The Molecular Regulatory Mechanism Of The Phosphate Two-component System On Daptomycin Biosynthesis In Streptomyces Roseosporus

Phosphate can function as the structural components of cells,and act as the signaling molecules for metabolism and its regulation.The morphological development and secondary metabolism for production of a number of antibiotics,pigments and immunomodulators are regulated by phosphate.The two component PhoR-PhoP system can respond to the low concentration of inorganic phosphate,and control the biosynthesis of series of secondary metabolites through intracellular signal transduction pathways.Streptomyces roseosporus has become the research highlights as the producing strain of daptomycin.However,researches on the regulatory biosynthesis of daptomycin by PhoR-PhoP have not yet been reported.We have found that PhoR-PhoP could positively regulate the production of daptomycin in S.roseosporus L30.The biosynthesis of daptomycin reduced to 1/20 of the parental strain after deletion of phoRP.Electrophoretic mobility shift assay(EMSA)showed that PhoP,the regulator of PhoR-PhoP system,could bind to the promoter of atrA,qRT-PCR data showed that the expression level of atrA in AphoRP mutant was much lower than wild type,which indicated that PhoR-PhoP system positively regulates daptomycin biosynthesis via AtrA.The PhoP,as an auto-regulator,could bind to its own promoter on the PHO boxes and positively regulate the expression of its own operon,suggested that PhoR-PhoP had a positive feedback regulatory mechanism,and continuously positively regulated the biosynthesis of daptomycin in secondary metabolism development.Our previous work has proved that pleiotropic regulator AdpA of A-factor signaling pathway can also bind to atrA promoter.The EMSA assays showed partial competitive relationship between Phop and AdpA.The binding sites of PhoP and AdpA were determined by DNase I footprinting assays,and 3bp overlapping region was identified.Moreover,GFP reporter system showed that PhoR-PhoP negatively regulated the gene expression of adpA,while the expression of phoRP was not influenced by AdpA.This article,with daptomycin as a model,for the first time showed the phosphate signaling pathway in regulating the biosynthesis of secondary metabolites.It is also revealed that PhoR-PhoP can strengthen its own regulation intensity through the positive feedback regulatory mechanism,and could perform cross-talk with hormone-triggered pathway at transcriptional and protein levels to harmonize the secondary metabolism development of Streptomyces.