Thiopeptides are ribosomally synthesized, posttranslationally modified peptides with potent activity against Gram-positives. However, only GE2270 has yielded semisynthetic derivatives under clinical investigations. The pbt gene cluster from the GE2270 producer Planobispora rosea was successfully expressed in the genetically tractable Nonomuraea ATCC39727. Gene deletions established that PbtO, PbtM1, PbtM2, PbtM3, and PbtM4 are involved in regiospecific hydroxylation and methylations of GE2270, leading to the generation of various derivatives with altered decorations. Further deletions established that PbtH and PbtG1 are involved in C-terminal amide and oxazoline formation, respectively. Surprisingly, preventing either step resulted in the accumulation of linear precursors in which the pyridine-generated macrocycle failed to form, and only one of the pyridine-forming serine residues had been dehydrated. Often, these linear precursors present a shortened C terminus but retain the full set of methylation and hydroxylation decorations.

Actinomycetes are known for their large genetic potential to produce several distinct classes of secondary metabolites. 1 While cultivating Actinomadura sp. DSMZ 13491, the producer of the cyclic heptapeptide GE23077, 2 we observed it produced an antibacterial activity. Despite being a potent and selective inhibitor of bacterial RNA polymerase, GE23077 is devoid of activity against bacterial cells, except for marginal activity against some Moraxella spp. 2 Here we report on the isolation, structure elucidation and properties of madurastatin C1 (1), the antimicrobial compound produced by this actinomycete.A 10-ml seed culture of Actinomadura sp. DSMZ 13491, prepared by inoculating a frozen cell stock into 30 ml of AFT medium (2% glucose, 0.2% yeast extract, 0.8% soybean meal, 0.5% tryptone, 0.1% NaCl, 0.4% CaCO 3 , adjusted to pH 7.2 with 0.1 N NaOH before sterilization) and incubating for 72-96 h, was transferred into 200 ml of fresh AFT medium. The production of antibacterial activity was monitored over time by depositing 10 ml of a microbial extract, prepared as described by Pozzi et al., 3 onto Muller Hinton Agar (Difco Laboratories, Detroit, MI, USA) plates, previously inoculated with 10 5 cfu ml À1 Staphylococcus aureus ATCC 6538P or Micrococcus luteus ATCC 10240. An inhibition halo was clearly observed on M. luteus plates after 24 h of cultivation, whereas only a weak activity was visible on the S. aureus plates. An HPLC analysis of the culture extract is shown in Figure 1a. A major peak was observed at 2.6 min with a m/z of 592 [M+H] + , whereas the two major congeners of GE23077, with m/z of 804 and 806, were observed at 1.8 min, as expected. 2 Minor peaks were also observed at 2.1 min, with m/z of 610 and 645. The 2.6-min metabolite was purified by medium pressure chromatography on a reverse-phase C18 RediSep RF column, using a CombiFlash RF Teledyne Isco Medium Pressure Chromatography System (Teledyne ISCO, Lincoln, NE, USA). The column was previously conditioned with a mixture of phase A (50 mM HCOONH 4 ), phase B (CH 3 CN) 95:5 (v/v), and eluted with a 15-min linear gradient from 5 to 95% phase B. The active fractions were collected, concentrated under vacuum and lyophilized, yielding 13 mg of purified 1 (estimated at least 95% pure by NMR).NMR and MS analyses of 1 indicated high similarity with the siderophores named madurastatins. 4 1 H-and 13 C-NMR data (Table 1) indicated the presence of an ortho disubstituted benzene, as confirmed by bidimensional experiments showing four aromatic protons with a TOCSY correlation. In particular, a doublet at 7.61 p.p.m. had a COSY correlation with a pseudo-triplet at 6.82 p.p.m., and a doublet at 6.89 p.p.m. correlated with a pseudotriplet at 7.34 p.p.m., whereas the pseudo-triplet signals intercorrelated. HMBC experiments demonstrated the disubstituted benzene to be a salicylic acid residue. Five aliphatic spin systems were also observed by using homo-and hetero-nuclear 2D-NMR experiments. COSY and TOCSY correlations evidenced the presence of an aziridine group...