title: Complementation of daptomycin dptA and dptD deletion mutations in trans and production of hybrid lipopeptide antibiotics.
abstract: Daptomycin is a lipopeptide antibiotic produced by Streptomyces roseosporus and recently commercialized as Cubicin (daptomycin-for-injection) for treatment of skin and skin-structure infections caused by Gram-positive pathogens. Daptomycin is synthesized by a non-ribosomal peptide synthetase (NRPS) encoded by three overlapping genes, dptA, dptBC and dptD. The dptE and dptF genes, immediately upstream of dptA, are likely to be involved in the initiation of daptomycin biosynthesis by coupling decanoic acid to the N-terminal Trp. Analysis of RT-PCR data suggests that dptE, dptF, dptA, dptBC, dptD and possibly other dpt genes are transcribed as one large message; however, it has been demonstrated that sequential translation of these genes from a long transcript is not essential for robust daptomycin production. The dptA and the dptD genes were deleted from the dpt gene cluster, and expressed from ectopic positions in the chromosome under the control of the strong constitutive ermEp* promoter to produce high levels of lipopeptides. This three-locus trans-complementation system was used to produce hybrid lipopeptide antibiotics by introducing the heterologous lptD and cdaPS3 genes from Streptomyces fradiae and Streptomyces coelicolor, respectively, to complement the DeltadptD mutation.
authors.affiliation: Cubist Pharmaceuticals Inc., 65 Hayden Avenue, Lexington, MA 02421, USA.
authors.forename: Marie-Françoise Coëffet-Le
authors.initials: MC
authors.lastname: Gal
authors.affiliation: Cubist Pharmaceuticals Inc., 65 Hayden Avenue, Lexington, MA 02421, USA.
authors.forename: Lisa
authors.initials: L
authors.lastname: Thurston
authors.affiliation: Cubist Pharmaceuticals Inc., 65 Hayden Avenue, Lexington, MA 02421, USA.
authors.forename: Paul
authors.initials: P
authors.lastname: Rich
authors.affiliation: Cubist Pharmaceuticals Inc., 65 Hayden Avenue, Lexington, MA 02421, USA.
authors.forename: Vivian
authors.initials: V
authors.lastname: Miao
authors.affiliation: Cubist Pharmaceuticals Inc., 65 Hayden Avenue, Lexington, MA 02421, USA.
authors.forename: Richard H
authors.initials: RH
authors.lastname: Baltz
mesh_terms: D000900:Anti-Bacterial Agents / Q000096:biosynthesis / Q000494:pharmacology; D001426:Bacterial Proteins / Q000235:genetics*; D017576:Daptomycin / Q000096:biosynthesis*; D017353:Gene Deletion; D015870:Gene Expression; D005816:Genetic Complementation Test; D008826:Microbial Sensitivity Tests; D010453:Peptide Synthases / Q000235:genetics*; D010957:Plasmids; D011401:Promoter Regions, Genetic; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics; D011995:Recombination, Genetic; D013211:Staphylococcus aureus / Q000187:drug effects; D013302:Streptomyces / Q000235:genetics* / Q000378:metabolism; D014158:Transcription, Genetic
chemical_list: D000900:Anti-Bacterial Agents; D001426:Bacterial Proteins; D011994:Recombinant Proteins; D010453:Peptide Synthases; D017576:Daptomycin