| Avermectins are a series of 16-membered macrocyclic lactones produced by Streptomyces avermitilis with potent anthelmintic and insecticidal activity. They are the most effective agricultural pesticides and antiparasitic agents, and used widely in medical, veterinary and agricultural fields.In addition to avermectins, S. avermitilis produces oligomycin, a strongly toxic compound. Gene deletion vector pXL05 was used to disrupt oligomycin polyketide synthase (PKS) encoding genes(olmA) in Streptomyces avermitilis CZ8-73, the producer of anthelmintic avermectins B and the cell growth inhibitor oligomycin. olmA gene cluster in the chromosome was displaced by deletion allele on the plasmid via double crossover. Four of disruptants were confirmed by Southern blotting. Shaking flask experiments and HPLC analyses showed that the four mutants no longer produced the toxic oligomycin, and only made four components of avermectins B, which were avermectin B1a, B1b, B2a, B2b. The yields of avermectins B in these mutants were separately equal to those in CZ8-73. This revealed that olmA genes deletion didn't affect the biosynthesis of avermectins. The deletion mutants were proved to be genetically stable, and thus might be promising strains in industrial production of avermectins B.Although S. avermitilis produces eight related components of avermectins, only two components, Bla and Bib, are available for the medical, veterinary and agricultural fields. In consideration of the colinearity between the activities of avermectin PKS domains and structure of the polyketide product, the DH2 domain of avermectin PKS, which corresponds to C-22,23 dehydration, appears to have partial dehydratase activity and this results in a mixture of C-22,23 double bonds ("1" components) and C23 hydroxy compounds ("2" components). In this study, we attempted to construct an engineering strain producing only avermectin Bl through the replacement of DNA encoding DH2-KR2 domains of the avermectin PKS (AveDH2-KR2) with DNA encoding DH2-KR2 domains from the pikromycin PKS in S. avermitilis Olm73-12, producing only avermectins B and no oligomycin. Gene replacement vector pXL201(pKC1139::5'flank+piA:DH2-KR2+3'flank) was used to transform 5. avermitilis Olm73-12 protoplasts. 160 double crossover mutants were obtained. Shaking flask experiments and HPLC analyses showed that 99 isolates still produced four components of avermectins B, in which the yields of avermectins B in 82 isolates were only about 0.1 ~2% of those produced by the parental strain Olm73-12. 2 of 82 isolates were confirmed to be the correct gene replacement mutants by PCR and sequence analysis. The mutant only producing avermectins Bl was not detected. This showed that the partial dehydratase activity of DH2 may be attributable to other reasons.Ivermectins (a mixture containing more than 80% of 22,23-dihydroavermectin Bla and less than 20% of 22,23-dihydroavermectin B1b), which have the same effective antiparasitic activities and the lower level of toxic side-effects than avermectins, are currently synthesized by chemically reducing the double bond between C22 and C23 of avermectins B1. The necessity of regiospecific hydrogenationresults in increased cost. Gene replacement vector pXL211(pKC1139:: 5'flank+pikA:DH4-ER4-KR4+ 3'flank) was used to substitute the DH and KR domains of module 2 of the avermectin PKS with a complete set of B-keto processing enzymes, consisting of the DH, ER and KR domains of module 4 from the pikromycin PKS in 5. avermitilis Olm73-12. 281 double crossover mutants were examined by fermentation experiments and HPLC analysis for production of polyketides. Besides avermectin B1a and B2a, a new compound (about l~4ug/mL) which showed a retention time and fragmentation patterns identical to those of the authentic sample of 22,23-dihydroavermectin Bla by HPLC and LC/MS analysis were detected in culture extracts of 27 isolates. The total amount of avermectins B were only about 0.5~2% of those produced by the parental strain Olm73-12. This study suggests that t...
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