Drysch A, El Massaoudi M, Wiechert W, de Graaf A A, Takors R
Institute of Biotechnology, Forschungszentrum Jülich GmbH, 52425 Jülich, Germany.
Biotechnol Bioeng. 2004 Mar 5;85(5):497-505. doi: 10.1002/bit.10915.
Using our recently developed sensor reactor approach, lysine-producing, nongrowing Corynebacterium glutamicum MH20-22B cells were subjected to serial (13)C-labeling experiments for flux analysis during the leucine-limited fed-batch production phase in a 300-L bioreactor. Based on two-dimensional (2D) nuclear magnetic resonance (NMR) measurements of (13)C-labeling patterns of cytoplasmic free metabolites, metabolic flux distributions in the central metabolism were successfully determined. Focusing on the highly concentrated metabolite L-glutamate, the working hypothesis was validated that the equilibration of labeling patterns in intracellular pools was much faster (up to 9.45 min) than the labeling period (3 h) used in the experiments. Analysis of anaplerotic reactions revealed that highly selective lysine production was accompanied by a significant reduction of decarboxylating reactions from 10 mol% to only 2 mol%, whereas PEP/pyruvate-carboxylating fluxes remained constant at about 40 mol% of consumed glucose. These results support the conclusion that an optimized C. glutamicum L-lysine producer should possess increased PEP carboxylase and/or pyruvate carboxylase activity combined with downregulated, decarboxylating fluxes consuming oxaloacetate/malate. The findings also illustrate the usefulness of the sensor reactor approach in the study of industrial fermentations.
利用我们最近开发的传感器-反应器方法，在300升生物反应器中，对不生长的产赖氨酸谷氨酸棒杆菌MH20-22B细胞进行了连续的¹³C标记实验，以分析亮氨酸限制补料分批生产阶段的代谢通量。基于对细胞质游离代谢物¹³C标记模式的二维核磁共振（NMR）测量，成功确定了中心代谢中的代谢通量分布。聚焦于高浓度代谢物L-谷氨酸，验证了工作假设，即细胞内池中标记模式的平衡比实验中使用的标记期（3小时）快得多（长达9.45分钟）。对回补反应的分析表明，高选择性赖氨酸生产伴随着脱羧反应从10 mol%显著降低至仅2 mol%，而PEP/丙酮酸羧化通量在消耗葡萄糖的约40 mol%时保持恒定。这些结果支持以下结论：优化的谷氨酸棒杆菌L-赖氨酸生产菌株应具有增强的PEP羧化酶和/或丙酮酸羧化酶活性，并下调消耗草酰乙酸/苹果酸的脱羧通量。这些发现还说明了传感器-反应器方法在工业发酵研究中的实用性。
