Daptomycin is a 13 amino acid, cyclic lipopeptide produced by a non-ribosomal peptide synthetase (NRPS) mechanism inStreptomyces roseosporus. A 128 kb region ofS. roseosporusDNA was cloned and verified by heterologous expression inStreptomyces lividansto 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 acidl-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 acidsl-3-methylglutamic acid and Kyn, were located next to the NRPS genes. The unexpected E-domain suggested that daptomycin would haved-Asn, rather thanl-Asn, as originally assigned, and this was confirmed by comparing stereospecific synthetic peptides and the natural product both chemically and microbiologically.

One of the main characteristics of the transmissible isoform of the prion protein (PrP^Sc) is its partial resistance to proteinase K (PK) digestion. Diagnosis of prion disease typically relies upon immunodetection of PK-digested PrP^Sc following Western blot or ELISA. More recently, researchers determined that there is a sizeable fraction of PrP^Sc that is sensitive to PK hydrolysis (sPrP^Sc). Our group has previously reported a method to isolate this fraction by centrifugation and showed that it has protein misfolding cyclic amplification (PMCA) converting activity. We compared the infectivity of the sPrP^Sc versus the PK-resistant (rPrP^Sc) fractions of PrP^Sc and analyzed the biochemical characteristics of these fractions under conditions of limited proteolysis. Our results show that sPrP^Sc and rPrP^Sc fractions have comparable degrees of infectivity and that although they contain different sized multimers, these multimers share similar structural properties. Furthermore, the PK-sensitive fractions of two hamster strains, 263K and Drowsy (Dy), showed strain-dependent differences in the ratios of the sPrP^Sc to the rPrP^Sc forms of PrP^Sc. Although the sPrP^Sc and rPrP^Sc fractions have different resistance to PK-digestion, and have previously been shown to sediment differently, and have a different distribution of multimers, they share a common structure and phenotype. Prion diseases are protein misfolding disorders. Different strains of prions are known to have variable resistance to proteinase K (PK) digestion. Furthermore, the same strain possesses both a PK sensitive (sPrP^Sc) and PK resistant (rPrP^Sc) aggregate of PrP. We developed methods to isolate the sPrP^Sc from rPrP^Sc fraction of the 263K strain of hamster-adapted scrapie. Both fractions were infectious, but have different physico-chemical properties. When we analyzed the lesion targets in the brain produced by each fraction they were essentially identical, suggesting that they were the same strain. The biochemical differences in the phenotypes of these two fractions are due to different sized multimers that share common structural properties. Furthermore, the comparison of the sensitive fractions of two hamster strains, 263K and Drowsy (Dy), showed strain-dependent differences in the ratios of the PK-sensitive to the PK-resistant forms of PrP^Sc.

Daptomycin and the A21978C antibiotic complex are lipopeptides produced by Streptomyces roseosporus and also in recombinant Streptomyces lividans TK23 and TK64 strains, when a 128 kbp region of cloned S. roseosporus DNA containing the daptomycin gene cluster is inserted site-specifically in the ϕC31 attB site. A21978C fermentation yields were initially much lower in S. lividans than in S. roseosporus, and detection was complicated by the production of host metabolites. However A21978C production in S. lividans was improved by deletion of genes encoding the production of actinorhodin and by medium optimization to control the chemical form of the calcium dependent antibiotic (CDA). This latter compound has not previously been chemically characterized as a S. lividans product. Adding phosphate to a defined fermentation medium resulted in formation of only the phosphorylated forms of CDA, which were well separated from A21978C on chromatographic analysis. Adjusting the level of phosphate in the medium led to an improvement in A21978C yield from 20 to 55 mg/l.