyze the cold shock effect using Corynebacterium glutamicum as a model microorganism. Using this apparatus together with a silicon pipe, it was possible to assay the leakage effect on a timescale starting at 1 s after cooling cell suspension. The high turnover rates not only require a rapid quenching technique, but also the correct application. Moreover, we succeeded in showing that even when the required appropriate setup of methanol quenching is not used, the metabolism is not stopped within the required timescale. By applying robust techniques like rapid sampling in combination with reproducible sample processing, we ensured fast and reliable metabolic inactivation during all steps.
KeywordsMethanol quenching Metabolome analysis Metabolomics Microstructure heat exchanger Rapid sampling
Nicotinamide adenine dinucleotide
Nicotinamide adenine dinucleotide (reduced form)
Nicotinamide adenine dinucleotide-phosphate
Nicotinamide adenine dinucleotide-phosphate (reduced form)
This project was financially supported by the German Ministry of Research and Education (BMBF, grant 0313703) and Evonik Degussa GmbH. The authors would like to thank Prof. Christian Wandrey for the excellent working conditions at the Institute of Biotechnology 2, Forschungszentrum Jülich GmbH.
- 26.Oldiges M. 2005. Dissertation thesis: Metabolomanalyse zur Untersuchung der Dynamik im Aromatenbiosyntheseweg in l-Phenylalanin Produzenten von Escherichia coli. Lebenswissenschaften/Life Science, ISBN 3-89336-380-7Google Scholar
- 35.WeusterBotz D (1999) Habilitation thesis: Die Rolle der Reaktionstechnik in der mikrobiellen Verfahrensentwicklung, Jülich. Lebenswissenschaften/Life Sciences, ISBN 3-89336-245-2Google Scholar