Cloning of bldAa and the ect on morphological differentiation and avermrctins production in Streptomyces avermitilis NRRL8165

Abstract:

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_a, has been identified in the sequenced genome of Streptomyces avermitilis MA4680.To investigate the function of bldA_a, genomic DNA of S. avermitilis NRRL8165 was digested with BamHⅠ and the 5～6kb was fractioned and ligated with the BamHⅠ digested E.coli plasmid vector pIJ4642 to yield a sub-library. A clone containing bldA_a and its flanking sequence was obtained by screening from this genome sub-library. pHL358, a bldA_a replacement plasmid, was constructed using the λRED mediated PCR-targeting technique, and conjugated into S. avermitilis NRRL8165.Three bldA-disruption mutant strains (named TW10) were obtained, which showed a bald phenotype, indicating that bldA_a 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_a. 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 bldA_a, which were consistent with the experimental results.