Metabolomics: quantification of intracellular metabolite dynamics
Metabolomics: quantification of intracellular metabolite dynamics
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DOI：
10.1016/s1389-0344(02)00003-5
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发表时间：
2002-06-01
期刊：
影响因子：
--
通讯作者：
Takors, R
中科院分区：
文献类型：
--
作者：
Buchholz, A;Hurlebaus, J;Takors, R
为生产初级和次级代谢产物而对微生物菌株进行合理的改良（“代谢工程”）需要对微生物代谢进行定量的了解。这个信息可以从动态发酵实验的过程。通过将底物脉冲施加到底物限制的稳态培养物，细胞代谢从其代谢稳态转移。借助快速采样和淬灭程序，在此过程中每秒可以采集4-5个样本，从而捕获对该刺激的代谢反应。可以使用一系列不同的技术提取和分析来自大肠杆菌代谢的30多种代谢物、核苷酸和辅因子，例如酶测定、HPLC和LC-MS方法。使用不同的底物作为限制和脉冲底物（葡萄糖，甘油），不同的代谢途径和底物摄取系统进行了研究。细胞内代谢物浓度随时间变化的曲线图可作为微生物代谢网络建模的数据基础。(C)2002 Elsevier Science B. V.保留所有权利。
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. (C) 2002 Elsevier Science B.V. All rights reserved.