PCR-targeted Streptomyces gene replacement identifies a protein domain needed for biosynthesis of the sesquiterpene soil odor geosmin

Streptomycetes are high G+C Gram-positive, antibiotic-producing, mycelial soil bacteria. The 8.7-Mb Streptomyces coelicolor genome was previously sequenced by using an ordered library of Supercos-1 clones. Here, we describe an efficient procedure for creating precise gene replacements in the cosmid clones by using PCR targeting and λ-Red-mediated recombination. The cloned Streptomyces genes are replaced with a cassette containing a selectable antibiotic resistance and oriT RK2 for efficient transfer to Streptomyces by RP4-mediated intergeneric conjugation. Supercos-1 does not replicate in Streptomyces , but the clones readily undergo double-crossover recombination, thus creating gene replacements. The…