Transcriptional regulatory network analysis and its application for increasing antibiotics production in Streptomyces coelicolor

Abstract

Transcriptional regulatory networks in Streptomyces coelicolor A3(2) have evolved to execute various cellular processes affecting antibiotic production. Several key transcriptional regulators involved in biosynthesis of secondary metabolites have been characterized, including ActII-ORF4, RedD, and CdaR. However, new regulators are still needed to be identified to understand the complex transcriptional regulatory networks delineating antibiotic production. Here, we identified a list of regulators controlled by AbsB acting as a higher level regulator in the antibiotic production. These potential regulators were identified to show different expression patterns in the absence of absB gene. RNase cleavage assay revealed that sco6808 transcripts, one of the potential regulators, are cleaved by AbsB. This result indicates that sco6808 is a direct target of AbsB. Therefore, our approach provides a comprehensive list of novel regulators controlled by AbsB, which serves as a basis for understanding multi-layered regulatory networks. Manipulation of multiple transcription regulators controlling non-redundant pathways related to antibiotics production would effectively increase the antibiotics production. Here, we present an approach that uses a combination of two transcriptional regulators that control independent pathways to increase the antibiotics production. AfsS is one of key master activators of antibiotics production. Using the microarray data of afsS disruption mutants, we first selected the regulators transcriptionally independent of afsS. Among them, we focused on sco4677, an antagonistic regulator of sigma factor F, and sco4228 (phoU), a phosphate transport system regulator. Using the combination of sco4677 with afsS (BG4677S) and phoU with afsS (BG4228S), we observed the intracellular actinorhodin production increased in BG4677S by approximately 11-folds higher and BG4228S by approximately 149-folds higher than wild type. Time-course microarray experiments were performed using ndgR deletion mutants for expanding the fragmented transcriptional regulatory network. Previously, NdgR was identified by DNA affinity capture assay (DACA) and mass spectrometric analysis as a global regulator that controls amino acids synthesis, quorum sensing and antibiotics production. In addition, we unveiled that NdgR also controls ABC transport system of glutamate, branched chain amino acid, phosphate and methionine, carbohydrate uptakes, carbohydrate metabolism in PTS, glycerol metabolism, cell stress response, and tolerance to toxic materials. Those roles were confirmed by EMSA, cell viability test, and the observation by confocal microscope and TEM images. From these results, we could accumulate the information about the role of the global regulator, NdgR, to the incomplete transcriptional regulatory network in Streptomyces coelicolor. In conclusion, we attempted to identify the transcriptional regulatory network and developed a capable method to increase the antibiotics production.