The Relationship Of Two Similar Regulators SAV576and SAV577for Avermectin Biosynthesis And Characterization Of SAV4189in Streptomyces Avermitilis

The important industry strain Streptomyces avermitilis produces avermectins, which are widely used in agriculture and veterinary. The complex regulatory mechanisms of avermectin production remain poorly understood. In this work, several genes which could control avermectin production were studied to help understand the complex regulatory network of avermectin biosynthesis and construct avermectin high producer through genetic manipulation.In previous work, transcription profiles of wild-type S. avermitilis ATCC31267and avermectin high producer76-02-e were determined by agilent expression microarray. In this study, Analysis of microarray data revealed162up-regulated and150down-regulated genes (2-fold or more in76-02-e relative to ATCC31267at2and6days in the process of fermentation). RT-qPCR was used to test some selected genes and the PCR results were mainly consistent with the microarray data.SAV576and SAV577are both TetR family transcriptional regulators, and SAV577is similar to SAV576. Based on previous work, the transcriptional start sites of SAV575and SAV576(target genes of both SAV576and SAV577) were determined by5’RACE assays and the promoter structures of SAV576and SAV577were predicted. DNase I footprinting assays revealed that SAV576and SAV577had the same binding sites on the bidirectional SAV575-SAV576promoter region, indicating the similar functions of SAV576and SAV577:they both indirectly downregulate avermectin biosynthesis by directly repressing the expression of SAV575which encodes a putative cytochrome P450/NADPH-ferrihemoprotein reductase. EMSA results showed that SAV576and SAV577proteins compete for the same binding regions, and the DNA binding affinity of SAV576is much stronger than that of SAV577. GST pull-down assays revealed that SAV576or SAV577interacts with itself to form a homodimer, but SAV576does not interact with SAV577to form a heterodimer in v/vo.These findings lead to the possibility that SAV576plays a dominant role over SAV577in repressing target genes including SAV575, and in inhibiting avermectin production in S. avermitilis.SAV4189, which encodes a putative MarR family transcriptional regulator, was upregulated most significantly in76-02-e according to the microarray data. Determination of avermectin production in SAV4189gene deletion, complementation, and overexpression strains implied that SAV4189regulates avermectin biosynthesis positively. RT-qPCR and EMSA assays revealed that SAV4189indirectly upregulates avermectin production and directly represses the expression of the transcriptional unit SAV4189-SAV4190(encodes a putative major facilitator superfamily efflux pump). The transcriptional start site of SAV4189was determined by5’RACE assay, and the promoter structure of SAV4189was thus predicted. DNase I footprinting assays showed that the protected regions of SAV4I89protein overlap the potential-10and-35regions of the SAV4189promoter, indicating that SAV4189negatively regulates SAV4189-SAV4190transcription by blocking the access of RNA polymerase to its promoter region. EMSA assays revealed that some antibiotic molecular such as hygromycin B, thiostrepton, bacitracin and ampicillin, could inhibit the DNA binding activity of SAV4189protein. RT-qPCR analysis showed that hygromycin B and thiostrepton, not bacitracin and ampicillin, could induce the expression of SAV4189-SAV4190in S. avermitilis. These findings indicate that hygromycin B and thiostrepton can act as ligands of SAV4189protein. Comparison of avermectin production in wild-type strain and SAV4189deletion mutant in the presence or absence of hygromycin B suggested that SAV4189is an important gene involved in the connection with response of environmental antibiotic stress and avermectin production. ChIP-Seq, EMSA and RT-qPCR assays revealed that SAV4202(encodes a putative secreted protein)and SAV4203(encodes a putative peptidoglycan-binding protein) are target genes of SAV4189, and the relationship between these genes and avermectin biosynthesis should be further explored. Deletion of SAV4190, the target gene of SAV4189, resulted in increased avermectin production, which was consistent with decreased avermectin production and upregulated SAV4190expression in SAV4189deletion mutant, suggesting that SAV4190gene product has a negative effect on avermectin biosynthesis by transporting some yet-unknown substrate(s).