Corynebacterium glutamicum is intensively used for the industrial large-scale (fed-) batch production of amino acids, especially glutamate and lysine. However, metabolic flux analyses based on 13C-labeling experiments of this organism have hitherto been restricted to small-scale batch conditions and carbon-limited chemostat cultures, and are therefore of questionable relevance for industrial fermentations. To lever flux analysis to the industrial level, a novel Sensor Reactor approach was developed (El Massaoudi et al., Metab. Eng., submitted), in which a 300-L production reactor and a 1-L Sensor Reactor are run in parallel master/slave modus, thus enabling 13C-based metabolic flux analysis to generate a series of flux maps that document large-scale fermentation courses in detail. We describe the successful combination of this technology with nuclear magnetic resonance (NMR) analysis, metabolite balancing methods and a mathematical description of 13C-isotope labelings resulting in a powerful tool for quantitative pathway analysis during a batch fermentation. As a first application, 13C-based metabolic flux analysis was performed on exponentially growing, lysine-producing C. glutamicum MH20-22B during three phases of a pilot-scale batch fermentation. By studying the growth, (co-) substrate consumption and (by-) product formation, the similarity of the fermentations in production and Sensor Reactor was verified. Applying a generally applicable mathematical model, which included metabolite and carbon labeling balances for the analysis of proteinogenic amino acid 13C-isotopomer labeling data, the in vivo metabolic flux distribution was investigated during subsequent phases of exponential growth. It was shown for the first time that the in vivo reverse C(4)-decarboxylation flux at the anaplerotic node in C. glutamicum significantly decreased (70%) in parallel with threefold increased lysine formation during the investigated subsequent phases of exponential growth.

中文翻译：

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通过使用新型传感器反应器方法对微生物碳通量分布进行连续映射来监测生产过程：基于II-（13）C标记的代谢通量分析和L-赖氨酸生产。

谷氨酸棒杆菌被广泛用于氨基酸，特别是谷氨酸和赖氨酸的工业大规模（补料）批量生产。但是，迄今为止，基于这种生物的13 C标记实验的代谢通量分析仅限于小批量生产和碳限制的恒化器培养，因此对于工业发酵的相关性存在疑问。为了将通量分析提高到工业水平，开发了一种新颖的传感器反应器方法（El Massaoudi等人，Metab。Eng。，已提交），其中并行运行了300升生产反应器和1升传感器反应器主/从模式，从而使基于13C的代谢通量分析能够生成一系列通量图，详细记录大规模发酵过程。我们描述了这项技术与核磁共振（NMR）分析，代谢物平衡方法以及13C同位素标记的数学描述的成功结合，从而为分批发酵过程中的定量途径分析提供了强大的工具。作为第一个应用，在中试分批发酵的三个阶段中，对以指数方式生长，生产赖氨酸的谷氨酸棒杆菌MH20-22B进行了基于13C的代谢通量分析。通过研究生长，（共）底物消耗和（副）产物形成，验证了生产和传感器反应器中发酵的相似性。应用包括代谢物和碳标记天平在内的普遍适用的数学模型来分析蛋白原氨基酸13C-同位素标记数据，在随后的指数增长阶段研究了体内代谢通量分布。首次表明，在研究的指数增长后续阶段中，谷氨酸棒杆菌无节制节点的体内反向C（4）-脱羧通量显着降低（70％），同时赖氨酸形成增加了三倍。