Repurposing the CRISPR-Cas9 system for dynamic control of cellular metabolism

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This project addresses a challenging problem in synthetic biology, namely the organization of metabolic enzymes into a sequential multi-enzyme cascade in order to improve the overall pathway performance. The approach used in this project is to repurpose the CRISPR (Clustered Regularly Interspaced Short Palindromic Repeat) Cas9 system as a generalizable platform for site-specific enzyme assembly and to demonstrate its utility for metabolic engineering and synthetic biology applications. The two collaborators offer complementary expertise in pathway engineering and protein engineering in order to pursue a highly innovative design concept to achieve this goal. The students trained on this project will be exposed to multi-disciplinary training in the context of research that is anticipated to significantly advance synthetic biology capabilities. The goal of this project is to develop a set of orthogonal catalytically inactive Cas9 (dCas9) proteins as a new tool for in vivo assembly of enzyme cascades in order to redirect cellular resources as desired. The end result is the ability to provide dynamic tuning of endogenous processes to balance the demands of cell health and pathway efficiency. This will be achieved by combining metabolite-responsive dCas9 expression and controlled dCas9 degradation to provide both fast sensing and actuating ability into an integrated synthetic design in order to modulate the overall output function. The investigators will demonstrate the feasibility of this capability by designing multi-enzyme cascades, one using the well-known mevalonate pathway and the other using the conversion of methanol to lactate. The ultimate goal is to combine the knowledge gained as a transformative approach toward the design of dynamic enzyme cascades for any metabolic pathway of interest.This award was co-funded by the Systems and Synthetic Biology (SSB) program in the Molecular and Cellular Biosciences (MCB) Division in the Biological Sciences Directorate and the Biotechnology and Biochemical Engineering (BBE) program of the Division of Chemical, Bioengineering, Environmental and Transport Systems (CBET) in the Engineering Directorate.