Streptomyces Autoregulator Biosensors from Natural Product Cluster-Situated Regulators

Abstract The soil dwelling bacteria Streptomyces is an abundant producer of numerous anticancer, antifungal, and antibiotic compounds (i.e. Natural Products, NPs). The sophisticated cellular machinery required to produce NPs is frequently regulated by quorum-sensing systems, consisting of cluster situated regulators (CSRs), such as TetR-like repressors, and small-molecule autoregulator (AR) ligands. Only a small fraction of bioinformatically predicted quorum-sensing AR circuits have been experimentally determined, and fewer still have been engineered as inducible expression systems for synthetic biology. This research details the development of eight CSR-based AR biosensors and the synthetic routes to their AR ligands. Overall, the AR biosensors exhibit a range of maximum activation, AR affinity, and AR selectivity. We examined crosstalk between noncognate CSRs and ARs, as well as the ability of CSRs to regulate alternative operators. Additionally, we establish these biosensors can be cocultured with Streptomyces for rapid analysis of AR production. Finally, we demonstrate the CSR-based biosensor vectors can be combined to create orthogonal signaling systems in bacterial coculturing or multi-input genetic circuits. Longterm, these Streptomyces AR biosensors will contribute to the elucidation of small molecule quorum sensing circuits employed by Streptomyces as well as increasing the complexity of genetic circuits used in industrial or agricultural settings.