The mannopeptimycins are a novel class of lipoglycopeptide antibiotics active against multidrug-resistant pathogens with potential as clinically useful antibacterials. This report is the first to describe the biosynthesis of this novel class of mannosylated lipoglycopeptides. Included here are the cloning, sequencing, annotation, and manipulation of the mannopeptimycin biosynthetic gene cluster from Streptomyces hygroscopicus NRRL 30439. Encoded by genes within the mannopeptimycin biosynthetic gene cluster are enzymes responsible for the generation of the hexapeptide core (nonribosomal peptide synthetases [NRPS]) and tailoring reactions (mannosylation, isovalerylation, hydroxylation, and methylation). The NRPS system is noncanonical in that it has six modules utilizing only five amino acid-specific adenylation domains and it lacks a prototypical NRPS macrocyclizing thioesterase domain. Analysis of the mannopeptimycin gene cluster and its engineering has elucidated the mannopeptimycin biosynthetic pathway and provides the framework to make new and improved mannopeptimycins biosynthetically.Multidrug-resistant gram-positive bacterial pathogens now exist, and their rising numbers are a major concern. The prevalence of methicillin-resistant Staphylococcus aureus (MRSA) and vancomycin-resistant enterococci (VRE) exemplifies the seriousness of this concern (22, 29). Finding novel antibiotics active against such drug-resistant gram-positive pathogens, however, is problematic (32, 42). The mannopeptimycins ( Fig. 1), which are produced by Streptomyces hygroscopicus NRRL 30439, represent a new class of lipoglycopeptide antibiotics with exceptional in vitro and in vivo antibacterial activities against MRSA, VRE, and penicillin-resistant Streptococcus pneumoniae (17,36). Studies with mannopeptimycin-␦ demonstrated that this antibiotic acts by blocking the transglycosylation reaction in cell wall biosynthesis mediated by lipid II binding. Mannopeptimycin-␦ inhibits gram-positive cell wall biosynthesis through a mechanism which does not compete or render it ineffective due to crossresistance with the vancomycin-type antibiotics (33, 36).The unique structure, mode of action, and bioactivity of the mannopeptimycins have generated much interest in identifying a therapeutic candidate from this new class of molecules (17,33,36). One semisynthetic analog, AC98-6446, of the natural mannopeptimycins is significantly more potent and effective than the natural mannopeptimycins, with MICs in the 15-to 60-ng/ml range against MRSA, VRE, penicillin-resistant Streptococcus pneumoniae, and glycopeptide-intermediate Staphylococcus aureus (10, 31). The success in improving on the natural mannopeptimycins argues strongly for broadening the effort of generating additional new mannopeptimycins through promising strategies such as combinatorial biosynthesis, mutasynthesis, and chemoenzymatic synthesis (4, 28). The establishment and success of such approaches hinge on the cloning of the mannopeptimycin biosynthetic gene cluster and elucidation of ...

The lomaiviticins are a family of cytotoxic marine natural products that have captured the attention of both synthetic and biological chemists due to their intricate molecular scaffolds and potent biological activities. Here we describe the identification of the gene cluster responsible for lomaiviticin biosynthesis in Salinispora pacifica strains DPJ-0016 and DPJ-0019 using a combination of molecular approaches and genome sequencing. The link between the lom gene cluster and lomaiviticin production was confirmed using bacterial genetics, and subsequent analysis and annotation of this cluster revealed the biosynthetic basis for the core polyketide scaffold. Additionally, we have used comparative genomics to identify candidate enzymes for several unusual tailoring events, including diazo formation and oxidative dimerization. These findings will allow further elucidation of the biosynthetic logic of lomaiviticin assembly and provide useful molecular tools for application in biocatalysis and synthetic biology.

Pseudopterogorgia elisabethae is a common inhabitant of Caribbean reefs and is a well-known source of diterpenes with diverse biological activities. Notably, this octocoral is the sole source of the pseudopterosin family of anti-inflammatory diterpenes and is harvested to supply commercial demand for these metabolites. We have characterized the composition of the bacterial community associated with P. elisabethae collected from Providencia Island, Colombia, using both culture-dependent and culture-independent approaches. Culture-independent analysis revealed that the bacterial communities were composed of eight phyla, of which Proteobacteria was the most abundant. At the class level, bacterial communities were dominated by Gammaproteobacteria (82-87 %). Additionally, operational taxonomic units related to Pseudomonas and Endozoicomonas species were the most abundant phylotypes consistently associated with P. elisabethae colonies. Culture-dependent analysis resulted in the identification of 40 distinct bacteria classified as Bacilli (15), Actinobacteria (12), Gammaproteobacteria (9), Alphaproteobacteria (3), and Betaproteobacteria (1). Only one of the 40 cultured bacteria was closely related to a dominant phylotype detected in the culture-independent study, suggesting that conventional culturing techniques failed to culture the majority of octocoral-associated bacterial diversity. To the best of our knowledge, this is the first characterization of the bacterial diversity associated with P. elisabethae.

Expression of biosynthetic pathways in heterologous hosts is an emerging approach to expedite production improvement and biosynthetic modification of natural products derived from microbial secondary metabolites. Herein we describe the development of a versatile Escherichia coli-Streptomyces shuttle Bacterial Artificial Chromosomal (BAC) conjugation vector, pSBAC, to facilitate the cloning, genetic manipulation, and heterologous expression of actinomycetes secondary metabolite biosynthetic gene clusters. The utility of pSBAC was demonstrated through the rapid cloning and heterologous expression of one of the largest polyketide synthase (PKS) and nonribosomal peptide synthetase (NRPS) biosynthetic pathways: the meridamycin biosynthesis gene cluster (mer). The entire mer gene cluster ( approximately 90 kb) was captured in a single pSBAC clone through a straightforward restriction enzyme digestion and cloning approach and transferred into Streptomyces lividans. The production of meridamycin (1) in the heterologous host was achieved after replacement of the original promoter with an ermE* promoter and was enhanced by feeding with a biosynthetic precursor. The success of heterologous expression of such a giant gene cluster demonstrates the versatility of BAC cloning technology and paves the road for future exploration of expression of the meridamycin biosynthetic pathway in various hosts, including strains that have been optimized for polyketide production.

are known for producing diverse secondary metabolites. Combining genomics with untargeted data-dependent tandem MS and molecular networking, we characterized the secreted metabolome of the tunicamycin producer NRRL 3882. The genome harbors 128 predicted biosynthetic gene clusters. We detected>1,000 distinct secreted metabolites in culture supernatants, only 22 of which were identified based on standards and public spectral libraries. adapts the secreted metabolome to cultivation conditions. A number of metabolites are produced iron dependently, among them 17 desferrioxamine siderophores aiding in iron acquisition. Eight previously unknown members of this long-known compound class are described. A single desferrioxamine synthesis gene cluster was detected in the genome, yet different sets of desferrioxamines are produced in different media. Additionally, a polyether ionophore, differentially produced by the calcimycin biosynthesis cluster, was discovered. This illustrates that metabolite output of a single biosynthetic machine can be exquisitely regulated not only with regard to product quantity but also with regard to product range. Compared with chemically defined medium, in complex medium, total metabolite abundance was higher, structural diversity greater, and the average molecular weight almost doubled. Tunicamycins, for example, were only produced in complex medium. Extrapolating from this study, we anticipate that the larger part of bacterial chemistry, including chemical structures, ecological functions, and pharmacological potential, is yet to be uncovered.

The mannopeptimycins (MPPs) are potent glycopeptide antibiotics that contain both D and L forms of the unique, arginine-derived amino acid beta-hydroxyenduracididine (betahEnd). The product of the mppO gene in the MPP biosynthetic cluster resembles several non-heme iron, alpha-ketoglutarate-dependent oxygenases, such as VioC and clavaminate synthase. The role of MppO in betahEnd biosynthesis was confirmed through inactivation of mppO, which yielded a strain that produced dideoxy-MPPs, indicating that mppO is essential for generating the beta-hydroxy functionality for both betahEnd residues. Characterization in vitro of recombinant His6-MppO expressed in E. coli revealed that MppO selectively hydroxylates the beta carbon of free L-enduracididine.

Two Gram-negative, facultatively anaerobic, rod-shaped bacteria, strains EF212T and PS125T, were isolated from the octocorals Eunicea fusca and Plexaura sp., respectively. EF212T was isolated from a specimen of E. fusca collected off the coast of Florida, USA, and PS125T was isolated from a specimen of Plexaura sp. collected off the coast of Bimini, Bahamas. Analysis of the nearly full-length 16S rRNA gene sequences showed that these novel strains were most closely related to Endozoicomonas montiporae CL-33T, E. elysicola MKT110T and E. numazuensis HC50T (EF212T, 95.6–97.2 % identity; PS125T, 95.1–96.4 % identity). DNA–DNA hybridization values among EF212T, PS125T, E. montiporae LMG 24815T and E. elysicola KCTC 12372T were far below the 70 % cut-off, with all values for duplicate measurements being less than 35 %. Both EF212T and PS125T required NaCl for growth and showed optimal growth at 2–3 % NaCl, 22-30 °C and pH 8.0. The predominant cellular fatty acids were summed feature 3 (C16 : 1ω6c and/or C16 : 1ω7c), summed feature 8 (C18 : 1ω6c and/or C18 : 1ω7c), C16 : 0 and C14 : 0. The DNA G+C content of EF212T was 48.6 mol% and that of PS125T was 47.5 mol%. In addition to the genotypic differences observed between the two novel strains and related type strains, phenotypic and chemotaxonomic experiments also revealed differences between strains. Thus, strains EF212T and PS125T represent novel species of the genus Endozoicomonas , for which the names Endozoicomonas euniceicola sp. nov. and Endozoicomonas gorgoniicola sp. nov., respectively, are proposed. The type strains are EF212T ( = NCCB 100458T = DSM 26535T) for Endozoicomonas euniceicola sp. nov. and PS125T ( = NCCB 100438T = CECT 8353T) for Endozoicomonas gorgoniicola sp. nov. An emended description of the genus Endozoicomonas is also provided to encompass differences observed in the results of genotypic, chemotaxonomic and phenotypic tests compared from the original and amended genus descriptions.

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