The potential of the diverse chemistries present in natural products (NP) for biotechnology and medicine remains untapped because NP databases are not searchable with raw data and the NP community has no way to share data other than in published papers. Although mass spectrometry techniques are well-suited to high-throughput characterization of natural products, there is a pressing need for an infrastructure to enable sharing and curation of data. We present Global Natural Products Social molecular networking (GNPS, http://gnps.ucsd.edu), an open-access knowledge base for community wide organization and sharing of raw, processed or identified tandem mass (MS/MS) spectrometry data. In GNPS crowdsourced curation of freely available community-wide reference MS libraries will underpin improved annotations. Data-driven social-networking should facilitate identification of spectra and foster collaborations. We also introduce the concept of ‘living data’ through continuous reanalysis of deposited data.

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The chemical diversity of natural products is fueled by the emergence and ongoing evolution of biosynthetic pathways in secondary metabolism [1][2][3][4][5] . However, co-evolution of enzymes as functional assemblies for metabolic diversification is not well understood, especially at the biochemical level. Here, two parallel enzyme assemblies with an extraordinarily high sequence identity form a β-branched cyclopropane in the curacin A (Cur), and a vinyl chloride group in the jamaicamide (Jam) pathways, respectively. The assemblies include a halogenase (Hal), a 3-hydroxy-3-methylglutaryl (HMG) enzyme cassette for β-branching and an enoyl reductase domain (ER). Bioinformatic analysis indicated that the corresponding genes were inserted into modular polyketide synthases (PKSs) via acyltransferase (AT) domain replacement. The Hal from CurA, and the dehydratases (ECH 1 s) and decarboxylases (ECH 2 s) within the HMG enzyme cassettes and ERs from both Cur and Jam were assessed biochemically to determine the mechanism of cyclopropane and vinyl chloride formation. Unexpectedly, the polyketide β-branching pathway was modified by introduction of a γ-chlorination step on (S)-HMG mediated by Cur Hal, a nonheme Fe II , α-ketoglutarate (α-KG)-dependent halogenase 6 . In a divergent scheme, Cur ECH 2 was found to catalyze formation of the α,β C=C enoyl thioester, whereas Jam ECH 2 formed a vinyl chloride moiety by selectively generating the corresponding β,γ C=C (enoyl thioester) of the 3-methyl-4-chloroglutaconyl decarboxylation product. A non-conserved Tyr 82 residue in Cur ECH 2

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An innovative approach was developed for the discovery of new natural products by combining mass spectrometric metabolic profiling with genomic analysis, and resulted in the discovery of the columbamides, a new class of di- and tri-chlorinated acyl amides with cannabinomimetic activity. Three species of cultured marine cyanobacteria, Moorea producens 3L, Moorea producens JHB and Moorea bouillonii PNG, were subjected to genome sequencing and analysis for their recognizable biosynthetic pathways, and this information was then compared with their respective metabolomes as detected by MS-profiling. By genome analysis, a presumed regulatory domain was identified upstream of several previously described biosynthetic gene clusters in two of these cyanobacteria, M. producens 3L and M. producens JHB. A similar regulatory domain was identified in the M. bouillonii PNG genome, and a corresponding downstream biosynthetic gene cluster was located and carefully analyzed. Subsequently, MS-based molecular networking identified a series of candidate products, and these were isolated and their structures rigorously established. Based on their distinctive acyl amide structure, the most prevalent metabolite was evaluated for cannabinomimetic properties and found to be a moderate affinity ligand for CB1.

Cyanobacteria possess the unique capacity to naturally produce hydrocarbons from fatty acids. Hydrocarbon compositions of thirty-two strains of cyanobacteria were characterized to reveal novel structural features and insights into hydrocarbon biosynthesis in cyanobacteria. This investigation revealed new double bond (2- and 3-heptadecene) and methyl group positions (3-, 4- and 5-methylheptadecane) for a variety of strains. Additionally, results from this study and literature reports indicate that hydrocarbon production is a universal phenomenon in cyanobacteria. All cyanobacteria possess the capacity to produce hydrocarbons from fatty acids yet not all accomplish this through the same metabolic pathway. One pathway comprises a two-step conversion of fatty acids first to fatty aldehydes and then alkanes that involves a fatty acyl ACP reductase (FAAR) and aldehyde deformylating oxygenase (ADO). The second involves a polyketide synthase (PKS) pathway that first elongates the acyl chain followed by decarboxylation to produce a terminal alkene (olefin synthase, OLS). Sixty-one strains possessing the FAAR/ADO pathway and twelve strains possessing the OLS pathway were newly identified through bioinformatic analyses. Strains possessing the OLS pathway formed a cohesive phylogenetic clade with the exception of three Moorea strains and Leptolyngbya sp. PCC 6406 which may have acquired the OLS pathway via horizontal gene transfer. Hydrocarbon pathways were identified in one-hundred-forty-two strains of cyanobacteria over a broad phylogenetic range and there were no instances where both the FAAR/ADO and the OLS pathways were found together in the same genome, suggesting an unknown selective pressure maintains one or the other pathway, but not both.

Filamentous marine cyanobacteria are extraordinarily rich sources of structurally novel, biomedically relevant natural products. To understand their biosynthetic origins as well as produce increased supplies and analog molecules, access to the clustered biosynthetic genes that encode for the assembly enzymes is necessary. Complicating these efforts is the universal presence of heterotrophic bacteria in the cell wall and sheath material of cyanobacteria obtained from the environment and those grown in uni-cyanobacterial culture. Moreover, the high similarity in genetic elements across disparate secondary metabolite biosynthetic pathways renders imprecise current gene cluster targeting strategies and contributes sequence complexity resulting in partial genome coverage. Thus, it was necessary to use a dual-method approach of single-cell genomic sequencing based on multiple displacement amplification (MDA) and metagenomic library screening. Here, we report the identification of the putative apratoxin. A biosynthetic gene cluster, a potent cancer cell cytotoxin with promise for medicinal applications. The roughly 58 kb biosynthetic gene cluster is composed of 12 open reading frames and has a type I modular mixed polyketide synthase/nonribosomal peptide synthetase (PKS/NRPS) organization and features loading and off-loading domain architecture never previously described. Moreover, this work represents the first successful isolation of a complete biosynthetic gene cluster from Lyngbya bouillonii, a tropical marine cyanobacterium renowned for its production of diverse bioactive secondary metabolites.

Naturally derived chemical compounds are the foundation of much of our pharmacopeia, especially in antiproliferative and anti-infective drug classes. Here, we report that a naturally derived molecule called carmaphycin B is a potent inhibitor against both the asexual and sexual blood stages of malaria infection. Using a combination of in silico molecular docking and in vitro directed evolution in a well-characterized drug-sensitive yeast model, we determined that these compounds target the β5 subunit of the proteasome. These studies were validated using in vitro inhibition assays with proteasomes isolated from Plasmodium falciparum. As carmaphycin B is toxic to mammalian cells, we synthesized a series of chemical analogs that reduce host cell toxicity while maintaining blood-stage and gametocytocidal antimalarial activity and proteasome inhibition. This study describes a promising new class of antimalarial compound based on the carmaphycin B scaffold, as well as several chemical structural features that serve to enhance antimalarial specificity.