Streptomycetes present a medically and industrially important source of metabolites with diverse activities. These bacteria contain numerous biosynthetic gene clusters encoding biosynthesis of numerous secondary metabolites. However, their biotechnological production remains challenging due to their unstable genome and specific life cycle, and apparent inactivity of the majority of biosynthetic gene clusters. Initially, strain optimization was based on random mutagenesis, but today recombinant gene-engineering technologies are also used. One of novel, still experimental approaches to improving a host is the deletion of unwanted biosynthetic gene clusters (GBG) or a larger part of the Streptomyces genome called "genome reduction". The use of the CRISPR-Cas9 tools, which we have additionally optimized for application in S. rimosus has proven particularly valuable in this area. The aim of the master's thesis is an attempt of circularization of the linear genome of S. rimosus and an attempt to delete the large plasmid using the CRISPR-Cas9 tool, thereby potentially obtaining improved strains of S. rimosus with improved properties. Initially, we successfully constructed two appropriate plasmid constructs, in order to achieve the planned genome reduction efforts. In the first attempt, we used a plasmid containing the corresponding homologous regions targeting the telomeric regions of the linear chromosome, with the aim to achieve chromosome circularization. The second experiment, using a plasmid without homologous regions targeting the center of the large linear plasmid, thus attempting to cause plasmid breakdown and deletion. The E. coli-Streptomyces conjugation method was used to introduce plasmid constructs into different strains of S. rimosus. After analysing the conjugants, we failed to observe strains with the desired modifications.
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