produces the lipopeptide antibiotic friulimicin, which is a cyclic peptide with

produces the lipopeptide antibiotic friulimicin, which is a cyclic peptide with one exocyclic amino acid linked to a branched-chain fatty acid acyl residue. acid, and 2,3-diaminobutyric acid. By using heterologous gene expression in and genes in the biosynthesis of 2,3-diaminobutyric acid by gene inactivation and subsequent feeding experiments. In recent years, the sequences of numerous gene clusters involved in the synthesis of many secondary metabolites have become available for comparison and exploitation. The programmed manipulation of genes encoding enzymes in the biosynthetic pathways offers promise for redesigning existing antibiotic structures to create antibiotics with new activities and the ability to overcome bacterial resistance (50). Therefore, each newly analyzed gene cluster represents a new tool for combinatorial biosynthesis and can provide FLAG tag Peptide IC50 information about the synthesis of unusual building blocks, such as nonproteinogenic amino acids, IFNA17 acyl residues, and sugar moieties. An interesting group of secondary metabolites for such experiments seems to be bioactive lipopeptides isolated from streptomycetes. So far, only a few biosynthetic gene clusters corresponding to these lipopeptides have been isolated and characterized, such as the clusters for calcium-dependent antibiotic (CDA) from (17), daptomycin from (26), and “type”:”entrez-protein”,”attrs”:”text”:”A54145″,”term_id”:”1083803″,”term_text”:”pirA54145 from (24). By targeted modification and gene exchange, it is possible to generate new lipopeptide structures (12, 25). Another member of this group of secondary metabolites is the antibiotic friulimicin that is produced by the actinomycete strains (4). The biosynthesis of this lipopeptide is usually catalyzed by nonribosomal peptide synthetases (NRPS) (15). The eight bioactive lipopeptides isolated from (4) consist of 10 amino acids that form a ring structure with one exocyclic amino acid linked to an acyl residue of various chain lengths. The acyl residue is usually a branched-chain fatty acid with a double bond. The structures of four of these lipopeptides are identical to those of known peptide antibiotics of the amphomycin group and possess aspartic acid at the exocyclic position. The other four lipopeptides have asparagine at this position and are known as friulimicins A to D. The peptide component of these friulimicins is usually characterized by unusual amino acids, such as l-threo- and d-erythro-2,3-diaminobutyric acid, d-pipecolinic acid, and l-threo–methylaspartic acid (48) (Fig. ?(Fig.1).1). The peptide also contains the conserved amino acid sequence DXDG, which is a putative calcium-binding site in the nonribosomally synthesized lipopeptides “type”:”entrez-protein”,”attrs”:”text”:”A54145″,”term_id”:”1083803″,”term_text”:”pirA54145 (24), daptomycin, and CDA and in the ribosomally assembled calmodulin (11) (Fig. ?(Fig.22). FIG. 1. Chemical structure of the lipopeptide antibiotic friulimicin B from HAG010964 (4) was studied. ATCC 6633 (ATCC, Manassas, VA) was used as a friulimicin-sensitive indicator organism. TOP 10 10 (Invitrogen, Karlsruhe, Germany) was used for cloning, and BL21(DE3)pLysS (Invitrogen) and TK23 (18) were used for heterologous expression. The methylase-negative strain ET12567(pUB307) (9) was used as the donor strain for intergeneric conjugation. Plasmid vectors used in this study were pMos(Amersham Biosciences, Freiburg, Germany), pBluescript (Stratagene, Amsterdam, The Netherlands), pLitmus28 (New England BioLabs, Frankfurt, Germany), pOK12 (49), and pUC19 (52). The cosmid library for was constructed with pOJ436 (6). The vector pRSETB (Invitrogen) and the streptomycete-shuttle vector pEM4 (30) were used for heterologous expression. The following resistance cassettes were used: the apramycin resistance cassette from pKC505 FLAG tag Peptide IC50 (32), the apramycin/resistance FLAG tag Peptide IC50 cassette from pEH13 (16), and the hygromycin resistance cassette from pHP45Hyg (20). pK18mob (37), the pK18mob derivative pDS401 carrying the apramycin resistance cassette (16), and pSET152 (6) were used for intergeneric conjugation. DNA sequencing and analysis. Cosmid 4E08 was shotgun sequenced. Overlapping DNA fragments of cosmids 18M80 and 5P02 were detected by Southern hybridization, subcloned, and sequenced. Sequences were analyzed with Clone Manager 7 (Scientific and Educational Software, Cary, NC), Artemis (33), Clustal X (47), GeneDoc (29), and the NRPS-PKS database (3). Open reading frames (ORFs) were analyzed using the codon usage table (28). Functional assignments were FLAG tag Peptide IC50 made using the BLAST program (2) to compare the deduced gene products with proteins of known functions listed in the NCBI database. Cosmids 18M80, 4E08, and 5P02 cover the regions to to genes were obtained by PCR using the primer pair O1/O2 for and the primer pair AP1/AP2 for (Table ?(Table1).1). The fragments were cloned into HindIII-EcoRI-digested vector pDS401, resulting in pCMF11 and pCMF10,.

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