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

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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.