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Abstract
Streptomyces is a Gram-positive, aerobic soil bacterium, has growth pattern similar with fungus. Streptomyces is well known for their ability to synthesize an enormous variety of antibiotics as secondary metabolites. Among them, S. avermitilils produces avermectins, a group of antiparasitic agents used in human and veterinary medicine. However, S. avermitilis also produces oligomycin, which is a potential toxic inhibitor of oxidative phosphorylation in mammalian cells. Therefore, we decided that disruption oligomycin synthetase gene to prevent co-production of oligomycin in S. avermitilis. To create plasmid for disruption, the smallest gene of oligomycin synthease gene cluster was obtained by PCR from S. avermitilis. And also to create plasmid for disruption, the gene of avermectin A synthase, AveD. Then, apramycin resistance gene was inserted in oligomycin synthase gene and avermectin A synthase gene for selection. After transformation of this plasmid, oligomycin synthase gene(olmA1) and avermectin A synthase gene(AveD) in the chromosome was displaced on the plasmid via homologous recombination. And next, to create plasmid for over expression, the gene of avermectin synthase aglycon. Used pQE30 vector to gene transfer.
As a result of this way, we obtain mutants(olmA1 and AveD) and transgenic cell that no longer not makes the toxic oligomycin and unusable avermectin but more amount of most useful avermectin B.