Sreening Of A Transcriptional Regulator DepR1 And Its Regulatory Mechanism On Daptomycin Biosynthesis In Streptomyces Roseosporus SW0702

Daptomycin is a cyclic lipopeptide antibiotic produced by Streptomyces roseosporus. It has a cyclic lipopeptide with 13 amino acids and a straight chain C10 fatty acid. Daptomycin can bind to the cell membrane of Gram-positive bacterial pathogens, and cause the release of the intracellular ions. Due to its cyclic lipopeptide structure and anti-bacterial activity, daptomycin has broad application prospects. Current research of daptomycin mainly focuses on the functional characterization of its structure genes and daptomycin structure modification based on combinational biosynthesis, chemo-enzymetic and genetic engineering. However, there is no report on the transcriptional regulation for daptomycin biosynthesis.Daptomycin is synthesized by a non-ribosomal peptide synthetase (NRPS) in Streptomyces roseosporus. The whole daptomycin gene cluster is co-transcribed from the promoter of dptE (dptEp). In this study, we used the DNA affinity technology combined with LC/MS/MS to isolate regulators which might directly interact with dptEp,82 proteins were identified, including DepRl, a TetR-family transcriptional regulator. EMS A assays showed that DepRl directly bound to dptE promoter and the binding sequence was determined by DNase I footprinting. Using eGFP reporter system, we found that DepRl could positively regulate dptEp activity. Wild type, depRl mutant and depRl over expression strain were fermented in YEME medium containing 4% glucose, and HPLC was used to detect daptomycin production. Deletion of depRl abolished daptomycin yield, while depRl over expression enhanced daptomycin production compared to the wild type strain. Gene disruption and over expression revealed that DepRl plays an important role in the morphological differentiation in Streptomyces roseosporus. Compared with the wild type strain, depRl mutants showed defect in sporulation, and the complementation strain could restore sporulation. EMSA assays showed DepRl also bound to its own promoter depRlp and DNase I footprinting assay revealed that the binding sites on depRl promoter contain a typical palindromic region. Using eGFP reporter system, we also showed that DepRl was positively auto-regulated.In conclusion, we identified a TetR family transcriptional regulator DepRl by DNA affinity technology, and the interaction between DepRl and dptEp was further confirmed. Meanwhile, we studied the roles of depRl in morphological differentiation, daptomycin production, and transcriptional atuo-regulation in Streptomyces roseosporus, which provides the theoretical foundation for the construction of daptomycin high-yielding industrial strains.