Integrated analysis of isopentenyl pyrophosphate (IPP) toxicity in isoprenoid-producing Escherichia coli
Integrated analysis of isopentenyl pyrophosphate (IPP) toxicity in isoprenoid-producing Escherichia coli
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DOI：
10.1016/j.ymben.2018.03.004
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发表时间：
2018-05-01
影响因子：
8.4
通讯作者：
Lee, Taek Soon
中科院分区：
文献类型：
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作者：
Georg, Kevin W.;Thompso, Mitchell G.;Lee, Taek Soon
异戊烯焦磷酸（IPP）毒性对工程微生物系统提出了挑战，因为它的形成在萜烯生物合成中是不可避免的。在这项工作中，我们开发了一个实验平台来研究 IPP 对产生异戊二烯醇的大肠杆菌的毒性。我们首先描述了对 IPP 积累的生理反应，证明 IPP 水平升高与生长抑制、细胞活力降低和质粒不稳定有关。我们表明，IPP 毒性选择途径“断裂”，利用蛋白质组学将磷酸甲羟戊酸激酶 (PMK) 的减少确定为可能的恢复机制。接下来，使用多组学数据，我们证明内源性大肠杆菌代谢在全球范围内受到 IPP 积累的影响，IPP 积累会减慢营养吸收、降低 ATP 水平并扰乱核苷酸代谢。我们还观察了 IPP 的细胞外积累，并提出了 IPP 可以通过大肠杆菌转运的初步证据，这些发现可能与类异戊二烯生物合成的研究广泛相关。最后，我们发现 IPP 积累导致 ApppI 的形成，ApppI 是 IPP 的核苷酸类似物，可能有助于观察到的毒性表型。这种对 IPP 应力的综合评估提出了减轻异戊二烯二磷酸毒性的潜在策略，并强调了改善 IPP 通量和高滴度类异戊二烯生产的可能工程目标。
Isopentenyl pyrophosphate (IPP) toxicity presents a challenge in engineered microbial systems since its formation is unavoidable in terpene biosynthesis. In this work, we develop an experimental platform to study IPP toxicity in isoprenol-producing Escherichia coll. We first characterize the physiological response to IPP accumulation, demonstrating that elevated IPP levels are linked to growth inhibition, reduced cell viability, and plasmid instability. We show that IPP toxicity selects for pathway "breakage", using proteomics to identify a reduction in phosphomevalonate kinase (PMK) as a probable recovery mechanism. Next, using multi-omics data, we demonstrate that endogenous E. coil metabolism is globally impacted by IPP accumulation, which slows nutrient uptake, decreases ATP levels, and perturbs nucleotide metabolism. We also observe the extracellular accumulation of IPP and present preliminary evidence that IPP can be transported by E. coil, findings that might be broadly relevant for the study of isoprenoid biosynthesis. Finally, we discover that IPP accumulation leads to the formation of ApppI, a nucleotide analog of IPP that may contribute to observed toxicity phenotypes. This comprehensive assessment of IPP stress suggests potential strategies for the alleviation of prenyl diphosphate toxicity and highlights possible engineering targets for improved IPP flux and high titer isoprenoid production.