Avermitilis Poly Of Ketone Bodies Synthase Genetically Modified

Streptomyces avermitilis polyketide synthase genes were reconstructed applying the principle of combinatorial biosynthesis. The heterozygosis PKS modular 2 gene was attained and through gene recombination the engineering strain could directly produce ivermectin. By means of suitable endonuclease sites and vectors, this two modular gene fragment—3.6kb gene fragment containing milbemycin PKS modular 2 DH2-ER2 gene and 2.6kb containing avermectin PKS modular 2 AT-DH-KR gene,were reconstructed, the temperature sensitive and conjugal transfer plasmid pLZ5 was obtained which contained the heterozygosis PKS modular 2 gene. pLZ5 is transformed into S.avermectilis SY310 and we obtained 45 double crossover mutants though single cross-over,double cross-over steps. Shaking flask experiments and HPLC analyses showed that 32 isolates don't produced components of avermectins ; 10 isolates still produced components of avermectins;remained 3 isolates produced new metabolic product.This resuls is expected to be detected.Inaddition to avermectins,S.avermitilis produces oligomycin—a strongly toxic compound.With the designed primers, the 0.7kb fragment containing olmA2 gene and similar-size fragment containing olmA4 gene were obtained.This two fragments they were confirmed by gene sequence analysis. By means of suitable endonuclease sites and vectors, This two fragments were reconstructed as a temperature sensitive and conjugal transfer plasmid -called pZL5 .Finally genedeletion vector pZL5 was used to disrupt oligomycin polyketide synthase (PKS) encoding genes(olmA)in Streptomyces avermitilis SY310, the producer of anthelmintic avermectins and the cell growth inhibitor oligomycin. olmA2 gene -olmA4 gene in the chromosome was displaced by deletion allele on the plasmid via single and double crossover. We obtained 50 disruptents SY312.Shaking flask experiments and HPLC analyses showed that the 5 mutants no longer produced the toxic oligomycin, and only made components of avennectins.contract to SY310, Titer of The deletion mutants is not changed. This revealed that olmA genes deletion didn't affect the biosynthesis of avermectins. The deletion mutants were proved relatively stable, and thus might be promising strains.