Tools4Cells: Multi-kingdom acetyl-CoA sensing for real-time reporting and control of metabolism

Acetyl-CoA is a building block for many high-value chemicals produced by microbes and is often overproduced to improve large-scale production of these chemicals. Consequently, the ability to monitor acetyl-CoA levels during bioproduction would significantly help efforts to maximize the yield of these chemicals. The goal of this project is to develop a system for real-time monitoring of Acetyl-CoA in microbes and to exploit this new capability to modulate production of high-value chemicals. This project will also facilitate outreach activities to local high school through existing programs available at the University of Delaware. A protein-based biosensor that allows rapid, sensitive, and dynamic AcCoA is needed to provide a deeper understanding of the physiological roles of AcCoA in promoting and regulating cellular activities. The central framework here takes advantage of the natural and engineered protein-protein interaction to create the first real-time biosensor and metabolic dynamic regulation system that is responsive to AcCoA, which is one of the most important and central metabolites. This project will demonstrate that this AcCoA-specific interaction can be repurposed to create conditional split fluorescent reporters and dCas9 transcriptional regulators that are responsive to intracellular AcCoA levels. Protein variants that are activated by different levels of AcCoA will be designed to provide a broader range of dynamic control of pathway efficiency. AcCoA dynamic regulators will be deployed in both E. coli and Y. lipolytica to show its broad utility. The tools developed here could be transferred to other organisms, as directly demonstrated in Y. lipolytica, and used to address fundamental questions about control and regulation of metabolism in numerous cell types. This proposed effort, therefore, has the potential to broadly impact a wide range of AcCoA derived molecules relevant to onshoring critical supply chains. 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. NSF Award ID: 2540435 | Program: 01002627DB NSF RESEARCH & RELATED ACTIVIT | Principal Investigator: Mark Blenner | Institution: University of Delaware, NEWARK, DE | Award Amount: $1,000,000 View on NSF Award Search: https://www.nsf.gov/awardsearch/show-award/?AWD_ID=2540435 View on Research.gov: https://www.research.gov/awardapi-service/v1/awards/2540435.html