NSF Award
2428204
The use of engineered organisms to sustainably produce valuable chemicals is a promising approach to reduce environmental impact and costs. However, natural metabolic processes of the organism often limit production of desired compounds. This project aims to understand and modify key enzymes that control cellular metabolism to enable more efficient production of high-value chemicals. Undergraduate students from diverse STEM backgrounds will be trained in cutting-edge techniques to study enzyme structures and behavior. By collaborating with researchers in India, the team will test these engineered enzymes in living organisms under various conditions. The project's outcomes will provide a toolkit of modified enzymes that can be used to enhance the production of valuable compounds, offering cheaper and greener synthesis methods for a wide range of industries. Furthermore, this project will offer insight into the basic underpinnings that control cellular metabolic state and thus have potential applications to fields beyond biomanufacturing, such as medicine, agriculture, and environmental science. The project also includes the training of undergraduates in cryo-EM data analysis through a dedicated computational course. <br/><br/>The project aims to develop transhydrogenase systems to address redox imbalance bottlenecks in metabolic engineering. Focusing on the recently discovered autoregulated GltAB-GudB complex in Bacillus subtilis, the project will investigate its impact on stationary phase biosynthetic potential and physiology. This unusually large complex, nearly half the size of a ribosome, regulates glutamate metabolism through a sophisticated allosteric control system. Using time-resolved cryo-electron microscopy (cryo-EM), metabolite-protein interaction screening, and kinetic assays, the project aims to elucidate the structural mechanisms underlying autoregulation of the complex. By bridging the gap between in vivo and in vitro enzyme activity measurements, the project intends to develop a biophysical model of GltAB-GudB regulation to enable rational tuning of its regulatory features. This knowledge will be applied to engineer the GltAB-GudB system and explore alternative transhydrogenase systems to enhance the biosynthesis of compounds limited by redox imbalance, such as free fatty acids (biodiesel precursors) and shikimate (pharmaceutical precursors). Ultimately, the project plans to elucidate key elements of metabolic regulation and develop a modular redox balancing toolkit to boost anabolic biosynthetic pathways in metabolic engineering applications.<br/><br/>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.
