Systems Analyses Reveal the Resilience of Escherichia coli Physiology during Accumulation and Export of the Nonnative Organic Acid Citramalate.
Systems Analyses Reveal the Resilience of Escherichia coli Physiology during Accumulation and Export of the Nonnative Organic Acid Citramalate.
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系统分析揭示了大肠杆菌生理学在非天然有机酸柠苹果酸盐积累和输出过程中的恢复能力。
DOI：
10.1128/msystems.00187-19
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发表时间：
2019
期刊：
影响因子：
6.4
通讯作者：
Webb J
中科院分区：
文献类型：
--
作者：
Webb J
细菌细胞工厂的生产力经常受到重组蛋白合成和碳到产物转化所施加的压力的影响，但在系统水平上对这些生物过程知之甚少。在大肠杆菌中产生非自然代谢物柠檬酸酯需要表达一个编码柠檬酸酯合成酶的基因。多组学分析的发酵产生25克升−1柠檬酸盐进行了揭示其生产力的原因。表达柠檬酸酯合成酶或灭活酶的大肠杆菌的高密度补料发酵的代谢物、转录物、蛋白质和脂质谱相似。两种发酵都显示鞭毛基因下调，伴侣蛋白IbpA和IbpB上调，表明这些反应是由于重组蛋白的合成而不是柠檬酸盐的产生。柠檬酸盐的产生不会扰乱代谢物池，除了增加细胞内丙酮酸池。对柠檬酸盐的基因表达变化有限；一般的应激反应规则都没有被激活。转录因子活性的建模表明，柠檬酸盐引发了gadw介导的酸反应，GadY和RprA调节小RNA （sRNA）表达的变化支持了这一观点。虽然观察到膜脂组成的变化，但没有一个是柠檬酸盐生产所特有的。柠檬酸盐发酵的系统分析表明，大肠杆菌有能力适应重组蛋白和柠檬酸盐生产的资源重定向，这表明它是制造有机酸的绝佳底盘选择。柠檬酸盐是一种有吸引力的生物技术靶标，因为它是甲基丙烯酸甲酯的前体，甲基丙烯酸甲酯用于制造有机玻璃和其他高价值产品。工程大肠杆菌菌株能够产生高滴度的柠檬酸盐，尽管必须表达外源酶和耐受非本地生化物质的存在。对柠檬酸酯发酵进行了系统分析，以揭示支撑其生产力的原因。这表明大肠杆菌很容易适应代谢资源向重组蛋白和柠檬酸盐生产的重定向，并表明大肠杆菌是制造类似的小极性外来分子的良好基础。
Productivity of bacterial cell factories is frequently compromised by stresses imposed by recombinant protein synthesis and carbon-to-product conversion, but little is known about these bioprocesses at a systems level. Production of the unnatural metabolite citramalate in Escherichia coli requires the expression of a single gene coding for citramalate synthase. Multiomic analyses of a fermentation producing 25 g liter−1citramalate were undertaken to uncover the reasons for its productivity. Metabolite, transcript, protein, and lipid profiles of high-cell-density, fed-batch fermentations of E. coli expressing either citramalate synthase or an inactivated enzyme were similar. Both fermentations showed downregulation of flagellar genes and upregulation of chaperones IbpA and IbpB, indicating that these responses were due to recombinant protein synthesis and not citramalate production. Citramalate production did not perturb metabolite pools, except for an increased intracellular pyruvate pool. Gene expression changes in response to citramalate were limited; none of the general stress response regulons were activated. Modeling of transcription factor activities suggested that citramalate invoked a GadW-mediated acid response, and changes in GadY and RprA regulatory small RNA (sRNA) expression supported this. Although changes in membrane lipid composition were observed, none were unique to citramalate production. This systems analysis of the citramalate fermentation shows that E. coli has capacity to readily adjust to the redirection of resources toward recombinant protein and citramalate production, suggesting that it is an excellent chassis choice for manufacturing organic acids.IMPORTANCECitramalate is an attractive biotechnology target because it is a precursor of methylmethacrylate, which is used to manufacture Perspex and other high-value products. Engineered E. coli strains are able to produce high titers of citramalate, despite having to express a foreign enzyme and tolerate the presence of a nonnative biochemical. A systems analysis of the citramalate fermentation was undertaken to uncover the reasons underpinning its productivity. This showed that E. coli readily adjusts to the redirection of metabolic resources toward recombinant protein and citramalate production and suggests that E. coli is an excellent chassis for manufacturing similar small, polar, foreign molecules.