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Rapid Media Transition: An Experimental Approach for Steady State Analysis of Metabolic Pathways

机译:快速媒体转换:代谢途径稳态分析的实验方法

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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 in fed-batch mode under non-stationary conditions, which cannot be realized in chemostat cultures. A novel experimental setup-rapid media transition-enables steady state perturbation of metabolism on a time scale of several minutes in 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.
机译:通常,在条件明确的生理条件下,以固定的外部速率在连续培养物中进行微生物代谢的稳态分析。但是,大多数工业生物过程是在非平稳条件下以分批补料模式运行的,这在化学恒温器培养中无法实现。一种新颖的实验装置,快速的培养基过渡过程可在几分钟的时间范围内使代谢过程稳定地与生物过程并行运行。为此,将细胞从生产过程中分离出来,并转移到具有改进环境条件的实验室规模的搅拌槽反应器中。在来自补料分批过程的大肠杆菌的四个快速培养基转化实验中,对该新方法进行了实验评估。我们测试了在中央代谢各个点进入不同碳源的反应。在所有情况下,所施加的底物(葡萄糖,琥珀酸酯,乙酸盐和丙酮酸)都立即被细胞利用。细胞外速率和代谢组学数据表明短期培养过程中代谢稳定。化学计量分析显示细胞内通量的分布,这与所施加的碳源有很大不同。对于某些反应,通量的变化可能与代谢物浓度的变化相关。

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