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

在新兴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定氮。固氮的工程工厂可以提高生物经济的可持续性。然而，我们缺乏预测电子载体的行为和特异性的知识，如铁氧还蛋白，这是驱动氮酶所需要的。目前迫切需要测试真核电子转移组分与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。本文提出的研究总体目标是利用细菌底盘快速定义真核生物电子传递组分如何参与向氮酶的电子传递，并为合成电子流途径的进化发明一个强大的平台。该研究旨在通过将一个学期的生物工程固氮项目纳入现有课程，并使用一种新的文化响应教学框架，从而与教育目标协同作用。该项目的中心假设是，向氮酶的电子转移是阻止将该酶引入真核系统的主要限制之一，但有可能选择真核电子转移组分的变体来克服这一瓶颈。为了验证这一假设，研究者建议开发一种新的工具来分析流向氮酶的电子流。该工具将使用细菌底盘在生理水平上测试真核电子转移组分，并进化这些电子转移组分以增强电子流向氮酶。这样的贡献意义重大，因为它可以更准确地预测植物细胞器内的氮酶功能，并为优化电子向氮酶的转移建立一个强大的平台。这不仅会使工程植物固定氮的目标更容易实现，而且还会进一步加深我们对电子流决定因素的理解，以及如何为生物技术目的优化电子转移途径。该项目由分子和细胞生物科学部的系统和合成生物学集群支持。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。

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.