Phosphoenolpyruvate Transporter Enables Targeted Perturbation During Metabolic Analysis of L-Phenylalanine Production With Escherichia coli.
Phosphoenolpyruvate Transporter Enables Targeted Perturbation During Metabolic Analysis of L-Phenylalanine Production With Escherichia coli.
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DOI：
10.1002/biot.201700611
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发表时间：
2018-05
影响因子：
4.7
通讯作者：
Julia Tröndle;C. Albermann;Michael Weiner;G. Sprenger;D. Weuster‐Botz
中科院分区：
文献类型：
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作者：
Julia Tröndle;C. Albermann;Michael Weiner;G. Sprenger;D. Weuster‐Botz
通常通过添加底物来干扰细胞代谢，用于生产生物体的代谢分析，但干扰研究仅限于研究细胞的内源性底物。本研究的目的是克服这一限制，使磷酸烯醇式丙酮酸（PEP）与大肠杆菌生产L-苯丙氨酸的扰动研究。在42 L规模的标准化补料分批生产工艺中应用过表达PEP转运蛋白变体（UhpT-D388 C）的生产菌株。在从生产过程中快速转移细胞培养基后，用葡萄糖和甘油作为补料分批碳源进行四次平行的20 min短期扰动实验。PEP在9分钟后加入，并立即被细胞消耗，最高达1.5 mmol gCDW-1 h-1。加入PEP后，L-苯丙氨酸的生产率提高了200%。这清楚地表明在所研究的L-苯丙氨酸生产菌株中存在细胞内PEP限制。因此，它表明，过表达特定的转运蛋白的分析原因，使外源性底物可作为扰动底物的生产过程中采样的细胞的代谢分析，从而允许一个非常有针对性的扰动全细胞代谢。
Usually perturbation of the metabolism of cells by addition of substrates is applied for metabolic analysis of production organisms, but perturbation studies are restricted to the endogenous substrates of the cells under study. The goal of this study is to overcome this limitation by making phosphoenolpyruvate (PEP) available for perturbation studies with Escherichia coli producing L-phenylalanine. A production strain overexpressing a PEP-transporter variant (UhpT-D388C) is applied in a standardized fed-batch production-process on a 42 L-scale. Four parallel short-term perturbation experiments of 20 min are performed with glucose and glycerol as fed-batch carbon sources after rapid media transition of cells from the production-process. PEP is added after 9 min and is immediately consumed by the cells with up to 1.5 mmol gCDW-1 h-1 . L-phenylalanine production rates increased by up to 200% after addition of PEP. This clearly indicates an intracellular PEP-limitation in the L-phenylalanine production strain under study. Thus, it is shown that overexpressing specific transporters for analytical reasons makes exogenous substrates available as perturbation substrates for metabolic analyses of cells sampled from production-processes and thereby allows a very targeted perturbation of whole-cell metabolism.