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

植物代谢是合成生物学的一个高度优先目标，具有解决从粮食生产到能源和环境等全球挑战的巨大潜力。然而，合成生物学策略在复杂真核生物中的成功应用面临着独特的挑战。特别是，在如何在纳米尺度上控制细胞过程的知识方面仍然存在很大的差距，这是工程设计的重要组成部分。该项目将开发新的基于电子显微镜的策略，以可视化和创建从植物细胞捕获的不稳定蛋白质复合物的结构模型。将建立超灵敏的方法，使用先进的蛋白质组学和植物验证技术严格鉴定这些复合物的蛋白质组分。这将为合成代谢建立一个重要的新框架。该项目还将在弗吉尼亚理工大学和宾夕法尼亚州立大学创建一个独特的跨学科/跨机构培训环境，吸引各级教育的学生。该跨学科项目将使植物中动态多酶复合物的研究成为可能。将建立一个新的集成平台，用于表征蛋白质组装体在植物细胞复杂的细胞环境中执行其生物学功能的行为。这套技术将集成先进的电子显微镜，蛋白质组学和3D建模功能，以实现纳米级的询问和理解定义细胞生物化学和最终生理功能的蛋白质的大型动态组装。植物专门的途径导致类黄酮，一个长期的目标合成生物学，将作为实验模型。这将产生的结果，可以很容易地在植物中验证，并作为一个模板，其他生化系统的研究。总而言之，这项工作将创造一个强大的新能力，以解决目前合成生物学设计中的关键信息差距。该奖项反映了NSF的法定使命，并已被认为是值得通过使用基金会的智力价值和更广泛的影响审查标准进行评估的支持。

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.