Directed Evolution of Quorum-Sensing Dependent Transcriptional Repressors for Programmed Intercellular Communication

One of the key challenges in the field of synthetic biology is to engineer not only single cells but also communities of cells that exhibit coordinated, population level behaviors. Synthetic biologists have employed components from acyl-homoserine lactone (AHL)-based quorum-sensing (QS) systems in their endeavors to build muticellular systems. To date, the regulators available in the toolbox of QS regulatory parts for engineering intercellular communication have been limited to transcriptional activators, most frequently the AHL-dependent activator LuxR. Transcriptional activators function by recruiting RNA polymerase (RNAp) to a promoter sequence and thus require specific interactions with RNAp. In contrast, transcriptional repressors bind to operator regions within a promoter and inhibit the initiation of transcription. To expand this toolbox of regulatory parts, we have engineered the AHL-dependent transcriptional repressor, EsaR, for use in synthetic microbial communities. Characterization of wild-type EsaR showed that it requires micromolar concentrations of AHL to achieve complete derepression, while the LuxR will activate gene expression in the presence of nanomolar concentrations of AHL. In order to generate QS repressors with sensitivities comparable to LuxR, we built libraries of esaR mutants and identified EsaR variants with increased AHL sensitivity using an ON/OFF screening system. We first screened for an absence of gene expression to identify EsaR variants that retained the ability to repress gene expression (OFF screening). We subsequently screened for reporter gene expression in the presence of nanomolar concentrations of AHL (ON screening). We have identified a number of EsaR variants that respond to a range of AHL concentrations, from 10 nM to 1 uM. These new regulatory parts will enable future work exploring the roles of different regulatory mechanisms and network architectures for intercellular communication in both natural and engineered multicellular systems.