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

一些关键分子在细胞内扮演着中心电子和能量载体的角色。烟酰胺腺嘌呤二核苷酸(NADH)传递电子，三磷酸腺苷(ATP)提供能量。最常见的生产三磷酸腺苷的方法需要在膜上建立pH梯度。由此产生的质子流通过嵌入的蛋白质再生三磷酸腺苷。这个项目将检验这样一个假设，即可以开发替代途径，在不需要膜、质子梯度或嵌入酶的情况下，从NADH氧化中再生ATP。这将需要将天然酶和工程酶结合起来，创建新的循环途径。这些途径将有助于揭示复制中心生物过程所需的设计规则。这项技术将对合成细胞的发展至关重要。此外，该项目将包括指导学生和扩大当地社区现有的外展活动。该项目将设计、建造和测试新的合成代谢途径，这些途径可以利用从燃料氧化中获得的能量再生ATP。燃料氧化途径可产生NADH形式的还原当量。然而，其他关键的操作，特别是那些涉及运动的操作，是由三磷酸腺苷的水解提供动力的。线粒体的氧化磷酸化提供了NADH氧化产生的大部分细胞ATP，这需要建立质子梯度。虽然这为细胞能量处理提供了灵活性，但复制这个系统在工程上是一个困难的挑战。我们假设氧化磷酸化可以被由激酶酶组成的新途径所取代。这些途径将使用燃料氧化反应来驱动NADH的再生，而NAD(H)的磷酸化将驱动ATP的再生。作为概念验证，将演示在简单脂质体中由甲醇氧化提供动力的ATP依赖反应(萤火虫荧光素酶)。这将是线粒体氧化磷酸化的一种新的能量转导替代方案。这一途径可以很容易地用于广泛的潜在燃料，支持许多潜在的未来合成细胞和生物应用。这一奖项反映了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。

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.