Studies On The Alteration Of Avermectin Biosynthesis Genes In Streptomyces Avermitilis

In this study, the avermectin-producing strain Streptomyces avermitilis was studied and the avermectin biosynthesis gene cluster in the genomic DNA of Streptomyces avermitilis S-2 was altered by the method of gene engineering.Insertion inactivation of aveD gene in the cluster by introducing apramycin resistance gene into aveD gene resulted in the disappearance of "A" components of avermectins. When aveC gene was inactivated by the same way, four "1" components were lost and only "2" components , the potential precursor of ivermectin , were accumulated.In order to obtain a DNA fragment containing aveD gene, 5'end primer and 3'end primer, consisting of 18 and 20 nucleotides respectively , were designed and synthesized. A 2.2 kb DNA fragment containing aveD gene was obtained by PCR when genomic DNA of Streptomyces avermitilis S-2 was used as a template.A 1.5 kb apramycin resistance fragment was inserted into Nru I site of aveD gene and the inactivated aveD gene fragment was then introduced into MCS region of pHJL401-an E.coli/Streptomyces shuttle vector with conjugation function ( containing oriT gene). As a result of above procedures, a recombinant plasmid pID03 was obtained.Based on a 3.1kb Pst I fragment of genomic DNA of a wildS.avermitilis,a 1.5 kb apramycin resistance fragment was inserted into Sph I site of aveC gene in the 3.1 kb fragment, then a recombinant plasmid pC05 was obtained by introducing above inactivated aveC fragment into MCS region of pHJL401.Competent cells of ET12567 were transformed by recombinant plasmid pID03 and pC05 respectively. The transformants were cultivated and the harvested cells were used in the procedure of conjugation by which the recombinant plasmid pID03 and pC05 were introduced into Streptomyces avermitilis S-2 respectively.Double cross-over strains AveD24 and AveC9 were obtained after growth of several generations on MYM plate with or without antibiotics selection.Genomic DNA of double cross-over strains AveD24 and AveC9 were extracted and Southern hybridization were performed. Results of hybridization confirmed that the 1.5 kb apramycin resistance fragments were inserted into a 3.4 kb BamH I genomic DNA fragment containing aveD gene and a 3.0 kb Not I genomic DNA fragment containing aveC gene respectively.The analysis of fermentation products of strain AveD24 showed that strain AveD24, as expected, was unable to produce "A" components, but only "B" components. Furthermore, it produced 2 unusual components D24-1 and D24-2. It was basically confirmed, by HPLC and MS, that D 24-1 was oligomycin A ; while D24-2 was 5-oxo-avermectin la.The analysis of fermentation products of strain AveC9 showed that strain AveC9, as predicted, had lost its ability to synthesize "1 "components, but kept its ability to synthesize "2" components.