Actinoplanes friuliensis produces the lipopeptide antibiotic friulimicin, which is a cyclic peptide with one exocyclic amino acid linked to a branched-chain fatty acid acyl residue. The structural relationship to daptomycin and the excellent antibacterial performance of friulimicin make the antibiotic an attractive drug candidate. The complete friulimicin biosynthetic gene cluster of 24 open reading frames from A. friuliensis was sequenced and analyzed. In addition to genes for regulation, self-resistance, and transport, the cluster contains genes encoding peptide synthetases, proteins involved in the synthesis and linkage of the fatty acid component of the antibiotic, and proteins involved in the synthesis of the nonproteinogenic amino acids pipecolinic acid, methylaspartic acid, and 2,3-diaminobutyric acid. By using heterologous gene expression in Escherichia coli, we provide biochemical evidence for the stereoselective synthesis of L-pipecolinic acid by the deduced protein of the lysine cyclodeaminase gene pip. Furthermore, we show the involvement of the dabA and dabB 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 information about the synthesis of unusual building blocks, such as nonproteinogenic amino acids, 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 Streptomyces coelicolor (17), daptomycin from Streptomyces roseosporus (26), and A54145 from Streptomyces fradiae (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 Actinoplanes friuliensis. This compound is highly active against multidrug-resistant gram-positive bacteria, such as methicillinresistant Staphylococcus aureus strains (4). The biosynthesis of this lipopeptide is catalyzed by nonribosomal peptide synthetases (NRPS) (15).The eight bioactive lipopeptides isolated from A. friuliensis (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 a branched-chain fatty acid with a ⌬cis3 double bond. The ...

The molecular basis of orchid flower development is accomplished through a specific regulatory program in which the class B MADS-box AP3/DEF genes play a central role. In particular, the differential expression of four class B AP3/DEF genes is responsible for specification of organ identities in the orchid perianth. Other MADS-box genes (AGL6 and SEP-like) enrich the molecular program underpinning the orchid perianth development, resulting in the expansion of the original “orchid code” in an even more complex gene regulatory network. To identify candidates that could interact with the AP3/DEF genes in orchids, we conducted an in silico differential expression analysis in wild-type and peloric Phalaenopsis. The results suggest that a YABBY DL-like gene could be involved in the molecular program leading to the development of the orchid perianth, particularly the labellum. Two YABBY DL/CRC homologs are present in the genome of Phalaenopsis equestris, PeDL1 and PeDL2, and both express two alternative isoforms. Quantitative real-time PCR analyses revealed that both genes are expressed in column and ovary. In addition, PeDL2 is more strongly expressed the labellum than in the other tepals of wild-type flowers. This pattern is similar to that of the AP3/DEF genes PeMADS3/4 and opposite to that of PeMADS2/5. In peloric mutant Phalaenopsis, where labellum-like structures substitute the lateral inner tepals, PeDL2 is expressed at similar levels of the PeMADS2-5 genes, suggesting the involvement of PeDL2 in the development of the labellum, together with the PeMADS2-PeMADS5 genes. Although the yeast two-hybrid analysis did not reveal the ability of PeDL2 to bind the PeMADS2-PeMADS5 proteins directly, the existence of regulatory interactions is suggested by the presence of CArG-boxes and other MADS-box transcription factor binding sites within the putative promoter of the orchid DL2 gene.

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