阿维链霉菌NRRL8165中bldA_a的克隆及其对形态分化与阿维菌素合成的影响

Cloning of bldA_a and the effect on morphological differentiation and avermectins production in Streptomyces avermitilis NRRL8165

bldA编码天蓝色链霉菌中唯一有效识别UUA亮氨酸密码的tRNA(Leu)UUA。通过构建阿维链霉菌NRRL8165基因组亚文库,筛选得到含有阿维链霉菌bldAa及其侧翼序列的克隆。利用λRED介导的PCRtargeting技术构建了bldAa的基因置换质粒pHL358,将其跨属接合转移进入阿维链霉菌NRRL8165,筛选得到bldAa基因置换菌株TW10。TW10表现为光秃表型,表明bldAa调控阿维链霉菌的形态分化。摇瓶发酵TW10菌株并对发酵产物进行HPLC分析,发现TW10菌株均不合成阿维菌素组分,提示阿维菌素的合成受bldAa调控;考察阿维菌素生物合成基因簇,其中aveA3和aveR含有TTA密码,它们的翻译可能受bldAa调控,与实验结果一致。

bldA encodes the only tRNA that efficiently translates the rare UUA leucine codon in Streptomyces coelicolor, bldA inactivation leaded to defection in morphological development and production of two of four known antibiotics in S. coelicolor. A bldA homologue, termed bldA,, has been identified in the sequenced genome of Streptomyces avermitilis MA4680. To investigate the function of bldAα, genomic DNA of S. avermitilis NRRL8165 was digested with BamH Ⅰ and the 5 - 6kb was fractioned and ligated with the BamH Ⅰ digested E. coli plasmid vector pIJ4642 to yield a sub-library. A clone containing bldA, and its flanking sequence was obtained by screening from this genome sub-library, pHL358, a bldA, replacement plasmid, was constructed using the λRED mediated PCR-targeting technique, and conjugated into S. avermitilis NRRLS165.Three bldAdisruption mutant strains （named TW10） were obtained, which showed a bald phenotype, indicating that bldAα, controlled the morphological differentiation of S. avermitilis. HPLC analysis of the TW10 fermentation culture showed that TW10 did not synthesize avermectins anymore, suggesting that the synthesis of avermectins were dominated by bldA,. There are TTA codons within aveA3 and aver of the avermectin biosynthesis gene cluster, suggesting that the translation of the two genes may depend on bldAα, which were consistent with the experimental results.