The Regulatory Roles Of AlmR Ⅰ And AlmR Ⅱ In The Biosynthesis Of Autolytimycin In Streptomyces Autolyticus

In recent years, targeting heat shock protein Hsp90 is becoming one of the most exciting fields in cancer research. Geldanamycin (GM) and several closely related compounds that target Hsp90, classified as benzoquinone ansamycin antibiotics, have drawn increasing interest. GM and its analogues inhibit the ATP-dependent functions of Hsp90, which causes rapid degradation of some key proteins in cancer cells and thus leads to cytostatic or apoptosis of cancer cells.In our previous work, we discovered a new GM analogue, called autolytimycin, from the secondary metabolites of a new species of actinomycetes-Streptomyces autolyticus. Autolytimycin has been shown to possess potent anti-cancer activity. Although promising as an anti-cancer drug, the lack of knowledge on the biosynthesis of autolytimycin severely hampered the work trying to increase its yield or generate novel structural related compounds through genetic engineering. To understand the regulation of autolytimycin biosynthesis in Streptomyces autolysis, we studied the function of two LuxR-family regulators AlmRⅠand AlmRⅡin this work.almRⅠand almRⅡgenes were PCR amplified by using the chromosomal DNA of Streptomyces autolyticus as template, and then cloned into an expression vector pET28a. AlmRⅠand AlmRⅡwere expressed, and AlmRⅠprotein was purified. To exploit the cellular target of AlmRⅠ, bioinformatics analysis was performed on the gene cluster responsible for autolytimycin biosynthesis. Several genes that are postulated to be involved in the biosynthesis of autolytimycin were found to contain the possible binding site of LuxR-family regulators. The promoter regions of these genes were PCR amplified, and then EMSA was carried out to study the binding of the purified AlmRⅠprotein to these regions. The results showed that AlmRⅠcould bind to the promoter region of almK gene when supernatant from the cultures was added, suggesting that AlmRⅠprotein may regulate the expression of almK gene in the presence of an unkown inducing signal. Meanwhile, the lack of binding of AlmRⅠto the promoter of almM, almF and almO genes under similar conditions indicated that AlmRⅠmight not be involved in regulating the expression of these genes.The purpose of this study is to understand the function of almRⅠand almRⅡin the process of autolytimycin biosynthesis, which would shed lights on the regulatory mechanism of autolytimycin biosynthesis. This work provided an entry point for further studies that aim to increase the production of autolytimycin or generate novel compounds through genetic engineering.