Oldiges M, Kunze M, Degenring D, Sprenger G A, Takors R
Institute of Biotechnology, Forschungszentrum Jülich GmbH, 52425 Jülich, Germany.
Biotechnol Prog. 2004 Nov-Dec;20(6):1623-33. doi: 10.1021/bp0498746.
Using a concerted approach of biochemical standard preparation, analytical access via LC-MS/MS, glucose pulse, metabolic profiling, and statistical data analysis, the metabolism dynamics in the aromatic amino acid pathway has been stimulated, monitored, and analyzed in different tyrosine-auxotrophic L-phenylalanine-producing Escherichia coli strains. During the observation window from -4 s (before) up to 27 s after the glucose pulse, the dynamics of the first five enzymatic reactions in the aromatic amino acid pathway was observed by measuring intracellular concentrations of 3-deoxy-d-arabino-heptulosonate 7-phosphate DAH(P), 3-dehydroquinate (3-DHQ), 3-dehydroshikimate (3-DHS), shikimate 3-phosphate (S3P), and shikimate (SHI), together with the pathway precursors phosphoenolpyruvate (PEP) and P5P, the lumped pentose phosphate pool as an alternative to the nondetectable erythrose 4-phosphate (E4P). Provided that a sufficient fortification of the carbon flux into the pathway of interest is ensured, respective metabolism dynamics can be observed. On the basis of the intracellular pool measurements, the standardized pool velocities were calculated, and a simple, data-driven criterion--called "pool efflux capacity" (PEC)--is derived. Despite its simplifying system description, the criterion managed to identify the well-known AroB limitation in the E. coli strain A (genotype delta(pheA tyrA aroF)/pJF119EH aroF(fbr) pheA(fbr) amp) and it also succeeded to identify AroL and AroA (in strain B, genotype delta(pheA tyrA aroF)/pJF119EH aroF(fbr) pheA(fbr) aroB amp) as promising metabolic engineering targets to alleviate respective flux control in subsequent L-Phe producing strains. Furthermore, using of a simple correlation analysis, the reconstruction of the metabolite sequence of the observed pathway was enabled. The results underline the necessity to extend the focus of glucose pulse experiments by studying not only the central metabolism but also anabolic pathways.
通过采用生化标准制备、液相色谱-串联质谱（LC-MS/MS）分析、葡萄糖脉冲、代谢谱分析和统计数据分析的协同方法，在不同的酪氨酸营养缺陷型L-苯丙氨酸生产大肠杆菌菌株中，对芳香族氨基酸途径中的代谢动力学进行了刺激、监测和分析。在从葡萄糖脉冲前-4秒到脉冲后27秒的观察窗口内，通过测量细胞内3-脱氧-D-阿拉伯庚酮糖酸-7-磷酸（DAH(P)）、3-脱氢奎尼酸（3-DHQ）、3-脱氢莽草酸（3-DHS）、莽草酸-3-磷酸（S3P）和莽草酸（SHI）的浓度，以及途径前体磷酸烯醇丙酮酸（PEP）和磷酸戊糖（P5P），作为无法检测到的赤藓糖-4-磷酸（E4P）的替代物的总戊糖磷酸池，观察了芳香族氨基酸途径中前五个酶促反应的动力学。只要确保有足够的碳通量强化进入目标途径，就可以观察到相应的代谢动力学。基于细胞内池测量，计算了标准化池速度，并得出了一个简单的数据驱动标准——称为“池流出能力”（PEC）。尽管该标准简化了系统描述，但它成功地识别出大肠杆菌菌株A（基因型delta(pheA tyrA aroF)/pJF119EH aroF(fbr) pheA(fbr) amp）中众所周知的AroB限制，并且还成功地将AroL和AroA（在菌株B中，基因型delta(pheA tyrA aroF)/pJF119EH aroF(fbr) pheA(fbr) aroB amp）识别为有前景的代谢工程靶点，以减轻后续L-苯丙氨酸生产菌株中的相应通量控制。此外，通过简单的相关性分析，实现了对观察到的途径代谢物序列的重建。结果强调了不仅要研究中心代谢，还要研究合成代谢途径，以扩大葡萄糖脉冲实验重点的必要性。
