The Regulatory Mechanism Of Orf17 Gene In The Biosynthesis Of Geldanamycin In Streptomyces Autolyticus

Natural products produced by microorganisms play a very important role in human health.The diverse biological activity and richness of chemical structure of natural products provide good opportunity for developing new drugs.Streptomycetes are an important source of natural products.With the rapid development of DNA sequencing technology and genomics,more and more biosynthetic gene clusters for natural products are found in the genomes of Streptomycetes,which provides rich resources for studying their biosynthesis.Geldanamycin(GM),produced by Streptomyces autolyticus,possesses potent antitumor activity.GM also has good antivirus and immunomodulation activities.Studies show that geldanamycin targets Hsp90 by binding to the N terminal ATP/ADP domain of Hsp90,and specifically inhibits the ATPase activity of Hsp90,which in turn perturbs the signaling pathways of tumor cells.However,some details of geldanamycin biosynthesis and its regulatory mechanism are unclear.To further understand the regulatory mechanism of geldanamycin biosynthesis in Streptomyces autolyticus,the function and regulatory mechanism of a putative MarR family regulator,Orfl7,were studied in this work.The fermentation products of the wild-type strain and the orf17 mutant were analyzed.The production of geldanamycin in the orf17 mutant decreased,which suggested that orf17 gene might play a positive role in the biosynthesis of geldanamycin;wild-type orf17 gene was cloned and introduced into the orf17 mutant for complementary analysis to further confirm the regulatory role played by orf17 in the biosynthesis of geldanamycin;RT-PCR and Real-time PCR were used to analyze the expression of genes involved in the geldanamycin biosynthesis in the orf17 mutant,and it was found that the expression of almA ?,almF,almM,almH,almR?,almR?,and almR? decreased,implying that these genes are regulated by Orf17,directly or indirectly;Finally,the Orf17 protein was expressed and purified,and EMSAs were conducted to find the target gene(s)of Orfl7 protein.The above studies would help us to further understand the mechanism of regulation of geldanamycin biosynthesis in Streptomyces autolyticus and would lay the foundation for increase the productivity of geldanamycin and its analogs.