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

Abstract

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 lambda-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 antibiotic resistance cassettes are flanked by yeast FLP recombinase target sequences for removal of the antibiotic resistance and oriT(RK2) to generate unmarked, nonpolar mutations. The technique has been used successfully by >20 researchers to mutate around 100 Streptomyces genes. As an example, we describe its application to the discovery of a gene involved in the production of geosmin, the ubiquitous odor of soil. The gene, Sco6073 (cyc2), codes for a protein with two sesquiterpene synthase domains, only one of which is required for geosmin biosynthesis, probably via a germacra-1 (10) E,5E-dien-11-ol intermediate generated by the sesquiterpene synthase from farnesyl pyrophosphate.

Figure 1

Strategy for gene replacement in Streptomyces. (A) A gene replacement cassette containing aac (3)IV (Apra^R), oriT of RK2, and two FRT sites was amplified by PCR by using primers containing 39-nt 5′ homology extensions corresponding to regions flanking the S. coelicolor target gene (dotted lines a and b) and 20-nt 3′ homology to the unique priming sites P1 and P2 of the disruption cassette. (B) The PCR product from A was used to transform E. coli BW25113/pIJ790 (expressing the λ-Red recombination functions), containing the Supercos-1 cosmid with the cloned target gene (cyc2). (C) Apra^R transformants were selected, and the recombinant cosmid was identified by PCR and restriction analysis. (D) The potent methyl-specific restriction system of S. coelicolor was circumvented by introducing the recombinant cosmid into the methylation-deficient E. coli host ET12567/pUZ8002. The cosmid was mobilized in trans by pUZ8002 to Streptomyces by conjugation. (E) Selected exconjugants (Apra^R) were screened for Kan^S (loss of the Kan resistance gene neo), and the double cross-over allelic exchange was confirmed by PCR and Southern blot analysis. (F) E. coli DH5α cells containing pCP20 were transformed with the mutagenized cosmid DNA, and FLP synthesis was induced. (G) The loss of the disruption resistance marker was tested, and the cosmid was introduced by polyethylene glycol (PEG)-mediated transformation into the S. coelicolor mutant carrying a marked deletion within the gene of interest. (H) Kan^R transformants arising from single cross-over events were restreaked nonselectively and screened for the loss of both Kan^R and Apra^R, indicating a successful replacement by the in-frame deletion marked only by a “scar” sequence of 81 bp containing priming sites P1 and P2 (underlined), and one FRT site.

Figure 2

Sesquiterpene synthase homologues cyc1 and cyc2 of S. coelicolor. (A) Flanking genes and their proposed functions. The stop codon of cyc1 overlaps the start of SCO5223, suggesting the possibility of translational coupling of the two genes. SCO5223 encodes a cytochrome P450 enzyme that catalyzes sterol 14α-demethylation (35). cyc2 is flanked by SCO6072, encoding a 666-aa protein of unknown function, and SCO6074, encoding a 203-aa protein annotated as a possible integral membrane protein. (B) clustalx alignment of Cyc1 and the N- and C-terminal domains of Cyc2 (Cyc2N, Cyc2C) to the 337-aa sequence of the pentalenene synthase (PS) from Streptomyces UC5319 reveals two conserved terpenoid synthase motifs. The residues Asp-80, Asp-81, and Asp-84 within the sequence ⁷⁶FFLFDDLFD of pentalenene synthase are believed to form an Mg²⁺-binding site (36). Mg²⁺ is required to facilitate pyrophosphate departure in the first step of the cyclization reaction. Cyc1 contains all three conserved aspartate residues, whereas both the N- and the C-terminal domains of Cyc2 contain only the first two aspartate residues. Residues within a second sequence of pentalenene synthase (²¹⁷LLNDIASLEKE) are also thought to be associated with binding of Mg²⁺ ions and are conserved in Cyc1 and both domains of Cyc2.