| Recently, the abuse of antibiotics leads to serious drug resistance to many pathogens, whereas the selection of new kinds of antibiotics and discovery of its biosynthesis mechanisms contributes to reveal and provides rich resources for metabolic pathway engineering strategies.Bagremycin A and B,novel antibiotics from Streptomyces sp.Tii4128, show a moderate activity against fungus,Gram-positive bacteria and tumour cell and have potential application values in the fields of medicine and agriculture.In the present study, firstly, we obtained bagH、bagI and bagM deletion mutants, respectively,by using in-frame deletion,which were proved correctly in DNA and RNA levels. The result of HPLC/TOF-MS revealed that bagI was related to Bagremycin biosynthesis. Restoring Bagremycin production via bagI complementation.Secondly,the impacts of bagI mutants on mycelial growth,spore formation and pigment yields were explored throughout secondary metabolism.Results of scanning electron microscope displayed that bagI mutation delayed the hyphae growth and spore development,reduced pigment yields. However, the overexpresssion strain yields of Bagremycin A and Bagremycin B increased2.5-fold and2.6-fold in contrast to wild strain respectively.Thirdly,we investigated genes (un)related to Bagremycin biosynthesis by using real-time fluorescent quantitative PCR(Q-PCR).The result showed that bagI played the role of transcriptional activator in the course of Bagremycin biosynthesis,and the results of NCBI blast (SARP family transcriptional activator,51%identity) and sequence analysis gives a hint on this point.In the end,BagI protein was expressed in E.coli and certified by western blot.Based on it,EMSA was done. Although the desired results was not achieved,but it playes a certain implications for our understanding of the functions of BagI protein. |
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