(27bl) Leveraging the Carbon Storage Regulatory Network to engineer Complex Post-Transcriptional Gene Circuits

Historically, bacterial gene circuits have primarily relied upon transcriptional regulatory elements; including synthetic promoter libraries, engineered transcription factor proteins, and CRISPR-based systems. However, there is now a push to develop similar gene circuits using post-transcriptional regulatory systems as both maximize system response time and minimize cellular resource competition. Yet, there are few post-transcriptional systems that offer dynamic control over multiple heterologous targets, and bi-directional regulatory outcomes. With this in mind, the Carbon Storage Regulatory (Csr) Network of Escherichia coli serves as an ideal scaffold system to address current limitations, as it activates and represses hundreds of mRNA targets natively, as well as rapidly responds to environmental signals. Herein, we leverage the native Csr Network of Escherichia coli to create a semi-synthetic Buffer gate, and NOT gate for heterologous target regulation. We expand upon our initial designs to create a genetic toolbox for precise titration of gene expression over a 10-fold range. Additionally, we develop the OR, AND, NOR, and NAND multi-input logic gates derived from the original Buffer and NOT gates. Next, we apply this toolbox to optimize mevalonate production in E. coli as a downstream proof-of-concept. With our semi-synthetic gates, we produce mevalonate at titers five-fold greater than those using transcriptional control. Lastly, as the Csr Network is conserved throughout the Gammaproteobacteria class, we demonstrate these genetic circuits can be transferred across multiple organisms by leveraging the native components of the Csr Network within each organism.