Secondary metabolism in Streptomyces species is often extremely complex and heterogeneous. These bacteria can biosynthesize many bioactive compounds, of which many display antibacterial activity. Rapid identification of gene cluster and their annotation in Streptomyces genomes requires development of novel methods for gene manipulation. Gene manipulation methods and characterisation of biosynthetic pathways is on of approaches which can be applied for identification of novel bioactive compounds with interesting activities. Our work was carried out on S. rapamycinicus, known as producer of rapamycin, and we aimed to evaluate different approaches in gene disruption of known gene clusters present in S. rapamycinicus, such as rapamycin, elaiophylin, nigericin. Methods of molecular biology were used to design CRISPR-Cas based tools for DNA rearangement and plasmids for homologous recombination were constructed. We completed succesful deletion of the parts of gene clusters encoding rapamycin, elaiophylin or nigericin. Double rapamycin-elaiophylin knock-outs were also completed. During our work we also managed to prove funcionality of gusA reporter system in S. rapamycinicus as a tool for efficient genetic engineering.
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