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

Abstract

Streptomyces iranensis HM 35 is an alternative rapamycin producer to Streptomyces 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 in S. rapamycinicus. The approach depends upon efficient conjugational plasmid transfer from Escherichia coli to Streptomyces, and the failure of this step has frustrated its application to Streptomyces 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 Ca 2؉ ions in the conjugation media, we have achieved exconjugant formation for each of a series of gene deletions in S. iranensis HM 35. Among them were rapK, which generates the starter unit for rapamycin biosynthesis, and hutF, encoding a histidine catabolizing enzyme. The protocol that we have developed may allow efficient generation of targeted gene knockout mutants of Streptomyces species that are genetically difficult to manipulate. IMPORTANCE The developed protocol of conjugational plasmid transfer from Escherichia coli to Streptomyces iranensis may allow efficient generation of targeted gene knockout mutants of other genetically difficult to manipulate, but valuable, Streptomyces species. Since the discovery of streptomycin in 1943 (1), streptomycetes have been shown to produce thousands of compounds with possibly beneficial features, e.g., antibiotics, immunosuppressants, or anticancer drugs. Actinomycete-derived metabolites comprise over two-thirds of all known antibiotic compounds (2), and recent genome sequencing programs revealed that their biosynthesis potential has been underestimated. In their 8-to 12-Mb genomes, approximately 20 to 30 gene clusters encode the biosynthesis of secondary metabolites (3-5). One of the most important Streptomyces-derived compounds is the mechanistic target of rapamycin (mTOR) inhibitor rapamycin (sirolimus), a "billion dollar molecule" and, after cyclosporine, the most widely used immunosuppressant of microbial origin (6). Rapamycin was previously reported as an antifungal antibiotic produced by Streptomyces hygroscopicus ATCC 29253 (7), later renamed Streptomyces rapamycinicus (8). The rapamycin gene cluster in this strain has been sequenced (9), the biosynthetic pathway has been extensively characterized (10-14), and engineering of the cluster has yielded an impressive range of bioactive modified rapamycins (rapalogs) (15, 16), some in multigram amounts. So far, two other rapamycin-producing species are known: the taxonomically closely related Streptomyces iranensis HM 35 (5, 17) and Actinoplanes sp. strain N902-109 (18). To elucidate the molecular biology of rapamycin formation in these strains and to further exploit the physiological and pharmacological capability of rapamycin derivatives, genetic manipulation of these altern...

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