title: Daptomycin biosynthesis in Streptomyces roseosporus: cloning and analysis of the gene cluster and revision of peptide stereochemistry.
abstract: Daptomycin is a 13 amino acid, cyclic lipopeptide produced by a non-ribosomal peptide synthetase (NRPS) mechanism in Streptomyces roseosporus. A 128 kb region of S. roseosporus DNA was cloned and verified by heterologous expression in Streptomyces lividans to contain the daptomycin biosynthetic gene cluster (dpt). The cloned region was completely sequenced and three genes (dptA, dptBC, dptD) encoding the three subunits of an NRPS were identified. The catalytic domains in the subunits, predicted to couple five, six or two amino acids, respectively, included a novel activation domain and amino-acid-binding pocket for incorporating the unusual amino acid l-kynurenine (Kyn), three types of condensation domains and an extra epimerase domain (E-domain) in the second module. Novel genes (dptE, dptF) whose products likely work in conjunction with a unique condensation domain to acylate the first amino acid, as well as other genes (dptI, dptJ) probably involved in supply of the non-proteinogenic amino acids l-3-methylglutamic acid and Kyn, were located next to the NRPS genes. The unexpected E-domain suggested that daptomycin would have d-Asn, rather than l-Asn, as originally assigned, and this was confirmed by comparing stereospecific synthetic peptides and the natural product both chemically and microbiologically.
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: Marie-Françoise
authors.initials: MF
authors.lastname: Coëffet-LeGal
authors.affiliation: Cubist Pharmaceuticals, Inc., 65 Hayden Avenue, Lexington, MA 02421, USA.
authors.forename: Paul
authors.initials: P
authors.lastname: Brian
authors.affiliation: Cubist Pharmaceuticals, Inc., 65 Hayden Avenue, Lexington, MA 02421, USA.
authors.forename: Renee
authors.initials: R
authors.lastname: Brost
authors.affiliation: Cubist Pharmaceuticals, Slough, 545 Ipswich Road, Slough SL1 4EQ, UK.
authors.forename: Julia
authors.initials: J
authors.lastname: Penn
authors.affiliation: Cubist Pharmaceuticals, Slough, 545 Ipswich Road, Slough SL1 4EQ, UK.
authors.forename: Andrew
authors.initials: A
authors.lastname: Whiting
authors.affiliation: Cubist Pharmaceuticals, Slough, 545 Ipswich Road, Slough SL1 4EQ, UK.
authors.forename: Steven
authors.initials: S
authors.lastname: Martin
authors.affiliation: Cubist Pharmaceuticals, Slough, 545 Ipswich Road, Slough SL1 4EQ, UK.
authors.forename: Robert
authors.initials: R
authors.lastname: Ford
authors.affiliation: Cubist Pharmaceuticals, Inc., 65 Hayden Avenue, Lexington, MA 02421, USA.
authors.forename: Ian
authors.initials: I
authors.lastname: Parr
authors.affiliation: Cubist Pharmaceuticals, Inc., 65 Hayden Avenue, Lexington, MA 02421, USA.
authors.forename: Mario
authors.initials: M
authors.lastname: Bouchard
authors.affiliation: Cubist Pharmaceuticals, Inc., 65 Hayden Avenue, Lexington, MA 02421, USA.
authors.forename: Christopher J
authors.initials: CJ
authors.lastname: Silva
authors.affiliation: Cubist Pharmaceuticals, Slough, 545 Ipswich Road, Slough SL1 4EQ, UK.
authors.forename: Stephen K
authors.initials: SK
authors.lastname: Wrigley
authors.affiliation: Cubist Pharmaceuticals, Inc., 65 Hayden Avenue, Lexington, MA 02421, USA.
authors.forename: Richard H
authors.initials: RH
authors.lastname: Baltz
mesh_terms: D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D000900:Anti-Bacterial Agents / Q000096:biosynthesis* / Q000737:chemistry; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D001483:Base Sequence; D003001:Cloning, Molecular; D017124:Conserved Sequence; D017576:Daptomycin / Q000096:biosynthesis*; D008969:Molecular Sequence Data; D005810:Multigene Family; D016366:Open Reading Frames; D010453:Peptide Synthases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D010456:Peptides, Cyclic / Q000096:biosynthesis / Q000737:chemistry; D021122:Protein Subunits / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D017422:Sequence Analysis, DNA; D013237:Stereoisomerism; D013302:Streptomyces / Q000201:enzymology* / Q000235:genetics / Q000378:metabolism; D048170:Streptomyces lividans / Q000201:enzymology / Q000235:genetics / Q000378:metabolism
chemical_list: D000900:Anti-Bacterial Agents; D001426:Bacterial Proteins; D010456:Peptides, Cyclic; D021122:Protein Subunits; D010453:Peptide Synthases; C104385:non-ribosomal peptide synthase; D017576:Daptomycin