Tools4Cells: Realtime metabolite sensing and metabolon formation via biomolecular condensates

The aim of the research is to develop a new strategy for metabolite sensing and metabolite-induced enzyme localization; this will contribute to fundamental cellular knowledge and also improve the efficiency of bioprocesses. Living cells interact with their surroundings and with each other by recognizing specific chemical signatures in the environment, followed by logical processing of information to elicit required responses. This adaptability is achieved by a dynamic metabolic network that tightly regulates the activity of cellular components to adjust to fluctuating chemical signatures in the environment. A detailed real-time analysis of these chemical signatures (e.g. metabolites) would provide a deeper understanding of their physiological roles in promoting and regulating cellular activities, enabling more robust paradigms for cellular reprogramming. To achieve this, the investigators will exploit the reversibility of ligand-responsive transcription factor (LRTF)-DNA interaction as a new transformative framework to enable real-time metabolite sensing in live cells. The idea is to create synthetic LRTF-based protein devices that can translate metabolite-induced DNA binding into reversible protein condensate formation. Rapid switching in condensate formation leads to reversible compartmentalization of a cluster of reporters for highly sensitive, real-time biosensing. The reversible nature of LRTF-DNA interaction will also be 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. From educational and outreach perspectives, the research will span the core disciplines of biology, chemistry, and engineering, and will therefore provide ample opportunities for student training at all levels and in multiple areas. This project will also facilitate outreach activities to local high school teachers and students through existing programs available at the University of Delaware 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 will exploit the rapid and reversible binding of LRTFs to their cognate operator sequences as a transformative framework for real-time metabolite sensing. Inspired by the ease and efficiency of promoting condensate formation, the investigators will create a new metabolite-responsive strategy to reversibly sequester fluorescent protein reporters within the condensate compartment based on DNA binding for real-time biosensing. The reversible nature of assembly will also be 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 will impact 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.