Daptomycin biosynthesis in Streptomyces roseosporus: cloning and analysis of the gene cluster and revision of peptide stereochemistry

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 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 .