Rapid Media Transition: An Experimental Approach for Steady State Analysis of Metabolic Pathways
Rapid Media Transition: An Experimental Approach for Steady State Analysis of Metabolic Pathways
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DOI：
10.1002/btpr.290
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发表时间：
2010-01-01
影响因子：
2.9
通讯作者：
Weuster-Botz, Dirk
中科院分区：
文献类型：
--
作者：
Link, Hannes;Anselment, Bernd;Weuster-Botz, Dirk
通常，微生物代谢的稳态分析是在明确定义的生理条件下，在固定外部速率的连续培养中进行的。然而，大多数工业生物过程是操作它？在非稳态条件下的补料分批模式，这在恒化器培养中不能实现。一种新的实验设置-疯狂的媒体过渡-使稳态扰动的代谢时间尺度上的几分钟？与运行生物过程平行。为此，将细胞从生产过程中分离出来，并转移到具有改良环境条件的实验室规模搅拌罐反应器中。这种新的方法进行了评估，在四个快速的媒体过渡实验与大肠杆菌从补料分批过程。我们测试了在中枢代谢的不同点对不同碳源进入的反应。在所有情况下，所施加的底物（葡萄糖、琥珀酸盐、乙酸盐和丙酮酸盐）立即被细胞利用。细胞外速率和代谢组学数据表明在短期培养期间代谢稳定状态。化学计量分析揭示了细胞内通量的分布，该分布因所应用的碳源而显着不同。对于某些反应，通量的变化可能与代谢物浓度的变化相关。(C)2009年美国化学工程师学会生物技术。程序，26：1-10，2010
Commonly steady state analysis of microbial metabolism is performed under well defined physiological conditions in continuous cultures with fixed external rates. However, most industrial bioprocesses are operated it? fed-batch mode under non-stationary conditions, which cannot be realized in chemostat cultures. A novel experimental setup-rabid media transition-enables steady state perturbation of metabolism on a time scale of several minutes it? parallel to operating bioprocesses. For this purpose, cells are separated from the production process and transferred into a lab-scale stirred-tank reactor with modified environmental conditions. This new approach was evaluated experimentally in four rapid media transition experiments with Escherichia coli from a fed-batch process. We tested the reaction to different carbon sources entering at various points of central metabolism. In all cases, the applied substrates (glucose, succinate, acetate, and pyruvate) were immediately utilized by the cells. Extracellular rates and metabolome data indicate a metabolic steady state during the,short-term cultivation. Stoichiometric analysis revealed distribution of intracellular fluxes, which differs drastically subject to the applied carbon source. For some reactions, the variation of flux could be correlated to changes of metabolite concentrations. (C) 2009 American Institute of Chemical Engineers Biotechnol. Prog., 26: 1-10, 2010