Metabolic quenching of Corynebacterium glutamicum: efficiency of methods and impact of cold shock
Metabolic quenching of Corynebacterium glutamicum: efficiency of methods and impact of cold shock
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DOI：
10.1007/s00449-008-0280-y
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发表时间：
2009-08-01
影响因子：
3.8
通讯作者：
Oldiges, Marco
中科院分区：
文献类型：
--
作者：
Wellerdiek, Max;Winterhoff, Dajana;Oldiges, Marco
代表性和有效的细胞质浓度对于确保代谢组学分析结果的意义至关重要。在这方面最关键的一点是抽样本身。在比所研究的代谢物的转化率快得多的时间尺度上，代谢的快速和突然停止是值得的。这可以通过应用冷甲醇淬火结合可重复的，快速的，自动化的采样来实现。不幸的是，通过急剧的温度变化来抑制新陈代谢会导致所谓的冷休克或细胞泄漏效应。本文以谷氨酸棒状杆菌为模型微生物，采用微结构换热器对冷冲击效应进行了分析。将该装置与硅管一起使用，可以在冷却电池悬浮后1 s开始的时间尺度上测定泄漏效应。高周转率不仅需要快速淬火技术，而且需要正确的应用。此外，我们成功地表明，即使没有使用所需的适当的甲醇淬火设置，代谢也不会在所需的时间范围内停止。通过应用强大的技术，如快速采样与可重复的样品处理相结合，我们确保在所有步骤中快速可靠的代谢失活。
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.