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.

Daptomycin (Cubicin) is a lipopeptide antibiotic approved in the USA in 2003 for the treatment of skin and skin structure infections caused by Gram-positive pathogens. It is a member of the 10-membered cyclic lipopeptide family of antibiotics that includes A54145, calcium-dependent antibiotic (CDA), amphomycin, friulimicin, laspartomycin, and others. This review highlights research on this class of antibiotics from 1953 to 2005, focusing on more recent studies with particular emphasis on the interplay between structural features and antibacterial activities; chemical modifications to improve activity; the genetic organization and biosynthesis of lipopeptides; and the genetic engineering of the daptomycin biosynthetic pathway to produce novel derivatives for further chemical modification to develop candidates for clinical evaluation.

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.

Wild-type p53 plays a crucial role in the prevention of cancer. Since dysfunction of p53 can be caused by increased levels of the protein MDM2, small molecules which antagonize the interaction between these two proteins have potential in cancer therapy. The discovery and structure determination of a fungal metabolite, chlorofusin, which antagonizes the p53/MDM2 interaction are reported.

SummaryIn many peptide antibiotics, modified amino acids are important for biological activity. The amino acid 3-methyl-glutamic acid (3mGlu) has been found only in three cyclic lipopeptide antibiotics: daptomycin and the A21978C family produced by Streptomyces roseosporus, calcium-dependent antibiotic produced by Streptomyces coelicolor and A54145 produced by Streptomyces fradiae. We studied the non-ribosomal peptide synthetase genes involved in A21978C biosynthesis and the downstream genes, dptG, dptH, dptI and dptJ predicted to encode a conserved protein of unknown function, a thioesterase, a methyltransferase (MTase) and a tryptophan 2,3-dioxygenase respectively. Deletion of dptGHIJ reduced overall lipopeptide yield and led to production of a series of novel A21978C analogues containing Glu 12 instead of 3mGlu12. Complementation by only dptI, or its S. coelicolor homologue, glmT, restored the biosynthesis of the 3mGlu-containing compounds in the mutant. Compared with A21978C, the Glu12-containing derivatives were less active against Staphylococcus aureus. Further genetic analyses showed that members of the dptGHIJ locus cooperatively contributed to optimal A21978C production; deletion of dptH, dptI or dptJ genes reduced the yield significantly, while expression of dptIJ or dptGHIJ from the strong ermEp* promoter substantially increased lipopeptide production. The results indicate that these genes play important roles in the biosynthesis of daptomycin, and that dptI encodes a Glu MTase.