Cloning And Characterization Of The Citreamicins Biosynthetic Gene Cluster From Micromonospora Citrea NRRL18351

The citreamicins antibiotic is isolated from the fermentation liquid of Micromonospora citrea in1989by Carter. It belongs to the xanthene ketones antibiotics has important biological activity and can inhibit Gram-positive bacteria and anaerobic bacteria. In this paper, by extracting Micromonospora citrea NRRL18351total DNA, with the Sau3AI partially digested total DNA to obtain the desired size of the object fragments DNA at35-40kb, then connected with the carrier pJTU2554and packaging, transfection to the Escherichia coli EPI300, constructed a genomic library contains Micromonospora citrea NRRL18351gene fragment. Then, according to the antibiotics lysolipin, xantholipin and kigamicins which have been reported are similar to Citreamicins chemical structure. To design the degeneracy primers through homologous comparison. This primer using total DNA Micromonospora citrea NRRL18351as template for PCR amplification, homologous fragment can be obtained. Therefore, using this probe on the gene library screening (1996-well plates), screened out of seven positive clones were named1A3,1E5,7D1,11B12,15B8,16D4,19D8. In addition, in order to design degenerate primers to amplify the fragment Micromonospora citrea NRRL18351the genome and recovering as a labeled probe for the screening out of the seven positive clones were hybridized DNA. Meanwhile, screening out the seven positive clones were heterologously expressed to the expression strain S.lividanse K4by protoplast transformation method, but out of the transformed strains, and by culture fermentation broth were not detected in the produce Citreamicins antibiotic components, so guess which seven positive clones could not complete the synthesis Citreamicins antibiotic gene clusters or heterologous expression arising Citreamicins antibiotic production is too low to be detected.Therefore, in order to further explore whether these seven positive clones containing Citreamicins antibiotic biosynthetic gene cluster, the seven positive clones were digested with StuI, and using T4ligase occurred since their company and transformed into DH5a and Pick out the vector plasmid was self connected. And then the plasmid was digested with SpeI and XbaI and the objective fragment were recovered. The fragment was connected to the vector pJTU412, after the screening connected to the purpose of plasmids, the plasmid was digested by StuI and connected on Apra resistance gene as a selectable marker. Eventually constructed three knockout vectors are Pllx4, Pllx5and Pllx6. By conjugation, these three vectors and Micromonospora citrea NRRL18351knockout experiments was done in order to determine whether these plasmids which contain Citreamicins antibiotic synthetic gene clusters.