Lehrstuhl für Bioverfahrenstechnik, Technische Universität München, Garching 85748, Germany.
Biotechnol Prog. 2010 Jan-Feb;26(1):1-10. doi: 10.1002/btpr.290.
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.
通常，在连续培养中，在固定的外部速率下，在明确的生理条件下进行微生物代谢的稳态分析。然而，大多数工业生物过程在 fed-batch 模式下运行，处于非稳态条件下，而这在恒化器培养中无法实现。一种新的实验设置——快速培养基转换——使代谢在几分钟的时间尺度上能够与生物过程并行进行稳态扰动。为此，细胞从生产过程中分离出来，并转移到一个带有改良环境条件的实验室规模搅拌罐式反应器中。在四个来自 fed-batch 过程的大肠杆菌快速培养基转换实验中，对这种新方法进行了实验评估。我们测试了细胞对进入中心代谢不同点的不同碳源的反应。在所有情况下，细胞都立即利用了所施加的底物（葡萄糖、琥珀酸、乙酸盐和丙酮酸）。细胞外速率和代谢组数据表明，在短期培养过程中存在代谢稳态。代谢分析表明，细胞内通量的分布与所施加的碳源有很大的不同。对于一些反应，通量的变化可以与代谢物浓度的变化相关联。
