Daptomycin biosynthesis in Streptomyces roseosporus: cloning and analysis of the gene cluster and revision of peptide stereochemistry.
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.
Miao
Vivian
V
Cubist Pharmaceuticals, Inc., 65 Hayden Avenue, Lexington, MA 02421, USA.
Coëffet-LeGal
Marie-Françoise
MF
Cubist Pharmaceuticals, Inc., 65 Hayden Avenue, Lexington, MA 02421, USA.
Brian
Paul
P
Cubist Pharmaceuticals, Inc., 65 Hayden Avenue, Lexington, MA 02421, USA.
Brost
Renee
R
Cubist Pharmaceuticals, Inc., 65 Hayden Avenue, Lexington, MA 02421, USA.
Penn
Julia
J
Cubist Pharmaceuticals, Slough, 545 Ipswich Road, Slough SL1 4EQ, UK.
Whiting
Andrew
A
Cubist Pharmaceuticals, Slough, 545 Ipswich Road, Slough SL1 4EQ, UK.
Martin
Steven
S
Cubist Pharmaceuticals, Slough, 545 Ipswich Road, Slough SL1 4EQ, UK.
Ford
Robert
R
Cubist Pharmaceuticals, Slough, 545 Ipswich Road, Slough SL1 4EQ, UK.
Parr
Ian
I
Cubist Pharmaceuticals, Inc., 65 Hayden Avenue, Lexington, MA 02421, USA.
Bouchard
Mario
M
Cubist Pharmaceuticals, Inc., 65 Hayden Avenue, Lexington, MA 02421, USA.
Silva
Christopher J
CJ
Cubist Pharmaceuticals, Inc., 65 Hayden Avenue, Lexington, MA 02421, USA.
Wrigley
Stephen K
SK
Cubist Pharmaceuticals, Slough, 545 Ipswich Road, Slough SL1 4EQ, UK.
Baltz
Richard H
RH
Cubist Pharmaceuticals, Inc., 65 Hayden Avenue, Lexington, MA 02421, USA.
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
D000900:Anti-Bacterial Agents; D001426:Bacterial Proteins; D010456:Peptides, Cyclic; D021122:Protein Subunits; D010453:Peptide Synthases; C104385:non-ribosomal peptide synthase; D017576:Daptomycin