Capistruin is a 19-residue ribosomally synthesized lasso peptide encoded by the capABCD gene cluster in Burkholderia thailandensis. It is composed of an N-terminal 9-residue macrolactam ring, through which the 10-residue C-terminal tail is threaded. Using a heterologous capistruin production system in Escherichia coli, we have generated 48 mutants of the precursor protein CapA to gain insights into capistruin biosynthesis. Only 4 residues (Gly1, Arg11, Val12, and Ile13) of the lasso sequence were found to be critical for maturation. Tandem mass spectrometric fragmentation studies of capistruin F16A/F18A proved Arg15 to be responsible for the trapping of the C-terminal tail. Substituting Arg15 and Phe16 by alanine revealed a temperature-sensitive capistruin derivative, which unfolds into a branched cyclic peptide upon heating. In conclusion, our global mutagenic approach revealed a low overall specificity of the biosynthetic machinery and important structure-stability correlations.

Understanding the ribosome-independent biosynthesis of daptomycin assembly will provide opportunities for the generation of daptomycin derivatives with an altered pharmaceutical spectrum to address upcoming daptomycin-resistant pathogens. Herein, the structural properties of daptomycin, its biosynthesis, recent efforts for the generation of structural diversity, and its proposed mode of action are discussed.The rapid bacterial acquisition of resistance to conventional antibiotics represents an increasing challenge in treating infections with the contemporary drug arsenal (1). Especially the rise of drug-resistant Gram-positive pathogens, exemplified by methicillin-resistant Staphylococcus aureus (MRSA) 2 or vancomycin-resistant enterococci, underlines the urgent demand for antibiotics with alternative modes of action (2). The discovery of natural products as antibacterial drugs had a drastic impact on fatality rates and gave rise to a variety of antibacterial drug classes (3-5). Although the effort for the identification of new bioactive compounds has drastically increased in the past decades, only three new antibacterial classes have been approved by the Food and Drug Administration since the 1970s, one of them being daptomycin (Cubicin, Cubist Pharmaceuticals) (6). Daptomycin, a decanoic acid-inheriting acidic lipopeptide, was isolated as a member of an antibiotic complex, termed A21978C factors, from cultures of Streptomyces roseosporus (7). Daptomycin is a nontopically used natural lipopeptide antibiotic approved by the Food and Drug Administration in 2003 for the treatment of skin and skin structure infections caused by Gram-positive pathogens and for the treatment of bacteremia and right-sided endocarditis caused by S. aureus strains and MRSA in 2006 (8). Daptomycin does not meet non-inferiority criteria for the treatment of community-acquired pneumonia (9). Its low efficacy against community-acquired pneumonia is considered to be due to inhibition by pulmonary surfactants (10). Recently, Eisenstein et al. (11) provided an interesting historical overview on how daptomycin became available to the market. As the development of daptomycin resistance in Enterococcus faecium and S. aureus has been reported, concerns about decreasing clinical effectiveness of daptomycin will have to be addressed (12)(13)(14). Comprehensive knowledge about the biosynthesis of the lead compound offers the opportunity to enhance the structural diversity and the corresponding bioactivity of daptomycin (15)(16)(17)(18). In this minireview, the structural and functional properties of daptomycin as a member of the acidic lipopeptide family are presented, with focus on daptomycin biosynthesis. In addition, recent efforts and advances in the generation of novel daptomycin derivatives by means of genetic engineering and chemoenzymatic approaches are highlighted. In the last part of this minireview, the mode of action (MOA) of daptomycin is discussed in detail. Daptomycin, a Prototype of the Acidic Lipopeptide FamilyDaptomycin is a member...

Lysobactin (katanosin B) is a macrocyclic depsipeptide, displaying high antibacterial activity against human pathogens. In this work, we have identified and characterized the entire biosynthetic gene cluster responsible for lysobactin assembly. Sequential analysis of the Lysobacter sp. ATCC 53042 genome revealed the lysobactin gene cluster to encode two multimodular nonribosomal peptide synthetases. As the number of modules found within the synthetases LybA and LybB directly correlates with the primary sequence of lysobactin, a linear logic of lysobactin biosynthesis is proposed. Investigation of adenylation domain specificities in vitro confirmed the direct association between the synthetases and lysobactin biosynthesis. Furthermore, an unusual tandem thioesterase architecture of the LybB termination module was identified. Biochemical characterization of the individual thioesterases in vitro provides evidence that solely penultimate thioesterase domain mediates the cyclization and simultaneous release of lysobactin.

In this work we report the isolation, structural characterization, and the genetic analysis of the biosynthetic origin of rhodochelin, a unique mixed-type catecholate-hydroxamate siderophore isolated from Rhodococcus jostii RHA1. Rhodochelin structural elucidation was accomplished via MS(n)- and NMR-analysis and revealed the tetrapeptide to contain an unusual ester bond between an L-δ-N-formyl-δ-N-hydroxyornithine moiety and the side chain of a threonine residue. Gene deletions within three putative biosynthetic gene clusters abolish rhodochelin production, proving that the ORFs responsible for rhodochelin biosynthesis are located in different chromosomal loci. These results demonstrate the efficient cross-talk between distantly located secondary metabolite gene clusters and outline new insights into the comprehension of natural product biosynthesis.

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