具有产生安莎类抗生素潜能的放线菌的分子筛选

摘要

目的 建立一种高通量筛选具有产生安莎类抗生素潜能的放线菌的分子筛选方法.原理 3-氨基-5-羟基-苯甲酸(AHBA)是安莎类抗生素生物合成的前体.在通过氨基莽草酸途径生物合成AHBA的过程中,AHBA合酶是催化5-氨基-3-脱氢-5-脱氧莽草酸形成AHBA的一个特异性关键酶.AHBA合酶基因的存在可以作为放线菌菌株具有合成安莎类抗生素能力的一个必要而非充分的条件.AHBA合酶基因高度保守,通过PCR方法可以建立一种针对AHBA合酶基因存在与否的、具有安莎类抗生素产生潜能的放线菌菌株高通量筛选方法.方法 根据已知AHBA合酶序列的相似性,设计了针对AHBA合酶基因中高度保守的一段755bp片段大小的PCR筛选寡核苷酸引物.用于从土壤中分离的未知放线菌高通量筛选.结论 从1900株土壤分离的未知放线菌中筛选获得33株AHBA合酶基因阳性菌株,即具有安莎类抗生素产生潜能的放线菌菌株.%3-amino-5-hydroxybenzoic acid (AHBA) is the specific starter unit for all ansamycin biosyn-thesis. AHBA synthase is the unique terminal enzyme converting 5-amino-3-dehydro-5-deoxyshikimate intoAHBA in the aminoshikimate pathway specifically for AHBA biosynthesis. AHBA synthase gene could be usedas a specific, easy-detected gene-marker of ansamycin producing potential of actinomycetes. Multi-alignmentof known AHBA synthases involved in ansamycin biosynthesis indicated that AHBA synthases are highly con-served. In this paper, a pair of oligonucleotides was designed successfully for PCR amplification/detectionspecifically of the 755bp portion of the AHBA synthase genes from unspecified actinomycetes strains. Usingthis pair of oligonucleotides, a high-throughput PCR screening method for potential ansamycin producers fromactinomycetes strains was established. Of the 1900 unspecified soil-isolated strains screened, 33 strains con-tained AHBA synthase gene. These strains were likely to be potential ansamycin producing strains.