Engineered Gram-Positive Based Quorum Sensing for Metabolic Control in Escherichia coli

Quorum sensing (QS) is a cell-to-cell communication system that allows microbial communities to collaborate and function as a collective. QS functions as a population-dependent regulator by producing signals that scale with cell concentration, allowing surrounding cells to recognize the signal and activate the associated genes at a certain population density. Though many regulatory systems have been characterized, much of the engineering focus has been on a small subset of the expansive QS circuits that exist within nature. To expand the available QS circuits for use in Escherichia coli, two Gram-positive systems were identified as useful candidates: the Agr system, from the therapeutically relevant Staphylococcus aureus, and the Com system, from the model Gram-positive organism Bacillus subtilis. These QS systems were implemented and improved for functionality by modifying the expression strength of circuit components. Each system displayed tight control of their cognate promoters with the Com system reaching a final dynamic range of 2.27 ± 0.05, while the Agr system was improved to a dynamic range of 4.05 ± 0.43. The Agr system was then applied to downregulate endogenous genes tyrA, pheA, trpE, ppc, and pabB via CRISPRi. This regulation strategy allowed for the production of salicylic acid in E. coli MG1655 by diverting metabolic flux toward the target pathway, demonstrating the utility of Agr as a tightly regulated control system in E. coli.