Design of a genetically programmable artificial cell system for biocatalysis

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The machinery that drives protein synthesis can be purified intact from cells. Using this machinery, protein can be produced at the direction of synthetic DNA outside of a cell. This opens up a wide range of opportunities. Cell activities can be isolated and studied. Artificial systems can be designed to perform a limited number of cell functions. A cell-like system could be fabricated that produces a single chemical or pharmaceutical. The goal of this project is to encase cell-sized compartments inside polymer membranes. The compartments will contain protein synthesis machinery and DNA. The enzymes that are created will produce valuable chemicals from simple starting materials. DNA for different cell systems will be mixed and matched to allow rapid prototyping of artificial cell configurations. The project will provide training and mentoring opportunities for high-school, undergraduate and graduate students. STEM outreach activities aimed at PreK-12 students will be pursued using resources available locally.Advances in cell-free systems are enabling the bottom-up design of synthetic cells. These cells could be comprised of a genetically encoded multi-enzyme cascade. Spatial organization could be designed to mimic cellular environments. Rapid prototyping of biocatalytic cascades is a key capability of these systems. Cell-like systems would be specifically designed to meet biocatalytic process requirements. These would include robustness, operational stability and catalytic efficiency of enzymes, and membrane porosity for co-factor, substrate and product transport. The design of such, genetically programmable artificial cell-like systems for in vitro cascade biocatalysis has not yet been accomplished and poses challenges that this project will address. A framework will be developed for the design, fabrication and operation of a robust artificial cell system for multi-enzyme cascade catalysis. An industrially relevant redox cascade will constitute the model system. A robust chassis will be built with a composite membrane, enforced by a genetically programmed protein-based cell-wall that contain pores/channels that facilitate membrane transport. Biocatalysts will be expressed inside the compartments along with protein based macromolecular structures for spatial organization of enzymes and control microenvironments. Knowledge gained in this project will serve as a framework for the bottom-up design of a synthetic cell chassis with genetically programmable subcellular features and membrane properties. The artificial cells developed in this project will allow experimental testing and further development of mathematical models proposed for compartmentalized systems. Results from these studies will advance our understanding of biological systems and provide guidance for the rational design of synthetic cells for biomanufacturing and other applications.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.