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Metabolomics: quantification of intracellular metabolite dynamics.

机译：代谢组学：定量细胞内代谢动力学。

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摘要

The rational improvement of microbial strains for the production of primary and secondary metabolites ('metabolic engineering') requires a quantitative understanding of microbial metabolism. A process by which this information can be derived from dynamic fermentation experiments is presented. By applying a substrate pulse to a substrate-limited, steady state culture, cellular metabolism is shifted away from its metabolic steady state. With the aid of a rapid sampling and quenching routine it is possible to take 4-5 samples per second during this process, thus capturing the metabolic response to this stimulus. Over 30 metabolites, nucleotides and cofactors from Escherichia coli metabolism can be extracted and analysed using a range of different techniques, for example enzymatic assays, HPLC and LC-MS methods. Using different substrates as limiting and pulse-substrates (glucose, glycerol), different metabolic pathways and substrate uptake systems are investigated. The resulting plots of intracellular metabolite concentrations against time serve as a data basis for modelling microbial metabolic networks.
机译：为生产初级和次级代谢产物（“代谢工程”）而合理改良微生物菌株，需要对微生物代谢有定量的了解。提出了可以从动态发酵实验中获得该信息的过程。通过将底物脉冲应用于底物受限的稳态培养，细胞代谢会从其代谢稳态转移。借助快速采样和淬灭程序，在此过程中每秒可以采集4-5个样品，从而捕获对这种刺激的代谢反应。可以使用多种不同的技术，例如酶促测定，HPLC和LC-MS方法，提取和分析来自大肠埃希氏菌代谢的30多种代谢物，核苷酸和辅助因子。使用不同的底物作为限制底物和脉冲底物（葡萄糖，甘油），研究了不同的代谢途径和底物摄取系统。细胞内代谢物浓度随时间变化的结果图可作为建立微生物代谢网络模型的数据基础。

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