Collaborative Research: NSF/MCB: Repurposing metabolite-responsive aptamers for real-time sensing and dynamic control of Cas6-mediated metabolon assembly

The aim of the research is to develop novel strategies for metabolite sensing and metabolite-induced enzyme localization; this will contribute to fundamental cellular knowledge and improve the efficiency of bioprocesses that are associated with synthetic biology. In nature, many microorganisms have evolved to survive across different growth-permissive conditions. This adaptability is achieved through a highly coordinated metabolic network that tightly regulates the activity of cellular components at the required level in order to adjust to fluctuating nutrient conditions. A detailed analysis of these metabolites would provide a deeper understanding of their physiological roles in promoting and regulating cellular processes. To achieve this, the research exploits the reversible reconstitution of split RNA aptamer fragments for real-time metabolite sensing. The same reversible split aptamer assembly design is also exploited to create dynamic metabolons to reveal insights into yeast metabolism for optimizing product synthesis. The tool sets developed are easily transferrable to other eukaryotes such as mammalian cells to address fundamental questions about regulation and rewiring of metabolism. The research spans the core disciplines of biology, chemistry, and engineering, in providing ample opportunities for student training at all levels and in multiple areas. This project also facilitates outreach activities to local high school teachers and students through existing programs available at the University of Delaware and UC Irvine. Real-time quantification of intracellular metabolites is essential for our ability to interrogate, understand, and engineer metabolism in a range of biological systems. This project exploits the reversible reconstitution of split RNA aptamer fragments as a new framework for real-time metabolite sensing. Using a Cas6-mediated protein-RNA assembly strategy, real-time probing of metabolite-induced split RNA aptamer reconstitution is monitored using the reversible assembly of a split fluorescent protein reporter. The reversible nature of split aptamer assembly is also exploited to create dynamic metabolons for metabolite-responsive control of metabolism that is useful for a wide range of fundamental studies and synthetic biology applications. This research impacts the field of synthetic biology by creating a new method for real-time metabolite sensing and for metabolite-mediated dynamic assembly of metabolons in many organisms of interest. 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.