RoL: EAGER: DESYN-C3: Mimicking Mitochondria: Developing Synthetic Pathways to Power Pseudo-Cell Functions using Diverse Fuel Resources

RoL：EAGER：DESYN-C3：模仿线粒体：开发利用不同燃料资源为伪细胞功能提供动力的合成途径

• 批准号: 1844254
• 负责人: Scott Banta
• 金额: $ 30万
• 依托单位: Columbia University
• 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: 2018-10-01 至 2021-08-31
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=1844254&HistoricalAwards=false
• 关键词: RoL; EAGER; DESYN; C3; Mimicking

A few key molecules act as central electron and energy carriers inside cells. Nicotinamide adenine dinucleotide (NADH) transfers electrons and adenosine triphosphate (ATP) supplies energy. The most common method to produce ATP requires a pH gradient to be established across a membrane. The resulting flow of protons through an embedded protein regenerates ATP. This project will test the hypothesis that alternative pathways could be developed that can regenerate ATP from NADH oxidation without the need for a membrane, proton gradient, or embedded enzyme. This will require combining native and engineered enzymes to create novel cyclic pathways. These pathways will help uncover design rules required to replicate a central biological process. This technology will be critical to the development of synthetic cells. In addition, the project will involve mentoring of students and expanding existing outreach activities in the local community.This project will design, build and test novel synthetic metabolic pathways that could regenerate ATP using energy obtained from fuel oxidation. Fuel oxidation pathways can generate reducing equivalents in the form of NADH. However other critical operations, especially those involving motion, are powered by the hydrolysis of ATP. Oxidative phosphorylation in mitochondria supplies the majority of cellular ATP from NADH oxidation and this requires the establishment of a proton gradient. Although this supplies flexibility for cellular energy processing, replicating this system presents a difficult engineering challenge. We hypothesize that oxidative phosphorylation could be replaced with novel pathways composed of kinase enzymes. These pathways will use fuel oxidation reactions to drive NADH regeneration and NAD(H) phosphorylation will drive ATP regeneration. As a proof-of-concept, an ATP-dependent reaction (firefly luciferase) powered by methanol oxidation in simple liposomes will be demonstrated. This will represent a novel energy transduction alternative to mitochondrial oxidative phosphorylation. This pathway could be easily adopted to use a wide range of potential fuels, supporting many potential future synthetic cell and biology applications.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.

一些关键分子充当细胞内部的中央电子和能量载体。烟酰胺腺嘌呤二核苷酸（NADH）转移电子和三磷酸腺苷（ATP）提供能量。产生ATP的最常见方法需要在膜上建立pH梯度。质子通过嵌入蛋白再生的ATP产生的流动。 该项目将检验以下假设：可以开发出可以从NADH氧化中再生ATP的替代途径，而无需膜，质子梯度或嵌入式酶。这将需要组合天然和工程酶以创建新型的环状途径。 这些途径将有助于发现复制中心生物学过程所需的设计规则。该技术对于合成细胞的发展至关重要。此外，该项目将涉及指导学生并扩大当地社区中现有的外展活动。该项目将设计，构建和测试新型的合成代谢途径，这些途径可以使用从燃料氧化中获得的能量重新生成ATP。燃料氧化途径可以以NADH的形式产生减少等效物。 然而，其他关键操作，尤其是涉及运动的操作，是由ATP水解提供的。 线粒体中的氧化磷酸化提供了大多数来自NADH氧化的细胞ATP，这需要建立质子梯度。 尽管这为蜂窝能量处理提供了灵活性，但复制该系统却带来了困难的工程挑战。 我们假设可以用由激酶酶组成的新途径代替氧化磷酸化。 这些途径将使用燃料氧化反应来驱动NADH再生，而NAD（H）磷酸化将驱动ATP再生。 作为概念验证，将证明由甲醇氧化供电的简单脂质体中供电的ATP依赖性反应（萤火虫荧光素酶）。 这将代表线粒体氧化磷酸化的新型能量转导替代品。 该途径可以轻松地采用多种潜在的燃料，支持许多潜在的未来合成细胞和生物学应用。该奖项反映了NSF的法定任务，并通过使用基金会的知识分子和更广泛的影响评估标准来评估NSF的法定任务。