An Efficient Method To Generate Gene Deletion Mutants of the Rapamycin-Producing Bacterium Streptomyces iranensis HM 35

Streptomyces iranensis HM 35 is an alternative rapamycin producer toStreptomyces rapamycinicus . Targeted genetic modification of rapamycin-producing actinomycetes is a powerful tool for the directed production of rapamycin derivatives, and it has also revealed some key features of the molecular biology of rapamycin formation inS. rapamycinicus. The approach depends upon efficient conjugational plasmid transfer fromEscherichia coli toStreptomyces , and the failure of this step has frustrated its application toStreptomyces iranensis HM 35. Here, by systematically optimizing the process of conjugational plasmid transfer, including screening of various media, and by defining optimal temperatures and concentrations of antibiotics and Ca2+ ions in the conjugation media, we have achieved exconjugant formation for each of a series of gene deletions inS. iranensis HM 35. Among them wererapK , which generates the starter unit for rapamycin biosynthesis, andhutF , encoding a histidine catabolizing enzyme. The protocol that we have developed may allow efficient generation of targeted gene knockout mutants ofStreptomyces species that are genetically difficult to manipulate.IMPORTANCE The developed protocol of conjugational plasmid transfer fromEscherichia coli toStreptomyces iranensis may allow efficient generation of targeted gene knockout mutants of other genetically difficult to manipulate, but valuable,Streptomyces species.