Bacteria of the genus Streptomyces are industrially and medically important producers of biologically active compounds. Although classical cloning methods based on homologous recombination have already been developed for their genetic engineering, newer methods such as those with the Cas enzymes bring some potential improvements and therefore sensibly complement the set of molecular tools for this genus. We chose to use two different Cas nucleases that we found the most interesting after reviewing available literature: Cas9 from Streptococcus pyogenes and Cas12a from Francisella novicida. To express them, we assembled four segregationally unstable plasmids, using each nuclease to attempt a deletion of the same two genes on the genome of Streptomyces rimosus, an industrially important producer of oxytetracycline. The first target was the region of the oxyA and oxyB genes, which encode key enzymes of the polyketide synthase in the pathway of oxytetracycline biosynthesis. The second target was the gene for secretory tripeptidyl aminopeptidase, one of the more abundant extracellular proteases in S. rimosus. Plasmid constructs with CRISPR-Cas system components were prepared using modern cloning methods based on homologies such as overlap PCR, NEBuilder® HiFi/Gibson Assembly® and SliCE cloning. Plasmid constructs were first transformed into Escherichia coli by electroporation, followed by conjugation from E. coli into S. rimosus. After applying diverse culturing methods, successful deletions of both target genes were confirmed by PCR and sequencing of the amplified DNA fragment of S. rimosus mutants.
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