Engineered protein nanocompartments for in vivo and in vitro multi-step enzyme catalysis

智力优势：空间组织是所有生物系统功能的核心。因此，将类似的原理应用于重组微生物有机体的工程代谢途径的组织已经成为一种新兴的策略，以提高这种生物生产设计系统的效率。研究人员在之前的工作中已经证明，在大肠杆菌中表达时，构成肠沙门氏菌Eut隔室外壳的蛋白质也可以形成多面体隔室。研究还发现，只有一种壳蛋白是形成隔室和靶向蛋白进入这些结构所必需和充分的。这些标准提供了一种潜在的直接策略，用于工程和生产包裹货物蛋白质的纳米室。该项目将把这些发现提升到一个新的水平，并设计纳米室作为一个广泛的生物技术应用的通用平台。本研究的具体目标是：1)研究和优化大肠杆菌中纳米隔室的形成；2)设计一种将货物蛋白质装载到隔室中的通用系统；3)在大肠杆菌和溶液中对这种工程隔室中的多步骤酶过程进行概念验证。更广泛的影响：工程蛋白基纳米室将是非常理想的生物纳米结构，用于生物传感器和使用环境友好和可持续的生物过程的化学制造，以及药物靶向。此外，拟议的研究将为一名博士后研究员、一名研究生和几名本科生提供工程和生物科学方面的跨学科培训。该项目的研究将被整合到由PI指导的合成生物学课程中。来自不同学科的本科生以及来自当地高中的学生参加讲座和指导研究，并在学期结束时为秋季的iGEM竞赛制定一个夏季研究项目。事实上，这个项目的一些初步结果是作为iGEM夏季项目的一部分产生的。

Intellectual Merit: Spatial organization is central to the function of all biological systems. Consequently, application of similar principles to the organization of engineered metabolic pathways in recombinant microbial organisms has become an emerging strategy to increase the efficiencies of such designed systems for bioproduction. The investigator has shown inprevious work that proteins that make up the shells of Eut compartments from Salmonella enterica can form polyhedral compartments also when expressed in Escherichia coli. It was also discovered that only one of the shell proteins is necessary and sufficient for forming compartments, and also for targeting proteins into these structures. These criteria offer a potentially straightforward strategy for the engineering and production of nanocompartments with encapsulated cargo proteins. This project will take these finding to the next level and engineer nanocompartments as a versatile platform for a wide range of biotechnological applications. Specific objectives of this research are 1) investigation and optimization of nanocompartment formation in E. coli, 2) design of a universal system for cargo protein loading into compartments and 3) a proof-of-concept demonstration of a multi-step enzymatic process functioning in such engineered compartments in E. coli and in solution.Broader Impacts: Engineered protein-based nanocompartments would be highly desirable bio-nanostructures for applications as biosensors and for chemical manufacturing using environmentally benign and sustainable bioprocesses, as well as drug targeting. In addition, the proposed research will provide cross-disciplinary training in engineering and biological sciences for one postdoctoral researcher, a graduate student and several undergraduate students. Research from this project will be integrated in a course in Synthetic Biology directed by the PI. Undergraduate students from different disciplines as well as students from local high-schools participate in lectures and guided research, and at the end of the semester develop a summer research project for the iGEM competition in the fall. In fact, some of the initial results for this project were generated as part of an iGEM summer project.