engleski

The use of homologous recombination for cloning secondary metabolite clusters in Streptomyces

Secondary metabolite clusters are difficult to clone using purely in vitro genetic methods, because they are large (usually > 30 kb). We developed a strategy based on homologous recombination. The ends of the clusters are cloned using PCR and rare double crossover recombination events are selected using suitable marker genes. Recombination cassettes were constructed using the rpsL+ gene as a counter-selectable marker. The cassettes were shown to be stable in a circular plasmid based on SCP2 as well as in a newly constructed linear plasmid based on pSLA2. The system was used successfully to clone the actinorhodin cluster of S. lividans into the SCP2-based vector. The clones carrying the cluster on the plasmid overproduced the antibiotic and could be recognised by their blue colour. Cassettes were also constructed for the avermectin cluster of S. avermitilis and the rapamycin cluster of S. hygroscopicus.

S. lividans; S. avermitilis; S. hygroscopicus; actinorhodin cluster; avermectin cluster; rapamycin cluster; PCR; homologous recombination

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