Site-directed Improvement Of Effective Components Of Avermectins

Avermectins are a complex of eight Bla, B2a, Bib, B2b, Ala, A2a, Alb and A2b sixteen-membered macrolides. Among these components, Bla has the most effective antiparasitic activity. Through gene disruption, the biosynthetic pathway of avermectin's void components was blocked and only effective components were produced. C5 0-methyltransferase gene (aveD) and brach-chained-keto acid dehydrogenase gene (bkd) play key roles in biosynthesis of void components of avermectin.The gene aveD is located on the 3.4kb BamHl DNA fragment of S. avermitilis chromosome. The sub-library of approximate 3.4kb BamHl DNA fragment of S. avermitilis was constructed and the linked gene of aveD, aveF was isolated via PCR. The recombinant plasmid pADHOl, containing aveD, was abtained by using aveF as the probe. The sequence analysis and homogeneous comparison with GenBank indicated the cloned gene was identical to the aveD of S. avermitilis.Recombinant plasmid pDAlllS was constructed by inserting 1.7kb aprA into the Nrul site of aveD. Through subcloning aveD fragment into pHZ1358,the gene replacement shuttle vector, pHJ5803 (pHZ1358i '.aveD'. Iopr/1) was constructed and introduced into S. avermitilis via intergeneric conjugation between E. coli ET12567 (pUZ8002) and S. avermitilis. The double-crossed recombinant strain Bjbm0006 was selected after relaxed culture. The aveD gene of Bjbm0006 was proved to be disrupted via PCR. After LC-MS assay of fermentation broth of Bjbm0006, only four components were produced, which were identical to the four B components of the starting strain.Using Bjbm0006 as starting strain, plasmids pHJ5821 (pHZ1358: '.bkdABC'. \errn) and pHJ5816 (pHZ1358* '.bkdFGH'. \errn) were constructed via PCR for gene disruptions of bkdABC and bkdFGH respectively. It was proved that bkdABC gene disrupted mutant Bjbm5821 produced Bla, B2a and a novel compound while bkdFGH gene disrupted mutant Bjbm5816 accumulated exclusively the novel product.After crystalization, the novel compound was dissolved in deuterium instead acetone . Through assays of MS, ^-NMR, 13C-NMR and two-dimension NMR, the compound was attested to be oligomycin A. According to the structure of oligomycin A and avermectin aglycones, the biosynthetic pathway of oligomycin A was proposed. During bioactive detection of tests, it showed eminent inhibitory effect on A. niger, E. cinnamopurpureum and E. tonophilus while showed no effect on A. fumigatus, S. cerevisiae and C. tropicalis. Its inhibitory effect on Plutella xylostella was superior to avermectin, while effects on Tetranychus viennensis and Helicoverpa armigera were the same as avermectinThe results of fermentatire studyies of recombinant strains Bjbm0006, Bjbm5821 and Bjbm5816 revealed that the metabolic characteristics of recombinant strains weresimilar to the starting strain. The avermectin's synthetic pathway of Bjbm5821 was partially blocked and only 300u/ml avermectin was produced while the avermectin's synthetic pathway of Bjbm5816 was completely blocked and only oligomycin A was produced.