Myronovskyi M. Systems for gene cloning, inactivation and expression analysis in streptomycetes

Object of the research: systems for gene cloning, inactivation and expression in streptomycetes. The aim of the work: development of a new system for gene activity determination and expression regulation in streptomycetes, development of new effective system for gene cloning in S. sioyaensis Lv81. Methods: microbiological, molecular genetic, biochemical, mathematical statistics. Based on the beta-glucuronidase gene several vectors for the visual selection of gene-knockout streptomyces strains and studying of gene transcriptional and translational levels were created. A new system for regulated gene expression in streptomycetes based on archaebacteria Methanosarcina acetivorans genes which permit translation of UAG stop-codon was established. The systems for conjugal DNA transfer, gene inactivation and transposon mutagenesis in S. sioyaensis Lv81 were developed. It is shown that inactivation of either of sioA, sioB, sioG, sioH, sioJ, sioN, sioO, sioQ and sioS genes of S. sioyaensis Lv81 leads to the cessation of siomycin production. For primary metabolism genes which are involved in biosynthesis of siomycin have been found. According to our results, genome of S. sioyaensis Lv81 contains NRPS genes presumably involved in six different biosynthetic strategies. A new system for the detection of thiopeptide antibiotic producers was established. Area of use: applied genetics, biotechnology, microbiology.