Functional Analysis Of Transcriptional Factors AveT, AfsR And AfsS In Streptomyces Avermitilis

The important industry strain Streptomyces avermitilis produces avermectins, which are widely applied in the medical, agricultural, and veterinary fields. However, the detailed regulatory mechanisms of avermectin production remain unclear. In this work, the regulatory functions and mechanisms of a new TetR-family transcriptional regulator AveT and a global regulator of Streptomyces, AfsR, were studied. The investigations are important to help clarify the complex regulatory network of avermectin biosynthesis and provide the basis for rational construction of avermectin overproducers for industrial production.aveT （sav₃619） encodes a putative TetR-family transcriptional regulator. Shake-flask fermentation and morphological observation of aveT gene deletion, complementation, and overexpression strains implied that AveT regulates avermectin biosynthesis and morphological differentiation positively. RT-qPCR, EMSA and DNase I footprinting assays revealed that AveT indirectly stimulates avermectin production by affecting transcription of pathway-specific activator gene aveR. AveT directly repressed transcription of its own gene aveT, adjacent gene pepD2 （sav₃620, encoding a putative tricorn core peptidase）, ave M （sav₇490, encoding a putative transmembrane efflux protein） and sav₇491 （encoding a hypothetical protein） by binding to an 18 bp perfect palindromic sequence CGAAACGKTKYCGTTTCG （K:T or G; Y:T or C） within their promoter regions. AveT homologs are widely distributed among Streptomyces species. Overexpression of aveT gene in S. coelicolor M145 also stimulated antibiotic production and morphological differentiation, suggesting the general role of AveT homologs in control of antibiotic biosynthesis and morphological differentiation in Streptomyces. pepD2 had no effect on avermectin production, whereas aveM, the important target gene of AveT, had a striking negative effect on avermectin production and morphological differentiation. Overexpression of aveT and deletion of aveM in industrial strain A-178 provided by Qilu Pharmaceutical led to 22% and 42%increase of total avermectin yield in flask,35%and 48%increase of effective component B1a production in 100 L fermentor, indicating that manipulation of aveT and aveM expression is an effective strategy to improve avermectin production. EMSA results showed that C5-O-B1, the pathway precursor of avermectin B1, acts as an AveT ligand. Our findings indicate a positive-feedback regulatory mechanism of aveT expression and avermectin production by C5-O-B1 that ensures the irreversible production of avermectins and their appropriate concentration in cells.afsR encodes the response regulator of a conserved eukaryotic-type two-component system AfsK/R in Streptomyces. In S. coelicolor, AfsR positively regulates antibiotic biosynthesis and has cross regulation with phosphate metabolism and nitrogen metabolism. Fermentation and morphological observation of afsR gene deletion, complementation, and overexpression strains in 5. avermitilis implied that AfsR inhibits avermectin biosynthesis and morphological differentiation. This is the first report that AfsR negatively regulates antibiotic biosynthesis, suggesting the regulation mechanism of AfsR in S. avermitilis is different from that in model S. coelicolor. RT-qPCR, ChIP and EMSA assays showed that AfsR indirectly inhibits avermectin production by affecting transcription of aveR and directly activates transcription of afsS, avaRl and aco by binding to their promoter regions. EMSA results suggested that afsR may be directly regulated by B1dD. afsS is the important target gene of AfsR. In contrast to the positive regulatory function of AfsS on ACT and RED biosynthesis in S. coelicolor, fermentation of afsS gene deletion, complementation, and overexpression strains implied that AfsS negatively regulates avermectin biosynthesis. Immunoprecipitation assay suggested that SAV5905 and SucB （SAV6022） may interact with AfsS and this result requires further investigations.