Title: DAP-Seq Reveals Cluster-Situated Regulator Control of Numerous Streptomyces Natural Product Biosynthetic Genes

Natural products (NPs) are a rich source of therapeutic and agricultural compounds. Unfortunately, many promising metabolites are not expressed under the standard laboratory conditions. Deepening our understanding of the regulatory networks governing NP biosynthetic genes is essential to unlocking this hidden chemical diversity. One of the most common regulators of NP expression are TetR-family repressors encoded within NP biosynthetic gene clusters (BGCs). These repressors inhibit transcription of NP biosynthetic genes until interaction with their cognate small-molecule quorum-sensing ligands relieves this repression. While a small number of quorum-sensing cluster-situated regulators (qsCSRs) are known to control NP biosynthesis, the full extent of their regulatory scope has remained unclear due to limited genome-wide data. Using DNA Affinity Purification Sequencing (DAP-seq), we defined the predicted regulons for 84 qsCSR homologues across 78 Streptomyces strains. qsCSRs in this cohort exert influence across multiple cellular processes, with particularly strong impacts on other transcription factors throughout the genome. Approximately 30% of predicted NP BGCs contained qsCSR-regulated genes. In strains encoding multiple qsCSRs, we observed substantial overlap in the BGC regulation. Together, these results greatly expand the genomic landscape of qsCSR activity and provide a foundation for improved bioinformatic strategies to predict and interpret the regulatory control of NP biosynthesis.