Sterptomyces rochei4089的L基因阻断变株的构建及功能研究

运用同源重组技术破坏了一株格尔德霉素产生菌Sterptomyces rochei 4089的L基因,该基因编码氧化还原酶.以Sterptomyces rochei 4089基因组总DNA为模板,PCR扩增AHBA-KLM基因簇,采取Red/ET重组技术,构建L基因阻断质粒pKC1139-KLM-KmR.采用大肠杆菌与链霉菌的结合转移将阻断质粒含AHBA-KLM基因簇和Kan表达单元的3.0 kb线性片段转化Sterptomyces rochei 4089菌株,在卡纳霉素的平板上筛选卡纳霉素抗性转化子,经PCR检测分离到L基因阻断突变菌株.对原、变株的发酵液进行TLC和HPLC分析显示,Sterptomyces rochei 4089基因组中的L基因失活后,导致该菌株不能合成安莎类抗生素格尔德霉素.通过阻断L基因,为筛查这类放线菌产生安莎类抗生素提供了明确的组分指示作用.

Constrution of L gene disruption mutant by Red/ET recombination in Sterptomyces rochei 4089 and its functional analysis

The Sterptomyces rochei 4089 L gene encoding oxidoreductase was disrupted using homologous recombination technology. AHBA-KLM gene cluster fragment was amplified by PCR with Sterptomyces rochei 4089 genomic DNA as template and cloned into vector pKC1139. The selection marker, a Kanamycin resistance expression cassette, was inserted into the L gene resulting in the L disrupted plasmid pKC1139 -KLM-KmR by Red/ET recombination. The plasmid was used as transforming DNA for Sterptomyces rochei 4089 strain. The recipient Sterptomyces rochei 4089 contains an integrated Kanamycin resistance expression cassette. The transformauts were obtained by Kanamycin resistance selection, and then were screened by PCR. Transformant Sterptomyces rochei 4089 was identified to be the L gene disruption strain. The effect of L disruption Sterptomyces roehei 4089 on oxidoreduetase expression was analyzed by TLC and HPLC analysis. The result indicated that the disruption of L gene proved the production of geldanamycin in Sterptomyees roehei 4089 strain was related to L gene. Thus the disruption of L gene in a recipient strain would be helpful for preliminary mining ansamycins production in actinomycetes,