Cellular metabolism is capable of highly specific and efficient chemical synthesis at mild temperatures and pressures far beyond the capability of most synthetic chemical routes. Engineering specific pathways can be used to further improve the range of compounds that can be synthesized but it is a major challenge to achieve commercially viable productivity. To maximize productivity, it is crucial to fine-tuning pathway fluxes. The goal of this project is to develop a new transformative approach to modulate cell metabolism based on endogenous cellular information. An emerging strategy is the use of regulators that provide dynamic control of pathway fluxes. A recently discovered modified CRISPR based tool offers a unique approach for DNA targeting and transcriptional regulation. These new generation of dCas9 regulators can be used for dynamic gene repression and activation for many synthetic-biology and metabolic engineering applications. A new generation of toehold-gated dCas9 regulators governed by conditional sgRNA structures that are activated by toehold-mediated strand displacement will be created to provide simultaneous, orthogonal, and autonomous control of cellular metabolism. This new framework to design toehold-gated dCas9 regulators responsive to any endogenous mRNA will lay the foundation as a new transformative approach for implementing dynamic control of cellular metabolism.
Project 3. Redirecting cellular metabolism via synthetic toehold-gated dCas9 regulators (NSF)
