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

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.