Streptomyces Autoregulator Biosensors from Natural Product Cluster-Situated Regulators

The soil dwelling bacteriaStreptomycesis a prolific producer of bioactive 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 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 (10¹-10³ fold), AR affinity (0.03-111 μM), and AR selectivity. We examined crosstalk between noncognate CSRs and ARs, as well as the ability of CSRs to regulate alternative operators. Additionally, we established that these Escherichia coli chassis biosensors can be cocultured with Streptomyces for rapid analysis of AR production. Finally, we demonstrate that the AR biosensors can be combined to create 12 orthogonal signaling systems in E. coli coculturing or multi-input genetic circuits. In the long term, these AR biosensors will contribute to the elucidation of small molecule quorum sensing employed by Streptomyces for NP regulation. Additionally, these autoregulator-based biosensors provide ultrasensitive, robust-output platforms that can be adapted for quorum-sensing-regulated genetic circuits.