Metabolic quenching of Corynebacterium glutamicum: efficiency of methods and impact of cold shock

Abstract

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.

Stationary versus non-stationary 13C-MFA: A comparison using a consistent dataset

“…fructose-6-biphosphate (FBP). This discrepancy is (most) reasonably explained by the occurrence of metabolite leakage when applying the methanol quenching procedure (Wellerdiek et al, 2009).…”

“…Cryocultures were stored at −80 • C in LB-medium containing 10% (v v −1 ) glycerol. The media for precultures and main cultivation were based on modified CGXII media as described in (Wellerdiek et al, 2009). Precultures for 10% (v v −1 ) inoculation of the main culture were cultivated at 30 • C overnight.…”

Fast sampling for quantitative microbial metabolomics

“…Wellerdiek et al [31 ] applied a microstructure heat exchanger to analyze the cold shock effect on a time scale of seconds using Corynebacterium glutamicum as a model organism. They observed that fast cooling (within 220 ms) of cell suspension from 30 to 08C resulted in rapid and significant release (1.5-3-fold increase of the concentration in the supernatant) of central metabolites from the cells within 1.5 s or less.…”

Fast filtration for metabolome sampling of suspended animal cells

“…This is to avoid loss of metabolites caused by residual enzyme activity (de Koning and van Dam 1992) or quenching conditions. A standard quenching method for bacteria and yeast is cold methanol, though other protocols using a microstructure heat exchanger 3 or fast filtration are also applied (Wellerdiek et al 2009;Bolten and Wittmann 2008). Dietmair et al (2010) showed that using methanol, as proposed by Sellick et al (2009), causes increased cell permeabilization and leakage of intracellular components.…”