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SUMMARY: Conditions for the preparation of protoplasts from Streptomyces aureofaciens and transformation with several replicative and integrative plasmids were optimized. Of all the replicative plasmids used, carrying various Streptomyces replication origins, only plasmid pGM9 produced transformants, with a transformation efficiency of 2 × 103 (μg DNA)-1. When plasmid DNA isolated from S. aureofaciens was used, the transformation efficiency increased to 105 transformants (μg DNA)-1. Transformation of protoplasts with integrative plasmids containing part of the hrdA gene gave rise to transformants with an efficiency of approximately 30 transformants (μg DNA)-1. Integration frequency was strongly reduced when plasmid DNA was modified by dam or dcm methylase. By integrative transformation, via a double crossover, a stable null mutant of the hrdA gene was prepared. This mutation appeared to have no obvious effect on growth, morphology, differentiation and production of chlortetracycline.
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