Research On The Application Of Screening Ansamycins And Antibiotic Producers In Actinomycetes By The Conserved AHBA Synthase Gene Cluster

Biosynthetic studies of ansamycins indicate that the 3-amino-5-hydroxybenzoic acid (AHBA), which comprises the core structure (mC7N unit) of all ansamycins, is the common specific starter unit for ansamycin biosynthesis, followed by the sequential addition of extender units as acetate, malonate, and methoxymalonate to form a polyketide backbone catalyzed by type I polyketide synthsase (PKS), which then undergoes further downstream processing.According to the conserved regions of AHBA-KLM gene clusters involved in ansamycin biosynthesis, different sets of degenerate primers were designed and a PCR approach was used to detect AHBA-KLM gene clusters within the genomic DNA of 33 newly isolated actinomycete which were found harboring AHBA synthase genes.Then twenty two of them have the same gene-organization character with ansamycin producers. At the same time, all these newly obtained ABBA-KLM genes cluster were sequenced and submitted to GeneBank, and an unrooted neighbor-joining phylogenetic tree was constructed. Sequence analyses showed they exhibited more than 70% identity to each other. Therefor an universal targeting vector could be constructed which provided a rapid approach to mining the potential ansamycin producers from nature source.Universal L gene replacement vector with KanR gene selectable marker was constructed in KLM homologous regions by Red/ET recombination. As described this universal vector allows targeting L gene in 9 representative ansamycin potential strains. Thus it obviated the need for construction of double crossover recombination vector for each individual strain.All the gene replacement mutants produced less compounds than parent strains in the same fermentation condition. So this implied that the inactivated gene products were involved in the production of these compounds. In the end 3-20-1,3-27-1,24-100(18min),7-32(23min) and 8-32(23min) were the probably rifamycin-like products and 3-20-2,3-27-2,4-124(21min), 24-100(21min),4353(32min),4088(26min),24-59(14min),7-32(24min) and 8-32(24min) were the probably geldanamycin-like products by multi-measure analyse. And further chemical purification were done for defining their structure,4088(26min) and 3-27-1 were geldanamycin by LC-MS and rifamycin-SV by ESI-MS respectively.The work described here provided a rapid approach to mining the potential ansamycin producers from nature source. And it also might provide a new way for discovering AHBA-derived compounds with novel structures.