CAREER: Connecting eukaryotic electron transfer components to nitrogenase using a bacterial chassis

在新出现的合成生物学工具的支持下，我们对生物固氮的理解取得了重要进展，这表明我们比以往任何时候都更接近工程植物来固氮。通过工程植物来固定氮素可以提高生物经济的可持续性。然而，我们缺乏预测铁还蛋白等电子载体的行为和特异性的知识，这些电子载体是为固氮酶提供动力所必需的。迫切需要测试真核细胞的电子转移组分与固氮酶相互作用的能力，并测量细胞氧化还原环境的变化如何维持电子流。这项研究的总体目标是使用细菌底盘来快速定义真核电子传递组件如何参与固氮酶的电子传递，并发明一个强大的平台来进化合成电子流动途径。这项研究的目的是通过将一个为期一学期的项目结合到现有的课程中，利用一个新的文化响应性教学框架，将生物工程固氮纳入到教育目标中。该项目的中心假设是，电子转移到固氮酶是阻止这种酶引入真核系统的主要限制因素之一，但有可能在真核细胞电子转移成分中选择变体来克服这一瓶颈。为了验证这一假设，研究人员建议开发一种新的工具来分析电子流向固氮酶。这个工具将使用细菌底盘来测试真核细胞的生理水平的电子转移成分，并进化这些电子转移成分，以增强电子流向固氮酶。这一贡献将是重大的，因为它将能够更准确地预测植物细胞器内的固氮酶功能，并将为优化电子向固氮酶的转移建立一个强大的平台。这不仅将使工程植物固定氮的目标更容易实现，而且还将加深我们对电子流动的决定因素的理解，以及如何为生物技术目的优化电子转移途径。这个项目得到了分子和细胞生物科学部系统和合成生物学分部的支持。这个奖项反映了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。

Important advancements in our understanding of biological nitrogen fixation, bolstered by emerging synthetic biology tools, suggest we are closer than ever to engineering plants to fix nitrogen. Engineering plants to fix nitrogen could improve the sustainability of the bioeconomy. However, we lack the knowledge to predict the behavior and specificity of electron carriers such as ferredoxin, which are needed to power nitrogenase. There is a critical need to test eukaryotic electron transfer components for their ability to interact with nitrogenase and measure how changes in the cellular redox environment sustain electron flow. The overall objective of the research proposed here is to use a bacterial chassis to rapidly define how eukaryotic electron transfer components can participate in electron delivery to nitrogenase and invent a powerful platform for evolution of synthetic electron flow pathways. The research aims synergize with educational goals by incorporating a semester-long project that focuses on bioengineering nitrogen fixation into existing courses using a novel culturally responsive pedagogical framework.The central hypothesis for the project is that electron transfer to nitrogenase is one of the primary constraints preventing introduction of this enzyme into eukaryotic systems, but it is possible to select for variants in eukaryotic electron transfer components to overcome this bottleneck. To test this hypothesis, the investigator proposes to develop a new tool to analyze electron flow to nitrogenase. This tool will use a bacterial chassis to test eukaryotic electron transfer components at physiological levels and evolve these electron transfer components for enhanced electron flow to nitrogenase. Such a contribution would be significant because it would enable more accurate predictions regarding nitrogenase functionality within plant organelles and would establish a robust platform for optimizing electron transfer to nitrogenase. This would not only make the goal of engineering plants to fix nitrogen more attainable, but it would also further our understanding of the determinants of electron flow and how electron transfer pathways can be optimized for biotechnological purposes. This project is supported by the Systems and Synthetic Biology Cluster of the Division of Molecular and Cellular Biosciences.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.