DAP-seq Reveals Cluster-Situated Regulator Control of Numerous Streptomyces Natural Product Biosynthetic Genes

Abstract Natural products (NPs) are a rich source of therapeutic and agricultural compounds. Unfortunately, many promising metabolites are not expressed under standard laboratory conditions. Deepening our understanding of the regulatory networks governing NP biosynthetic genes is essential for unlocking this hidden chemical diversity. Cluster-situated regulators (CSRs) are transcription factors involved in the regulation of NPs, but their full regulatory range has remained elusive due to limited genome-wide data. Using DNA Affinity Purification Sequencing (DAP-seq), we defined the predicted regulons for 84 CSR homologs across 78 Streptomyces strains. CSRs in this cohort exerted influence across multiple cellular processes, with particularly strong impacts on other transcription factors throughout the genome. Approximately 30% of predicted NP biosynthetic gene clusters (BGCs) contained CSR-regulated genes. In strains encoding multiple CSR homologs, we observed substantial overlap in BGC regulation. Together, these results greatly expand the genomic landscape of CSR activity and provide a foundation for improved bioinformatic strategies to predict and interpret regulatory control of NP biosynthesis.