Orthogonal quorum sensing circuits enable dynamic regulation in Escherichia coli

Engineers have effectively employed quorum sensing (QS) in a variety of applications to dynamically regulate gene expression. Particular emphasis has been placed on the class of well-studied systems that use acyl homoserine lactones (AHL) as signaling molecules due to their ease of implementation, high expression level, and previous optimization efforts. However, many of these AHL systems respond to ligands with similar structures, causing crosstalk when combined in multi-layered regulation strategies. Here, we first confirmed the functional orthogonality of the previously identified Tra and Rpa quorum sensing circuits within a single strain of Escherichia coli MG1655 by analyzing the pairwise interactions of several AHL systems. The orthogonality of the systems allowed for independent tuning of two control strategies, which was then applied to the naringenin biosynthetic pathway. The Tra system was used to activate expression of tyrosine-ammonia lyase (TAL) and 4-coumaroyl-CoA ligase (4CL), controlling the expression of the upstream pathway. Meanwhile, Rpa dynamically downregulated competing pathways of native metabolism via CRISPRi to increase availability of malonyl-CoA. This multi-layered approach provided finely-tuned metabolic control that allowed for a combinatorial screening of optimal dynamic regulation. A strain library with varying promoter strengths was then built to test AHL induction timings and screened for target compound production. Naringenin production from this autoinducible method reached a final titer of 71.02 ± 3.96 mg/L in flask-scale fermentation.