|Silva, Christopher - Chris|
Submitted to: Microbiology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 3/15/2005
Publication Date: 5/20/2005
Citation: Miao, V., Coeffet-LeGal, M-F, Brian, P., Brost, R., Penn, J., Whiting, A., Martin, S. Ford, R., Parr, I., Bouchard, M., Silva, C.J., Wrigley, S.K., Baltz, R.H. 2005. Daptomycin biosynthesis in Streptomyces roseosporus: cloning and analysis of the gene cluster and revision of peptide stereochemistry. Microbiology. 151(5):1507-1523. Interpretive Summary: This work describes the gene sequence of the daptomycin gene cluster. The gene cluster encodes the proteins necessary to make the important antibiotic daptomycin. A detailed analysis of the sequence showed that the structure had been misassigned. One of the 13 amino acids, asparagine, was assigned the L stereochemistry. In fact, it had the D stereochemistry.
Technical Abstract: Daptomycin is a 13 amino acid, cyclic lipopeptide produced by a nonribosomal peptide synthetase (NRPS) mechanism in Streptomyces roseosporus. A 128 kp 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 5, 6, or 2 amino acids, respectively, included a novel activation domain and amino acid binding pocket for incorporating the ususal 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 acrylate the first amino acid, as well as other genes (dptI, dptJ) probably involved in supply of the non-proteinogenic amino acids L-3-methyl- glutamic 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.