Metabolomics and Genomics Enable the Discovery of a New Class of Nonribosomal Peptidic Metallophores from a Marine Micromonospora.

Although microbial genomes harbor an abundance of biosynthetic gene clusters, there remain substantial technological gaps that impair the direct correlation of newly discovered gene clusters and their corresponding secondary metabolite products. As an example of one approach designed to minimize or bridge such gaps, we employed hierarchical clustering analysis and principal component analysis (hcapca, whose sole input is MS data) to prioritize 109 marine Micromonospora strains and ultimately identify novel strain WMMB482 as a candidate for in-depth "metabologenomics" analysis following its prioritization. Highlighting the power of current MS-based technologies, not only did hcapca enable the discovery of one new, nonribosomal peptide bearing an incredible diversity of unique functional groups, but metabolomics for WMMB482 unveiled 16 additional congeners via the application of Global Natural Product Social molecular networking (GNPS), herein named ecteinamines A-Q (1-17). The ecteinamines possess an unprecedented skeleton housing a host of uncommon functionalities including a menaquinone pathway-derived 2-naphthoate moiety, 4-methyloxazoline, the first example of a naturally occurring Ψ[CH2NH] "reduced amide", a methylsulfinyl moiety, and a d-cysteinyl residue that appears to derive from a unique noncanonical epimerase domain. Extensive in silico analysis of the ecteinamine (ect) biosynthetic gene cluster and stable isotope-feeding experiments helped illuminate the novel enzymology driving ecteinamine assembly as well the role of cluster collaborations or "duets" in producing such structurally complex agents. Finally, ecteinamines were found to bind nickel, cobalt, zinc, and copper, suggesting a possible biological role as broad-spectrum metallophores.

Rapid Unambiguous Structure Elucidation of Streptnatamide A, a New Cyclic Peptide Isolated from A Marine-derived Streptomyces sp.

Cyclic peptides have been excellent source of drug leads. With the advances in discovery platforms, the pharmaceutical industry has a growing interest in cyclic peptides and has pushed several into clinical trials. However, structural complexity of cyclic peptides brings extreme challenges for structure elucidation efforts. Isotopic fine structure analysis, Nuclear magnetic resonance (NMR), and detailed tandem mass spectrometry rapidly provided peptide sequence for streptnatamide A, a cyclic peptide isolated from a marine-derived Streptomyces sp. Marfey's analysis determined the stereochemistry of all amino acids, enabling the unambiguous structure determination of this compound. A non-ribosomal peptide synthetase biosynthetic gene cluster (stp) was tentatively identified and annotated for streptnatamide A based on the in silico analysis of whole genome sequencing data. These analytical tools will be powerful tools to overcome the challenges for cyclic peptide structure elucidation and accelerate the development of bioactive cyclic peptides.

Bacillimidazoles A-F, Imidazolium-Containing Compounds Isolated from a Marine Bacillus.

Chemical investigations of a marine sponge-associated Bacillus revealed six new imidazolium-containing compounds, bacillimidazoles A-F (1-6). Previous reports of related imidazolium-containing natural products are rare. Initially unveiled by timsTOF (trapped ion mobility spectrometry) MS data, extensive HRMS and 1D and 2D NMR analyses enabled the structural elucidation of 1-6. In addition, a plausible biosynthetic pathway to bacillimidazoles is proposed based on isotopic labeling experiments and invokes the highly reactive glycolytic adduct 2,3-butanedione. Combined, the results of structure elucidation efforts, isotopic labeling studies and bioinformatics suggest that 1-6 result from a fascinating intersection of primary and secondary metabolic pathways in Bacillus sp. WMMC1349. Antimicrobial assays revealed that, of 1-6, only compound six displayed discernible antibacterial activity, despite the close structural similarities shared by all six natural products.

Bacillibactins E and F from a Marine Sponge-Associated Bacillus sp.

Chemical investigation of a marine sponge-associated Bacillus sp. led to the discovery of bacillibactins E and F (1 and 2). Despite containing the well-established cyclic triester core of iron-binding natural products such as enterobactin, bacillibactins E and F (1 and 2) are the first bacterial siderophores that contain nicotinic and benzoic acid moieties. The structures of the new compounds, including their absolute configurations, were determined by extensive spectroscopic analyses and Marfey's method. A plausible biosynthetic pathway to 1 and 2 is proposed; this route bears great similarity to other previously established bacillibactin-like pathways but appears to differentiate itself by a promiscuous DhbE, which likely installs the nicotinic moiety of 1 and the benzoic acid group of 2.

Pyridine-2,6-Dithiocarboxylic Acid and Its Metal Complexes: New Inhibitors of New Delhi Metallo ß-Lactamase-1.

Keywords: NDM-1 inhibitors; marine-derived Streptomyces sp.; carbapenem-resistant Enterobacteriaceae; metal chelators.

Bulbiferates A and B: Antibacterial Acetamidohydroxybenzoates from a Marine Proteobacterium, Microbulbifer sp.

Here we report the discovery of two new 3-acetamido-4-hydroxybenzoate esters, bulbiferates A (1) and B (2), isolated from Microbulbifer sp. cultivated from the marine tunicate Ecteinascidia turbinata. The structures of 1 and 2 were determined by analysis of 2D NMR and MS data. Additionally, three synthetic analogues (3-5), differing in ester sizes/lengths, were prepared for the purposes of evaluating potential structure-activity relationships; no clear correlations tying ester lengths to activity were evident. Bulbiferates A (1) and B (2) demonstrated antibacterial activity against both Escherichia coli (E. coli) and methicillin-sensitive Staphylococcus aureus (MSSA), whereas the synthetic analogues 3 and 4 displayed activity only against MSSA.

Phallusialides A-E, Pyrrole-Derived Alkaloids Discovered from a Marine-Derived Micromonospora sp. Bacterium Using MS-Based Metabolomics Approaches.

Integrating MS-based metabolomics approaches, LC-MS-PCA and molecular networking enabled the targeted isolation of five new pyrrole-derived alkaloids, phallusialides A-E (1-5), from a marine-derived Micromonospora sp. bacterium. The structures of 1-5 were elucidated by analysis of their HRMS, MS/MS, and NMR spectroscopic data. The absolute configuration of phallusialide A (1) was determined on the basis of comparisons of experimental and theoretically calculated ECD spectra. Compounds 1 and 2 exhibited antibacterial activity against methicillin resistant S. aureus (MRSA) and E. coli, with MIC values of 32 and 64 µg/mL, respectively, whereas 3-5 showed no antibacterial activity even at 256 µg/mL, yielding important SAR insights for this class of compounds.

Enhypyrazinones A and B, Pyrazinone Natural Products from a Marine-Derived Myxobacterium Enhygromyxa sp.

To date, studies describing myxobacterial secondary metabolites have been relatively scarce in comparison to those addressing actinobacterial secondary metabolites. This realization suggests the immense potential of myxobacteria as an intriguing source of secondary metabolites with unusual structural features and a wide array of biological activities. Marine-derived myxobacteria are especially attractive due to their unique biosynthetic gene clusters, although they are more difficult to handle than terrestrial myxobacteria. Here, we report the discovery of two new pyrazinone-type molecules, enhypyrazinones A and B, from a marine-derived myxobacterium Enhygromyxa sp. Their structures were elucidated by HRESIMS and comprehensive NMR data analyses. Compounds 1 and 2, which contain a rare trisubstituted-pyrazinone core, represent a unique class of molecules from Enhygromyxa sp.

Symbiosis-inspired approaches to antibiotic discovery.

Covering: 2010 up to 2017Life on Earth is characterized by a remarkable abundance of symbiotic and highly refined relationships among life forms. Defined as any kind of close, long-term association between two organisms, symbioses can be mutualistic, commensalistic or parasitic. Historically speaking, selective pressures have shaped symbioses in which one organism (typically a bacterium or fungus) generates bioactive small molecules that impact the host (and possibly other symbionts); the symbiosis is driven fundamentally by the genetic machineries available to the small molecule producer. The human microbiome is now integral to the most recent chapter in animal-microbe symbiosis studies and plant-microbe symbioses have significantly advanced our understanding of natural products biosynthesis; this also is the case for studies of fungal-microbe symbioses. However, much less is known about microbe-microbe systems involving interspecies interactions. Microbe-derived small molecules (i.e. antibiotics and quorum sensing molecules, etc.) have been shown to regulate transcription in microbes within the same environmental niche, suggesting interspecies interactions whereas, intraspecies interactions, such as those that exploit autoinducing small molecules, also modulate gene expression based on environmental cues. We, and others, contend that symbioses provide almost unlimited opportunities for the discovery of new bioactive compounds whose activities and applications have been evolutionarily optimized. Particularly intriguing is the possibility that environmental effectors can guide laboratory expression of secondary metabolites from "orphan", or silent, biosynthetic gene clusters (BGCs). Notably, many of the studies summarized here result from advances in "omics" technologies and highlight how symbioses have given rise to new anti-bacterial and antifungal natural products now being discovered.

Dedicated ent-kaurene and ent-atiserene synthases for platensimycin and platencin biosynthesis.

Platensimycin (PTM) and platencin (PTN) are potent and selective inhibitors of bacterial and mammalian fatty acid synthases and have emerged as promising drug leads for both antibacterial and antidiabetic therapies. Comparative analysis of the PTM and PTN biosynthetic machineries in Streptomyces platensis MA7327 and MA7339 revealed that the divergence of PTM and PTN biosynthesis is controlled by dedicated ent-kaurene and ent-atiserene synthases, the latter of which represents a new pathway for diterpenoid biosynthesis. The PTM and PTN biosynthetic machineries provide a rare glimpse at how secondary metabolic pathway evolution increases natural product structural diversity and support the wisdom of applying combinatorial biosynthesis methods for the generation of novel PTM and/or PTN analogues, thereby facilitating drug development efforts based on these privileged natural product scaffolds.

Expression of the platencin biosynthetic gene cluster in heterologous hosts yielding new platencin congeners.

Platensimycin (PTM) and platencin (PTN) are potent and selective inhibitors of bacterial and mammalian fatty acid synthases and have emerged as promising drug leads for both antibacterial and antidiabetic therapies. We have previously cloned and sequenced the PTM-PTN dual biosynthetic gene cluster from Streptomyces platensis MA7327 and the PTN biosynthetic gene cluster from S. platensis MA7339, the latter of which is composed of 31 genes encoding PTN biosynthesis, regulation, and resistance. We have also demonstrated that PTM or PTN production can be significantly improved upon inactivation of the pathway-specific regulator ptmR1 or ptnR1 in S. platensis MA7327 or MA7339, respectively. We now report engineered production of PTN and congeners in a heterologous Streptomyces host. Expression constructs containing the ptn biosynthetic gene cluster were engineered from SuperCos 1 library clones and introduced into five model Streptomyces hosts, and PTN production was achieved in Streptomyces lividans K4-114. Inactivation of ptnR1 was crucial for expression of the ptn biosynthetic gene cluster, thereby PTN production, in S. lividans K4-114. Six PTN congeners, five of which were new, were also isolated from the recombinant strain S. lividans SB12606, revealing new insights into PTN biosynthesis. Production of PTN in a model Streptomyces host provides new opportunities to apply combinatorial biosynthetic strategies to the PTN biosynthetic machinery for structural diversity.

Genome Mining and Metabolomics Unveil Pseudonochelin: A Siderophore Containing 5-Aminosalicylate from a Marine-Derived Pseudonocardia sp. Bacterium.

Pseudonochelin (1), a siderophore from a marine-derived Pseudonocardia sp. bacterium, was discovered using genome mining and metabolomics technologies. A 5-aminosalicylic acid (5-ASA) unit, not previously found in siderophore natural products, was identified in 1. Annotation of a putative psn biosynthetic gene cluster combined with bioinformatics and isotopic enrichment studies enabled us to propose the biosynthesis of 1. Moreover, 1 was found to display in vitro and in vivo antibacterial activity in an iron-dependent fashion.

Characterization of FdmV as an amide synthetase for fredericamycin A biosynthesis in Streptomyces griseus ATCC 43944.

Fredericamycin (FDM) A is a pentadecaketide natural product that features an amide linkage. Analysis of the fdm cluster from Streptomyces griseus ATCC 43944, however, failed to reveal genes encoding the types of amide synthetases commonly seen in natural product biosynthesis. Here, we report in vivo and in vitro characterizations of FdmV, an asparagine synthetase (AS) B-like protein, as an amide synthetase that catalyzes the amide bond formation in FDM A biosynthesis. This is supported by the findings that (i) inactivation of fdmV in vivo afforded the ΔfdmV mutant strain SB4027 that abolished FDM A and FDM E production but accumulated FDM C, a biosynthetic intermediate devoid of the characteristic amide linkage; (ii) FdmV in vitro catalyzes conversion of FDM C to FDM B, a known intermediate for FDM A biosynthesis (apparent K(m) = 162 ± 67 µM and k(cat) = 0.11 ± 0.02 min(-1)); and (iii) FdmV also catalyzes the amidation of FDM M-3, a structural analog of FDM C, to afford amide FDM M-6 in vitro, albeit at significantly reduced efficiency. Preliminary enzymatic studies revealed that, in addition to the common nitrogen sources (L-Gln and free amine) of class II glutamine amidotransferases (to which AS B belongs), FdmV can also utilize L-Asn as a nitrogen donor. The amide bond formation in FDM A biosynthesis is proposed to occur after C-8 hydroxylation but before the carbaspirocycle formation.

Actinopolysporins A-C and tubercidin as a Pdcd4 stabilizer from the halophilic actinomycete Actinopolyspora erythraea YIM 90600.

Our current natural product program utilizes new actinomycetes originating from unexplored and underexplored ecological niches, employing cytotoxicity against a selected panel of cancer cell lines as the preliminary screen to identify hit strains for natural product dereplication, followed by mechanism-based assays of the purified natural products to discover potential anticancer drug leads. Three new linear polyketides, actinopolysporins A (1), B (2), and C (3), along with the known antineoplastic antibiotic tubercidin (4), were isolated from the halophilic actinomycete Actinopolyspora erythraea YIM 90600, and the structures of the new compounds were elucidated on the basis of spectroscopic data interpretation. All four compounds were assayed for their ability to stabilize the tumor suppressor programmed cell death protein 4 (Pdcd4), which is known to antagonize critical events in oncogenic pathways. Only 4 significantly inhibited proteasomal degradation of a model Pdcd4-luciferase fusion protein, with an IC50 of 0.88±0.09 µM, unveiling a novel biological activity for this well-studied natural product.

Turbinmicin inhibits Candida biofilm growth by disrupting fungal vesicle-mediated trafficking.

The emergence of drug-resistant fungi has prompted an urgent threat alert from the US Centers for Disease Control (CDC). Biofilm assembly by these pathogens further impairs effective therapy. We recently identified an antifungal, turbinmicin, that inhibits the fungal vesicle-mediated trafficking pathway and demonstrates broad-spectrum activity against planktonically growing fungi. During biofilm growth, vesicles with unique features play a critical role in the delivery of biofilm extracellular matrix components. As these components are largely responsible for the drug resistance associated with biofilm growth, we explored the utility of turbinmicin in the biofilm setting. We found that turbinmicin disrupted extracellular vesicle (EV) delivery during biofilm growth and that this impaired the subsequent assembly of the biofilm matrix. We demonstrated that elimination of the extracellular matrix rendered the drug-resistant biofilm communities susceptible to fungal killing by turbinmicin. Furthermore, the addition of turbinmicin to otherwise ineffective antifungal therapy potentiated the activity of these drugs. The underlying role of vesicles explains this dramatic activity and was supported by phenotype reversal with the addition of exogenous biofilm EVs. This striking capacity to cripple biofilm assembly mechanisms reveals a new approach to eradicating biofilms and sheds light on turbinmicin as a promising anti-biofilm drug.

In vivo investigation of the roles of FdmM and FdmM1 in fredericamycin biosynthesis unveiling a new family of oxygenases.

Fredericamycin (FDM) A, a highly oxidized aromatic pentadecaketide natural product, exhibits potent cytotoxicity and has been studied as a new anticancer drug lead. The FDM biosynthetic gene cluster has been previously cloned from Streptomyces griseus ATCC 49344 and successfully expressed in the heterologous host Streptomyces albus J1074. The fdmM and fdmM1 genes code for two proteins with high sequence homology to each other but unknown function. In-frame deletion of each of the genes from the fdm cluster was accomplished in the S. albus host. Each mutant failed to produce FDM A and the key biosynthetic intermediate FDM E but produced various new metabolites, the titers of which were dramatically increased via overexpression of an fdm pathway-specific activator fdmR1. The DeltafdmM mutant strain accumulated three new compounds FDM M-1, FDM M-2, and FDM M-3, whereas the DeltafdmM1 mutant strain produced one new compound FDM M1-1. Isolation and structural characterization of these compounds enable us to propose that FdmM and FdmM1 catalyze the C-6 and C-8 hydroxylations for FDM biosynthesis, respectively. Homologs of FdmM and FdmM1 can be found in biosynthetic gene clusters of many other aromatic polyketides, ranging from dodecaketides to pentadecaketides, but to date all of them were annotated as proteins of unknown function. Based on the findings reported here for FdmM and FdmM1, we now propose similar functions for those proteins, and FdmM and FdmM1 therefore represent an emerging family of novel oxygenases responsible for hydroxylation of aromatic polyketide natural products.

iso-Migrastatin, migrastatin, and dorrigocin production in Streptomyces platensis NRRL 18993 is governed by a single biosynthetic machinery featuring an acyltransferase-less type I polyketide synthase.

iso-Migrastatin and related glutarimide-containing polyketides are potent inhibitors of tumor cell migration and their implied potential as antimetastatic agents for human cancers has garnered significant attention. Genome scanning of Streptomyces platensis NRRL 18993 unveiled two candidate gene clusters (088D and mgs); each encodes acyltransferase-less type I polyketide synthases commensurate with iso-migrastatin biosynthesis. Both clusters were inactivated by lambda-RED-mediated PCR-targeting mutagenesis in S. platensis; iso-migrastatin production was completely abolished in the DeltamgsF mutant SB11012 strain, whereas inactivation of 088D-orf7 yielded the SB11006 strain that exhibited no discernible change in iso-migrastatin biosynthesis. These data indicate that iso-migrastatin production is governed by the mgs cluster. Systematic gene inactivation allowed determination of the precise boundaries of the mgs cluster and the essentiality of the genes within the mgs cluster in iso-migrastatin production. The mgs cluster consists of 11 open reading frames that encode three acyltransferase-less type I polyketide synthases (MgsEFG), one discrete acyltransferase (MgsH), a type II thioesterase (MgsB), three post-PKS tailoring enzymes (MgsIJK), two glutarimide biosynthesis enzymes (MgsCD), and one regulatory protein (MgsA). A model for iso-migrastatin biosynthesis is proposed based on functional assignments derived from bioinformatics and is further supported by the results of in vivo gene inactivation experiments.

hcapca: Automated Hierarchical Clustering and Principal Component Analysis of Large Metabolomic Datasets in R.

Microbial natural product discovery programs face two main challenges today: rapidly prioritizing strains for discovering new molecules and avoiding the rediscovery of already known molecules. Typically, these problems have been tackled using biological assays to identify promising strains and techniques that model variance in a dataset such as PCA to highlight novel chemistry. While these tools have shown successful outcomes in the past, datasets are becoming much larger and require a new approach. Since PCA models are dependent on the members of the group being modeled, large datasets with many members make it difficult to accurately model the variance in the data. Our tool, hcapca, first groups strains based on the similarity of their chemical composition, and then applies PCA to the smaller sub-groups yielding more robust PCA models. This allows for scalable chemical comparisons among hundreds of strains with thousands of molecular features. As a proof of concept, we applied our open-source tool to a dataset with 1046 LCMS profiles of marine invertebrate associated bacteria and discovered three new analogs of an established anticancer agent from one promising strain.

Draft Genome Sequence of Pseudenhygromyxa sp. Strain WMMC2535, a Marine Ascidian-Associated Bacterium.

Pseudenhygromyxa WMMC2535, a representative of the myxobacteria (family Nannocystaceae), was isolated from a ragged sea hare in the Florida Keys, and its genome was sequenced using PacBio technology. The WMMC2535 genome sequence is the first of this genus and validates the notion that myxobacteria represent outstanding sources of structurally diverse natural products.

MS-Derived Isotopic Fine Structure Reveals Forazoline A as a Thioketone-Containing Marine-Derived Natural Product.

Forazoline A is a structurally complex PKS-NRPS hybrid produced by marine-derived Actinomadura sp. During the course of studies highlighting the application of IFS analysis as a powerful tool for natural products analysis, we were alerted to an earlier misinterpretation with respect to forazoline A structure elucidation. In particular, IFS reveals that forazoline A contains a thioketone moiety rarely seen in secondary metabolites and, thus, constitutes an even more intriguing structure than originally thought.