EAGER:Collaborative Research:Innovating technologies to inform synthetic plant metabolism through a new understanding of the cellular protein machinery

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Plant metabolism is a high priority target for synthetic biology, with significant potential to address global challenges ranging from food production to energy and the environment. However, the successful application of synthetic biology strategies in complex eukaryotes faces unique challenges. In particular, large gaps remain in the knowledge of how cellular processes are controlled at the nanoscale, which is an essential component of engineering design. The project will develop new electron microscopy-based strategies to visualize and create structural models of labile protein complexes captured from plant cells. Ultra-sensitive methods will be established to rigorously identify the protein components of these complexes using advanced proteomics and in planta validation techniques. This will establish a critical new framework for synthetic metabolism. The project will also create a unique interdisciplinary/inter-institution training environment at Virginia Tech and Pennsylvania State University that will engage students at all levels of education.This interdisciplinary project will enable the study of dynamic multi-enzyme complexes in plants. A new integrated platform will be established for characterizing protein assemblies in the act of performing their biological functions in the uniquely-complex cellular environment of the plant cell. This suite of technologies will integrate advanced electron microscopy, proteomics, and 3D modeling capabilities to enable the nanoscale-level interrogation and understanding of the large, dynamic assemblies of proteins that define cellular biochemistry, and ultimately physiological function. The plant specialized pathway leading to flavonoids, a long-standing target for synthetic biology, will serve as the experimental model. This will generate outcomes that can be readily validated in planta and serve as a template the study of other biochemical systems. Altogether, the work will create a powerful new capability to address a critical information gap in current synthetic biology design.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.