Drysch A, El Massaoudi M, Mack C, Takors R, de Graaf A A, Sahm H
Institute of Biotechnology, Research Center Jülich, Forschungszentrum Julich GmbH, 52425 Jülich, Germany.
Metab Eng. 2003 Apr;5(2):96-107. doi: 10.1016/s1096-7176(03)00005-3.
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.
谷氨酸棒杆菌被广泛用于氨基酸的工业大规模（补料）分批生产，尤其是谷氨酸和赖氨酸。然而，迄今为止，基于该生物体的13C标记实验的代谢通量分析仅限于小规模分批条件和碳限制恒化器培养，因此对于工业发酵的相关性存疑。为了将通量分析提升到工业水平，开发了一种新颖的传感器反应器方法（El Massaoudi等人，《代谢工程》，已提交），其中一个300升的生产反应器和一个1升的传感器反应器以主/从模式并行运行，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。我们描述了该技术与核磁共振（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，从而形成了一种用于分批发酵过程中定量途径分析的强大工具。作为首次应用，在中试规模分批发酵的三个阶段，对指数生长的产赖氨酸谷氨酸棒杆菌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，验证了生产反应器和传感器反应器中发酵的相似性。应用一个通用的数学模型，该模型包括用于分析蛋白质ogenic氨基酸13C同位素异构体标记数据的代谢物和碳标记平衡，研究了指数生长后续阶段的体内代谢通量分布。首次表明，在研究的指数生长后续阶段，谷氨酸棒杆菌回补节点处的体内反向C(4)-脱羧通量显著降低（70%），同时赖氨酸形成增加了三倍。
