[Cloning of bldAa and the effect on morphological differentiation and avermectins production in Streptomyces avermitilis NRRL8165].

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 bldA., 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 bldAa and its flanking sequence was obtained by screening from this genome sub-library. pHL358, a bldA, replacement plasmid, was constructed using the lambdaRED 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, 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 bldAa . 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 bldAa, which were consistent with the experimental results.