Quorum-sensing repressor-based tools for cell-cell communication in synthetic biology

Microbial communities that exhibit coordinated, population-dependent behaviors have been engineered for use in applications of biotechnology, therapeutics, bioprocessing, and synthetic biology. Intercellular communication enables these mixed cell populations to receive foreign signals, transfer into metabolic activities, and form interactions with each other. This process requires reliable communication pathways that function in single populations and also multi-species communities. Synthetic biology provides the platform to engineer biological systems at every life form. Biological components, like DNA, RNA, and proteins, are treated as parts and can be integrated to form novel systems with non-native functions. Therefore, natural intercellular communication systems can be modified or provide components to build artificial communication pathways such that communication is enabled in unique combinations of cell populations. However, limitations often arise upon the construction of modules due to a lack of available, well-characterized "tools" in synthetic biology. Quorum sensing (QS) is a type of cell-cell communication in bacteria via exchange of small chemical molecules. QS-mediated functions are regulated by QS transcription regulators, acting as activators or repressors, in response to the QS signal molecules as a function of local cell population. QS activators require direct interactions with RNA polymerase (RNAP) for gene activation and can only function with specific organization of the promoter. Unlike activators, QS repressors are "derepressed" by signal molecules and then released from the promoter sites, to allow binding of RNAP to initiate gene expression. Thus, QS repressors do not require interactions with RNAP. This thesis focuses on engineering the esa system, from Pantoea stewartii, where the cognate QS regulator, EsaR, can act as a repressor. The availability of QS repressors will enable construction of new modules, such as negative feedback, that are not feasible with the currently existing parts. Via directed evolution, we engineered the wild-type EsaR protein to increase the range of sensitivity to its native signal. We have also generated a new set of EsaR-dependent promoters that exhibit altered transcriptional levels and AHL-dependent responses. Taking advantage of the AHL-dependent DNA-binding activity of EsaR, we have built synthetic AND gates driven by both an extracellular signal and cell density. These new transcriptional repressors and promoters will enable construction of novel artificial density-dependent or multicellular systems for metabolic engineering applications.