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Metabolic Pinching for the Formation of Pyruvate- and Acetyl CoA-Derived Chemicals

丙酮酸和乙酰辅酶A衍生化学物质形成的代谢挤压

基本信息

批准号：
1802533
负责人：
Mark Eiteman
金额：
$ 30万
依托单位：
University of Georgia Research Foundation Inc
依托单位国家：
美国
项目类别：
Standard Grant
财政年份：
2018
资助国家：
美国
起止时间：
2018-09-01 至 2022-08-31
项目状态：
已结题

来源：
https://www.nsf.gov/awardsearch/showAward?AWD_ID=1802533&HistoricalAwards=false
关键词：
Metabolic Pinching Formation Pyruvate Acetyl

项目摘要

Cells can produce products ranging from fuels to pharmaceuticals. This is usually accomplished by overproducing enzymes involved in the product pathway. This pulls more material onto that path, decreasing the amount leading to cell growth. The cell tries to pull more material towards growth pathways. If it is not successful, the cell can stop growing and possibly die, ending all production. This project proposes to look at more balanced approaches to providing sufficient material for moderate cell growth while funneling a majority of the material and energy towards making products. The old approach can be described as an on/off light switch, while the new approach can be compared to using a dimmer to slightly adjust each pathway up or down. This will involve modifying the enzymes involved at the entrance to the production pathway. The research is complemented by educational and training opportunities for high school students and undergraduates. This will lead to a workforce better prepared to actively participate in the growing biomanufacturing sector.Bioproduct formation involves redirecting a substrate towards the target product and away from biomass generation. Operational strategies rely on an external sensor of metabolism. Dynamic, genetic control relies on the external or internal concentration of some triggering compound. Dynamic control might ultimately increase the metabolic burden by virtue of additional gene expression. It has also not been demonstrated to increase yield and productivity compared to operational and static approaches. This project will investigate a strategy to pinch metabolism by altering the intrinsic activities of key enzymes in central metabolism in the absence of trigger compounds. If successful, we think this approach would serve as a useful component in the toolbox of biochemical product formation. Specifically, the objective of this project is to alter kCAT and KM of pyruvate dehydrogenase component E1 and citrate synthase in Escherichia coli. This should increase the intracellular pools of pyruvate and acetyl CoA, increasing the flux through competing pathways. We will then experimentally assess strain variants for growth, metabolic flux distribution and the production of several biochemical compounds derived from pyruvate and/or acetyl CoA.This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.

细胞可以生产从燃料到药品的各种产品。这通常是通过过度生产产品途径中涉及的酶来实现的。这会将更多的物质吸引到这条道路上，减少导致细胞生长的数量。该细胞试图将更多的物质拉向生长途径。如果不成功，细胞可能会停止生长，甚至可能死亡，从而终止所有生产。该项目建议寻找更平衡的方法，为细胞适度生长提供足够的材料，同时将大部分材料和能量用于制造产品。旧的方法可以被描述为开/关灯开关，而新的方法可以被比作使用调光器来微调每条路径的上下。这将涉及修改在生产途径的入口处涉及的酶。这项研究还为高中生和本科生提供了教育和培训机会。这将使劳动力做好更好的准备，积极参与不断增长的生物制造部门。生物产品的形成涉及将底物重新定向到目标产品，而不是生物质生产。操作策略依赖于新陈代谢的外部传感器。动态的基因控制依赖于某些触发化合物的外部或内部浓度。动态控制最终可能会通过额外的基因表达而增加代谢负担。与操作和静态方法相比，它也没有被证明能提高产量和生产率。这个项目将研究一种策略，在没有触发化合物的情况下，通过改变中枢代谢中关键酶的内在活性来扼杀新陈代谢。如果成功，我们认为这种方法将成为生化产品形成工具箱中的一个有用组件。具体地说，本项目的目标是改变大肠杆菌中丙酮酸脱氢酶组分E1和柠檬酸合成酶的kCAT和Km。这应该会增加细胞内丙酮酸和乙酰辅酶A的池，通过竞争途径增加通量。然后，我们将对菌株的生长、代谢通量分布和丙酮酸和/或乙酰CoA衍生的几种生化化合物的生产进行实验评估。该奖项反映了NSF的法定使命，并通过使用基金会的智力优势和更广泛的影响审查标准进行评估，被认为值得支持。