Genetic engineering in Streptomyces roseosporus to produce hybrid lipopeptide antibiotics.
Daptomycin is a lipopeptide antibiotic produced by a nonribosomal peptide synthetase (NRPS) in Streptomyces roseosporus. The holoenzyme is composed of three subunits, encoded by the dptA, dptBC, and dptD genes, each responsible for incorporating particular amino acids into the peptide. We introduced expression plasmids carrying dptD or NRPS genes encoding subunits from two related lipopeptide biosynthetic pathways into a daptomycin nonproducing strain of S. roseosporus harboring a deletion of dptD. All constructs successfully complemented the deletion in trans, generating three peptide cores related to daptomycin. When these were coupled with incomplete methylation of 1 amino acid and natural variation in the lipid side chain, 18 lipopeptides were generated. Substantial amounts of nine of these compounds were readily obtained by fermentation, and all displayed antibacterial activity against gram-positive pathogens.
Miao
Vivian
V
Cubist Pharmaceuticals, Inc., 65 Hayden Avenue, Lexington, Massachusetts 02421, USA.
Coëffet-Le Gal
Marie-Francois
MF
Nguyen
Kien
K
Brian
Paul
P
Penn
Julia
J
Whiting
Andrew
A
Steele
Jonathan
J
Kau
David
D
Martin
Steve
S
Ford
Robert
R
Gibson
Trevor
T
Bouchard
Mario
M
Wrigley
Stephen K
SK
Baltz
Richard H
RH
D000595:Amino Acid Sequence; D000900:Anti-Bacterial Agents / Q000096:biosynthesis / Q000494:pharmacology*; D001483:Base Sequence; D003001:Cloning, Molecular; D017576:Daptomycin / Q000737:chemistry / Q000494:pharmacology; D005818:Genetic Engineering; D016908:Gram-Positive Bacterial Infections / Q000188:drug therapy; D008074:Lipoproteins / Q000096:biosynthesis / Q000737:chemistry / Q000494:pharmacology*; D008745:Methylation; D008969:Molecular Sequence Data; D005810:Multigene Family; D010453:Peptide Synthases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D010957:Plasmids; D021122:Protein Subunits; D013302:Streptomyces / Q000235:genetics / Q000378:metabolism*
D000900:Anti-Bacterial Agents; D008074:Lipoproteins; D021122:Protein Subunits; D010453:Peptide Synthases; C104385:non-ribosomal peptide synthase; D017576:Daptomycin