Microbiology 151 (2005), 1507-1523; DOI 10.1099/mic.0.27757-0
Microbiology 151 (2005), 1507-1523; DOI 10.1099/mic.0.27757-0
© 2005 Society for General Microbiology
Daptomycin biosynthesis in Streptomyces roseosporus: cloning and analysis of the gene cluster and revision of peptide stereochemistry
Vivian Miao1,
Marie-Françoise Coëffet-LeGal1,
Paul Brian1,
Renee Brost1,
Julia Penn2,
Andrew Whiting2,
Steven Martin2,
Robert Ford2,
Ian Parr1,
Mario Bouchard1,
Christopher J. Silva1,
Stephen K. Wrigley2 and
Richard H. Baltz1
1 Cubist Pharmaceuticals, Inc., 65 Hayden Avenue, Lexington, MA 02421, USA
2 Cubist Pharmaceuticals, Slough, 545 Ipswich Road, Slough SL1 4EQ, UK
Correspondence
Vivian Miao
vmiao{at}cubist.com
Richard H. Baltz
Rbaltz{at}cubist.com
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.
Abbreviations: A-domain, amino acid-activating domain; BAC, bacterial artificial chromosome; CDA, calcium-dependent antibiotic; C-domain, condensation domain; DMF, dimethylformamide; E-domain, epimerization domain; ESI LC-MS, electrospray ionization liquid chromatography-mass spectrometry; Fmoc, 9-fluorenylmethoxycarbonyl; Kyn, kynurenine; 3mGlu, 3-methylglutamic acid; NRPS, non-ribosomal peptide synthetase; OPfp, O-pentafluorophenyl; T-domain, thiolation domain; Te, thioesterase; TFA, trifluoroacetic acid; TIS, triisopropylsilane
The GenBank/EMBL/DDBJ accession number for the sequence of pCV1 is AY787762.
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