A major goal in natural product discovery programs is to rapidly dereplicate known entities from complex biological extracts. We demonstrate here that molecular networking, an approach that organizes MS/MS data based on chemical similarity, is a powerful complement to traditional dereplication strategies. Successful dereplication with molecular networks requires MS/MS spectra of the natural product mixture along with MS/MS spectra of known standards, synthetic compounds, or well-characterized organisms, preferably organized into robust databases. This approach can accommodate different ionization platforms, enabling cross correlations of MS/MS data from ambient ionization, direct infusion, and LC-based methods. Molecular networking not only dereplicates known molecules from complex mixtures, it also captures related analogs, a challenge for many other dereplication strategies. To illustrate its utility as a dereplication tool, we apply mass spectrometry-based molecular networking to a diverse array of marine and terrestrial microbial samples, illustrating the dereplication of 58 molecules including analogs.

The human skin is an organ with a surface area of 1.5-2 m 2 that provides our interface with the environment. The molecular composition of this organ is derived from host cells, microbiota, and external molecules. The chemical makeup of the skin surface is largely undefined. Here we advance the technologies needed to explore the topographical distribution of skin molecules, using 3D mapping of mass spectrometry data and microbial 16S rRNA amplicon sequences. Our 3D maps reveal that the molecular composition of skin has diverse distributions and that the composition is defined not only by skin cells and microbes but also by our daily routines, including the application of hygiene products. The technological development of these maps lays a foundation for studying the spatial relationships of human skin with hygiene, the microbiota, and environment, with potential for developing predictive models of skin phenotypes tailored to individual health.human skin | 3D mapping | mass spectrometry | 16S rRNA T he skin provides the interface between our internal molecular processes and the external environment. The stratum corneum (SC), the outer layer of the epidermis, is the most exposed organ of the human body and is composed of molecules derived from our own skin cells, our microbiota, and the environment (1). In addition, it may be possible that personal hygiene products, the clothing we wear, and the food we eat can affect the chemical composition of the skin (2, 3). The chemical environment of the skin is also critical for the development of microbial communities, yet there are no systematic workflows that can be used to correlate skin chemistry and microbes. Human skin microbiome inventories are changing our views of commensal organisms and their roles in establishing and maintaining the immune system and general epithelial health (4-7), but their role in biotransformation of skin molecules is as yet poorly characterized.Understanding the molecular topography of the surface of the SC will provide insights into the chemical environment in which microbes reside on the skin and how in turn they modify this environment. To understand the relationship between the chemical milieu and the microbial communities, we must develop workflows that map the chemistry and microbiology of the human skin and that determine the associations between them. The composition of the human skin microbiota has been correlated with skin anatomy, dividing into moist, dry, and sebaceous microenvironments, and can be influenced by beauty and hygiene products (2-9). Regions such as the face, chest, and back, areas with a high density of sebaceous glands, promote growth of lipophilic microorganisms such as Propionibacterium and Malassezia. In contrast, less exposed regions, such as the groin, axilla, and toe web, which are higher in temperature and moisture content, are colonized by Staphylococcus and Corynebacterium (7, 10, 11). Previous studies have reported mass spectrometry (MS) methods to identify compounds from human skin (12, 13). However, th...

In many macroorganisms, the ultimate source of potent biologically active natural products has remained elusive due to an inability to identify and culture the producing symbiotic microorganisms. As a model system for developing a meta-omic approach to identify and characterize natural product pathways from invertebrate-derived microbial consortia we chose to investigate the ET-743 (Yondelis®) biosynthetic pathway. This molecule is an approved anti-cancer agent obtained in low abundance (10−4–10−5% w/w) from the tunicate Ecteinascidia turbinata, and is generated in suitable quantities for clinical use by a lengthy semi-synthetic process. Based on structural similarities to three bacterial secondary metabolites, we hypothesized that ET-743 is the product of a marine bacterial symbiont. Using metagenomic sequencing of total DNA from the tunicate/microbial consortium we targeted and assembled a 35 kb contig containing 25 genes that comprise the core of the NRPS biosynthetic pathway for this valuable anti-cancer agent. Rigorous sequence analysis based on codon usage of two large unlinked contigs suggests that Candidatus Endoecteinascidia frumentensis produces the ET-743 metabolite. Subsequent metaproteomic analysis confirmed expression of three key biosynthetic proteins. Moreover, the predicted activity of an enzyme for assembly of the tetrahydroisoquinoline core of ET-743 was verified in vitro. This work provides a foundation for direct production of the drug and new analogs through metabolic engineering. We expect that the interdisciplinary approach described is applicable to diverse host-symbiont systems that generate valuable natural products for drug discovery and development.

Petrobactin, a virulence-associated siderophore produced by Bacillus anthracis, chelates ferric iron through the rare 3,4-isomer of dihydroxybenzoic acid (3,4-DHBA). Most catechol siderophores, including bacillibactin and enterobactin, use 2,3-DHBA as a biosynthetic subunit. Significantly, siderocalin, a factor involved in human innate immunity, sequesters ferric siderophores bearing the more typical 2,3-DHBA moiety, thereby impeding uptake of iron by the pathogenic bacterial cell. In contrast, the unusual 3,4-DHBA component of petrobactin renders the siderocalin system incapable of obstructing bacterial iron uptake. Although recent genetic and biochemical studies have revealed selected early steps in petrobactin biosynthesis, the origin of 3,4-DHBA as well as the function of the protein encoded by the final gene in the B. anthracis siderophore biosynthetic (asb) operon, asbF (BA1986), has remained unclear. In this study we demonstrate that 3,4-DHBA is produced through conversion of the common bacterial metabolite 3-dehydroshikimate (3-DHS) by AsbF-a 3-DHS dehydratase. Elucidation of the cocrystal structure of AsbF with 3,4-DHBA, in conjunction with a series of biochemical studies, supports a mechanism in which an enolate intermediate is formed through the action of this 3-DHS dehydratase metalloenzyme. Structural and functional parallels are evident between AsbF and other enzymes within the xylose isomerase TIM-barrel family. Overall, these data indicate that microbial species shown to possess homologs of AsbF may, like B. anthracis, also rely on production of the unique 3,4-DHBA metabolite to achieve full viability in the environment or virulence within the host.AsbF structure ͉ dehydratase ͉ siderophore ͉ virulence factor S iderophore production in pathogenic bacteria has gained considerable attention because of its crucial function in essential iron uptake by many microbes and the relevance of siderophore-associated proteins as molecular markers of various infectious agents (1). In Bacillus anthracis, the causative agent of anthrax, 2 siderophores, petrobactin and bacillibactin (Fig. 1A), play a significant role during iron acquisition (2-4), but only petrobactin is absolutely essential for full virulence within a mammalian host (5). This siderophore was initially discovered from the Gram-negative marine bacterium Marinobacter hydrocarbonoclasticus, whose genome bears a biosynthetic gene cluster homologous to the B. anthracis asb operon (2). Recent genetic and chemical analysis suggests that petrobactin biosynthesis may also be a prerequisite for virulence in related Bacillus species (6). These studies highlight the importance of elucidating the mechanisms of siderophore production in pathogenic microbes as a target for abrogating infection by organisms like B. anthracis, a rapidly virulent microbe with proven potential as a bioterrorism agent. Based on these factors, we have initiated studies to investigate key biosynthetic enzymes for petrobactin assembly in efforts to establish new antimicrobial targets t...

Summary The putative modular polyketide synthase (PKS) that prescribes biosynthesis of the bryostatin natural products from the uncultured bacterial symbiont of the marine bryozoan Bugula neritina possesses a discrete ORF (bryP) that encodes a protein containing tandem acyltransferase (AT) domains upstream of the PKS ORFs. BryP is hypothesized to catalyze in trans acylation of the PKS modules for polyketide chain elongation. To verify conservation of function, bryP was introduced into AT-deletion mutant strains of a heterologous host containing a PKS cluster with similar architecture, and polyketide production was partially rescued. Biochemical characterization demonstrated that BryP catalyzes selective malonyl-CoA acylation of native and heterologous acyl carrier proteins and complete PKS modules in vitro. The results support the hypothesis that BryP loads malonyl-CoA onto Bry PKS modules, and provide the first biochemical evidence of the functionality of the bry cluster.

A biocatalytic platform that employs the final two monomodular type I polyketide synthases (PKS) of the pikromycin pathway in vitro followed by direct appendage of D-desosamine and final C-H oxidation(s) in vivo was developed and applied toward the synthesis of a suite of 12-and 14-membered ring macrolide natural products. This methodology delivered both compound classes in thirteen steps (longest linear sequence) from commercially available (R)-Roche ester in >10% overall yields.

Saltwater culture of Myrothecium verrucaria, separated from a Spongia sp. collected in Hawaii, was a source of three new trichothecenes, 3-hydroxyroridin E (1a), 13′-acetyltrichoverrin B (2), and miophytocen C (3) and nine known related compounds (1b, 4, 5, 6, 7a, 7b, 8, 9a, and 9b). The stereostructures of the new compounds were established on the basis of 1D and 2D NMR spectral analyses and a chemical transformation. At the same time, the stereostructures of known compounds, 1b, 4, and 5 reported previously were also elucidated. All the compounds except 3 showed significant cytotoxicity against murine and human tumor cell lines. Moreover, the structure-activity relationships (SARs) were established from the results of the bioassay data.

Summary In vitro analysis of natural product biosynthetic gene products isolated from unculturable symbiotic bacteria is necessary to probe the functionalities of these enzymes. Herein, we report the biochemical characterization of BryR, the 3-hydroxy-3-methylglutaryl (HMG)-CoA synthase (HMGS) homolog implicated in β-branching at C13 and C21 of the core ring system from the bryostatin metabolic pathway (Bry). We confirmed the activity of BryR using two complementary methods, radio-SDS PAGE and Fourier Transform Ion Cyclotron Resonance-Mass Spectrometry (FTICR-MS). The activity of BryR depended on pairing of the native acetoacetyl-BryM3 acceptor acyl carrier protein (ACP) with an appropriate donor acetyl-ACP from a heterologous HMGS cassette. Additionally, the ability of BryR to discriminate between various ACPs was assessed using a surface plasmon resonance (SPR)-based protein-protein binding assay. Our data suggest that specificity for a protein-bound acyl group is a distinguishing feature between HMGS homologs found in PKS or PKS/NRPS biosynthetic pathways and those of primary metabolism. These findings reveal an important example of molecular recognition between protein components that are essential for biosynthetic fidelity in natural product assembly and modification.