[Objective] The aim of this study was to produce Streptomyces avermitilis strain with site-directed mutagenesis in aveD gene,and so as to provide theoretical basis for genetic breeding of S.avermitilis.[Method] PCR-dr...[Objective] The aim of this study was to produce Streptomyces avermitilis strain with site-directed mutagenesis in aveD gene,and so as to provide theoretical basis for genetic breeding of S.avermitilis.[Method] PCR-driven overlap extension was conducted for the site-directed mutagenesis in aveD gene;the mutated aveD gene then was used to construct vector pDC3（pKC1139∷aveD） via molecular manipulations like in vitro enzyme digestion and ligation;the vector pDC3（pKC1139∷aveD） was then introduced to aveD deletion mutant 489 of avermectin-producing strain S.avermitilis 76-9.[Result] Mutant strain 536 of site-directed mutagenesis of S.avermitilis 76-9 was obtained by homologous recombination.The sequencing results show that the sixty-ninth base C in aveD-coding region of mutant 536 was substituted by T,and the corresponding amino acid Thr was mutated to be Ile.[Conclusion] This study laid basis for the development of strains specifically producing avermectin B.展开更多
基金Supported by Special Fund for Doctors in Universities in Hebei Province(B2003201)~~
文摘[Objective] The aim of this study was to produce Streptomyces avermitilis strain with site-directed mutagenesis in aveD gene,and so as to provide theoretical basis for genetic breeding of S.avermitilis.[Method] PCR-driven overlap extension was conducted for the site-directed mutagenesis in aveD gene;the mutated aveD gene then was used to construct vector pDC3（pKC1139∷aveD） via molecular manipulations like in vitro enzyme digestion and ligation;the vector pDC3（pKC1139∷aveD） was then introduced to aveD deletion mutant 489 of avermectin-producing strain S.avermitilis 76-9.[Result] Mutant strain 536 of site-directed mutagenesis of S.avermitilis 76-9 was obtained by homologous recombination.The sequencing results show that the sixty-ninth base C in aveD-coding region of mutant 536 was substituted by T,and the corresponding amino acid Thr was mutated to be Ile.[Conclusion] This study laid basis for the development of strains specifically producing avermectin B.
