In Streptomyces rimosus M527, the oxytetracycline (OTC) biosynthetic gene cluster is not expressed under laboratory conditions. In this study a reported-guided mutant selection (RGMS) procedure was used to activate the cluster. The double-reporter plasmid pAGT was constructed in which gusA encoding a β-glucuronidase and tsr encoding a thiostrepton resistance methyltransferase were placed under the control of the native promoter of oxyA gene (PoxyA ). Plasmid pAGT was introduced and integrated into the chromosome of S. rimosus M527 by conjugation, yielding initial strain M527-pAGT. Subsequently, mutants of M527-pAGT were generated by using ribosome engineering technology. The mutants harboring activated OTC gene cluster were selected based on visual observation of GUS activity and thiostrepton resistance. Finally, mutant M527-pAGT-R7 was selected producing OTC in a concentration of 235.2 mg/L. In this mutant transcriptional levels of oxysr genes especial oxyAsr gene were increased compared to wild-type strain S. rimosus M527. The mutant M527-pAGT-R7 showed antagonistic activities against Gram-negative and Gram-positive strains. All data indicate that the OTC gene cluster was successfully activated using the RGMS method.
Keywords: Streptomyces rimosus M527; activation of silent gene cluster; double-reporter-guided; oxytetracycline; ribosome engineering.
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