Sunday, July 29, 2007
P117
Enhancement of avermectin production in Streptomyces avermitilis
Kim In woo, Yeon Woo Ryu, and Won Kyu Lee. Dept of Molecular Science and Technology, Ajou University, San 5, Wonchon-dong, Yeongtong-gu, Suwon, 443-749, Korea., Suwon, South Korea
Avermectin is a secondary metabolite with powerful anthelmintic and insecticidal activities, thus it is used as an efficient agent in the field of agriculture and animal health. Avermetin can be produced by polyketide pathway in S. avermitilis. Also oligomycin is a polyketide compound produced by S. avermitilis. It was reported that disruption of avermatin production resulted in increasing of oligomycin production. This result showed that there is a relationship between avermectin and oligomycin productions.
In this research, oligomycin production was disrupted to increase avermectin production. To create plasmid for disruption, the smallest gene of oligomycin synthetase gene cluster was obtained by PCR from S. avermitilis chromosome. Then, apramycin resistance gene was inserted in oligomycin synthetase gene for selection. After transformation of this plasmid, oligomycin synthetase gene (olmA5) in the chromosome was replaced with disruption cassette on the plasmid via homologous recombination. As a result of this gene replacement, we obtained mutants (olmA5::apra) that no longer makes the oligomycin compounds. And the mutants confirmed by PCR & HPLC analysis. Although no significant increment of avermectin production in the mutant strain was observed at this point, we are applying other effects such as SAM gene expression which is reported as a helper of secondary metabolites production, random mutation and medium optimization for further increment of avermectin production in S. avermitilis.
We are in progress of combination of those effects to increase avermectin production and overall enhancement will be discussed.
In this research, oligomycin production was disrupted to increase avermectin production. To create plasmid for disruption, the smallest gene of oligomycin synthetase gene cluster was obtained by PCR from S. avermitilis chromosome. Then, apramycin resistance gene was inserted in oligomycin synthetase gene for selection. After transformation of this plasmid, oligomycin synthetase gene (olmA5) in the chromosome was replaced with disruption cassette on the plasmid via homologous recombination. As a result of this gene replacement, we obtained mutants (olmA5::apra) that no longer makes the oligomycin compounds. And the mutants confirmed by PCR & HPLC analysis. Although no significant increment of avermectin production in the mutant strain was observed at this point, we are applying other effects such as SAM gene expression which is reported as a helper of secondary metabolites production, random mutation and medium optimization for further increment of avermectin production in S. avermitilis.
We are in progress of combination of those effects to increase avermectin production and overall enhancement will be discussed.
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