Friulimicin B is a naturally occurring cyclic lipopeptide, produced by the actinomycete Actinoplanes friuliensis, with excellent activity against gram-positive pathogens, including multidrug-resistant strains. It consists of a macrocyclic decapeptide core and a lipid tail, interlinked by an exocyclic amino acid. Friulimicin is water soluble and amphiphilic, with an overall negative charge. Amphiphilicity is enhanced in the presence of Ca 2؉ , which is also indispensable for antimicrobial activity. Friulimicin shares these physicochemical properties with daptomycin, which is suggested to kill gram-positive bacteria through the formation of pores in the cytoplasmic membrane. In spite of the fact that friulimicin shares features of structure and potency with daptomycin, we found that friulimicin has a unique mode of action and severely affects the cell envelope of gram-positive bacteria, acting via a defined target. We found friulimicin to interrupt the cell wall precursor cycle through the formation of a Ca 2؉ -dependent complex with the bactoprenol phosphate carrier C 55 -P, which is not targeted by any other antibiotic in use. Since C 55 -P also serves as a carrier in teichoic acid biosynthesis and capsule formation, it is likely that friulimicin blocks multiple pathways that are essential for a functional gram-positive cell envelope.

Seven complete genes and one incomplete gene for the biosynthesis of the glycopeptide antibiotic balhimycin were isolated from the producer, Amycolatopsis mediterranei DSM5908, by a reverse-cloning approach and characterized. Using oligonucleotides derived from glycosyltransferase sequences, a 900-bp glycosyltransferase gene fragment was amplified and used to identify a DNA fragment of 9,882 bp. Of the identified open reading frames, three (oxyA to -C) showed significant sequence similarities to cytochrome P450 monooxygenases and one (bhaA) showed similarities to halogenase, and the genesbgtfA to -C showed similarities to glycosyltransferases. Glycopeptide biosynthetic mutants were created by gene inactivation experiments eliminating oxygenase and glycosyltransferase functions. Inactivation of the oxygenase gene(s) resulted in a balhimycin mutant (SP1-1) which was not able to synthesize an antibiotically active compound. Structural analysis by high-performance liquid chromatography–mass spectrometry, fragmentation studies, and amino acid analysis demonstrated that these oxygenases are involved in the coupling of the aromatic side chains of the unusual heptapeptide. Mutant strain HD1, created by inactivation of the glycosyltransferase gene bgtfB, produced at least four different compounds which were not glycosylated but still antibiotically active.

Gene-inactivation studies point to the involvement of OxyB in catalyzing the first oxidative phenol coupling reaction during glycopeptide antibiotic biosynthesis. The oxyB gene has been cloned and sequenced from the vancomycin producer Amycolatopsis orientalis, and the hemoprotein has been produced in Escherichia coli, crystallized, and its structure determined to 1.7-Å resolution. OxyB gave UV-visible spectra characteristic of a P450-like hemoprotein in the low spin ferric state. After reduction to the ferrous state by dithionite or by spinach ferredoxin and ferredoxin reductase, the CO-ligated form gave a 450-nm peak in a UV-difference spectrum. Addition of putative heptapeptide substrates to resting OxyB produced type I changes to the UV spectrum, but no turnover was observed in the presence of ferredoxin and ferredoxin reductase, showing that either the peptides or the reduction system, or both, are insufficient to support a full catalytic cycle. OxyB exhibits the typical P450-fold, with helix L containing the signature sequence FGHGXHXCLG and Cys 347 being the proximal axial thiolate ligand of the heme iron. The structural similarity of OxyB is highest to P450nor, P450terp, CYP119, and P450eryF. In OxyB, the F and G helices are rotated out of the active site compared with P450nor, resulting in a much more open active site, consistent with the larger size of the presumed heptapeptide substrate.

The entire simocyclinone biosynthetic cluster (sim gene cluster) from the producer Streptomyces antibioticus Tü6040 was identified on six overlapping cosmids (1N1, 5J10, 2L16, 2P6, 4G22, and 1K3). In total, 80.7 kb of DNA from these cosmids was sequenced, and the analysis revealed 49 complete open reading frames (ORFs). These ORFs include genes responsible for the formation and attachment of four different moieties originating from at least three different pools of primary metabolites. Also in the sim gene cluster, four ORFs were detected that resemble putative regulatory and export functions. Based on the putative function of the gene products, a model for simocyclinone D8 biosynthesis was proposed. Biosynthetic mutants were generated by insertional gene inactivation experiments, and culture extracts of these mutants were analyzed by high-performance liquid chromatography. Production of simocyclinone D8 was clearly detectable in the wild-type strain but was not detectable in the mutant strains. This indicated that indeed the sim gene cluster had been cloned.Simocyclinone D8 (Fig. 1) is produced by Streptomyces antibioticus Tü6040. It is active against gram-positive bacteria and also shows distinct cytostatic activities against human tumor cell lines (39,40,46). Simocyclinone D8 consists of four different moieties, an angucyclic polyketide core, a deoxyhexose (D-olivose), a tetraene side chain, and a halogenated aminocoumarin. The aromatic polyketide moiety is characterized by a large number of unusually placed hydroxyl groups and an oxiran bridge at positions C-12a and C-6a. It contains a Cglycosidically linked D-olivose at position C-9. Attached to the 4-OH group of D-olivose is an acetyl group, and attached to the 3-OH group is a tetraene side chain. Both are linked to the deoxysugar by ester bonds. The final amino-coumarin moiety is linked to the tetraene chain by an amide bond, resulting in an unusual polyene-amide structure. Features that distinguish simocyclinone from other angucycline antibiotics are the enormous size of the molecule and the fact that it originates from at least three different pools of primary metabolites. S. antibioticus Tü6040 also produces other simocyclinones, which can be seen as intermediates of simocyclinone D8. These compounds include simocyclinones of the A-series, the B-series, and the C-series, consisting either of the polyketide moiety (series A), the polyketide moiety plus D-olivose (series B), and the polyketide moiety plus D-olivose plus the tetraene side chain (series C) (40). Genetic engineering and combinatorial biosynthesis in bacteria provide an important new tool for drug discovery and drug design (16,19,24). Knowledge of the sequence and function of genes involved in the biosynthesis of natural products is prerequisite for this new approach. In the present study we describe the isolation of the simocyclinone biosynthetic gene cluster. Sequencing of the entire gene cluster revealed the presence of 49 open reading frames (ORFs) probably involved in simocyclinone bios...

Model mutants. The biosynthesis of glycopeptide antibiotics must be understood before it can be reprogrammed to generate altered antibiotics. Based on a detailed HPLC‐ESI‐MS analysis of linear and cyclic peptide intermediates of balhimycin biosynthesis mutants, a new model for glycopeptide assembly is suggested (see figure). We propose that the three central oxidative cyclizations by P450‐dependant monooxygenases occur during peptide assembly before cleavage from the nonribosomal peptide synthetase complex.