Collaborative Research: Synthetic methane fixation cascades based on engineered membrane vesicles for biofuel cell applications

石油和天然气开采技术的进步使主要由甲烷组成的天然气得到广泛使用。在这些操作过程中，大量的甲烷泄漏到大气中。井场通常是偏远和孤立的。标准的捕集和处理技术一般不适用于这些情况。这个项目的目标是将甲烷转化为电能。将产生人工酶级联，将甲烷完全氧化为二氧化碳，并在此过程中产生电子。这些电子将用于燃料电池。该项目将包括一个教育和推广计划，扩大学生获得基于项目的学习的机会。开发一个综合的教学、研究和课程开发平台将吸引研究生、本科生和高中生，特别是那些代表性不足的群体。由于缺乏有效的酶级联反应，在环境温度下将大部分浪费的甲烷转化为更有价值的产品存在很大差距。为了解决这个问题，提出了在膜囊泡上使用有组织的四酶级联将甲烷固定在甲醇上，并结合甲醇氧化以释放电子的概念。在甲烷营养细菌中发现的膜结合酶会在温和条件下选择性地将甲烷转化为甲醇。这是该方法的基础。采用新型混合酶-合成生物学方法，利用直接从宿主膜上分离的酶结合囊泡，人工酶级联可以将甲醇完全氧化为二氧化碳并产生用于燃料电池的电子。具体来说，将蛋白质支架结合到甲烷固定酶结合的膜囊泡上，将使三种脱氢酶的组装成为可能，从而将甲烷依次转化为二氧化碳。三种脱氢酶与甲烷固定酶的共定位将促进酶之间的协同作用，因为底物通道，导致总电流密度增强。级联酶的另一个好处是能够改善脱氢酶和甲烷固定酶之间NADH和NAD+的转移，从而提高催化效率。预期的结果是一个新的平台来生成合成膜囊泡系统，用于甲烷固定二氧化碳，为生物燃料电池提供动力，并作为其他膜结合酶系统的技术平台。该奖项反映了美国国家科学基金会的法定使命，并通过使用基金会的知识价值和更广泛的影响审查标准进行评估，被认为值得支持。

Advances in oil and gas extraction techniques have made natural gas, composed primarily of methane, widely available for use. Large quantities of methane leak into the atmosphere during these operations. Well sites are often remote and isolated. Standard capture and treatment technologies are not generally feasible to apply in these cases. The objective of this project is to convert methane to electric power. Artificial enzyme cascades will be created to completely oxidize methane to CO2, generating electrons in the process. The electrons will be used in fuel cell applications. This project will include an education and outreach program that expands student access to project-based learning. Developing an integrated teaching, research, and curriculum development platform will engage graduate students, undergraduate, and high school students, particularly those in underrepresented groups.There is a big gap on converting mostly wasted methane to more valuable products under ambient temperature due to the lack of efficient enzyme cascades. To address this problem, the concept of fixing methane to methanol coupled to oxidation of methanol for electron release using an organized four-enzyme cascade on membrane vesicles is proposed. The membrane-bound enzyme found in methanotrophic bacteria will selectively convert methane to methanol under mild conditions. This is the foundation of the approach. Employing new hybrid enzyme-synthetic biology artificial enzyme cascades, using the enzyme-bound vesicles isolated directly from the host membranes, should completely oxidize methanol to CO2 and generate electrons for fuel-cell applications. Specifically, conjugating a protein scaffold onto the methane fixing enzyme-bound membrane vesicles will enable the assembly of three dehydrogenases for the sequential conversion of methane to carbon dioxide. Co-localization of the three dehydrogenases with the methane fixing enzyme will promote the synergistic action between the enzymes due to substrate channeling, resulting in an enhancement in the overall current density. One added benefit of cascading the enzymes is to enable the improved transfer of NADH and NAD+ between dehydrogenase and the methane fixing enzyme for higher catalytic efficiency. The expected result is a new platform to generate synthetic membrane vesicles system for methane fixation to CO2 to power biofuel cells, and to serve as a technology platform for other membrane-bound enzyme systems.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.