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Skiba, MA, Sikkema AP, Moss NA, Lowell AN, Su M, Sturgis RM, Gerwick L, Gerwick WH, Sherman DH, Smith JL.  2018.  Biosynthesis of t-Butyl in apratoxin A: functional analysis and architecture of a PKS loading module. Acs Chemical Biology. 13:1640-1650.   10.1021/acschembio.8b00252   AbstractWebsite

The unusual feature of a t-butyl group is found in several marine-derived natural products including apratoxin A, a Sec61 inhibitor produced by the cyanobacterium Moorea bouillonii PNG 5-198. Here, we determine that the apratoxin A t-butyl group is formed as a pivaloyl acyl carrier protein (ACP) by AprA, the polyketide synthase (PKS) loading module of the apratoxin A biosynthetic pathway. AprA contains an inactive "pseudo" GCN5-related N-acetyltransferase domain (Psi GNAT) flanked by two methyltransferase domains (MT1 and MT2) that differ distinctly in sequence. Structural, biochemical, and precursor incorporation studies reveal that MT2 catalyzes unusually coupled decarboxylation and methylation reactions to transform dimethylmalonyl-ACP, the product of MT1, to pivaloyl-ACP. Further, pivaloyl-ACP synthesis is primed by the fatty acid synthase malonyl acyltransferase (FabD), which compensates for the Psi GNAT and provides the initial aryl-transfer step to form AprA malonyl-ACP. Additionally, images of AprA from negative stain electron microscopy reveal multiple conformations that may facilitate the individual catalytic steps of the multienzyme module.

Shao, CL, Mou XF, Cao F, Spadafora C, Glukhov E, Gerwick L, Wang CY, Gerwick WH.  2018.  Bastimolide B, an antimalarial 24-membered marine macrolide possessing a tert-butyl group. Journal of Natural Products. 81:211-215.   10.1021/acs.jnatprod.7b00917   AbstractWebsite

We reported previously the discovery of the potent antimalarial 40-membered macrolide bastimolide A (1) from the tropical marine cyanobacterium Okeania hirsute. Continued investigation has led to the discovery of a new analogue, bastimolide B (2), a 24-membered polyhydroxy macrolide with a long aliphatic chain and unique terminal tertbutyl group. Its complete structure was determined by a combination of extensive spectroscopic methods and comparative analysis of its methanolysis products with those of bastimolide A. A methanolysis mechanism for bastimolide A is proposed, and one unexpected isomerization product of the C2-C3 double bond, 2-(E)-bastimolide A (3), was obtained. Bastimolide B (2) showed strong antimalarial activity against chloroquine-sensitive Plasmodium falciparum strain HB3. A preliminary investigation of the structure activity relationship based on six analogues revealed the importance of the double bond as well as the 1,3-diol and 1,3,5-triol functionalities.

Moss, NA, Leao T, Glukhov E, Gerwick L, Gerwick WH.  2018.  Collection, Culturing, and Genome Analyses of Tropical Marine Filamentous Benthic Cyanobacteria. Marine Enzymes and Specialized Metabolism, Pt A. 604( Moore BS, Ed.).:3-43., San Diego: Elsevier Academic Press Inc   10.1016/bs.mie.2018.02.014   Abstract

Decreasing sequencing costs has sparked widespread investigation of the use of microbial genomics to accelerate the discovery and development of natural products for therapeutic uses. Tropical marine filamentous cyanobacteria have historically produced many structurally novel natural products, and therefore present an excellent opportunity for the systematic discovery of new metabolites via the information derived from genomics and molecular genetics. Adequate knowledge transfer and institutional know-how are important to maintain the capability for studying filamentous cyanobacteria due to their unusual microbial morphology and characteristics. Here, we describe workflows, procedures, and commentary on sample collection, cultivation, genomic DNA generation, bioinformatics tools, and biosynthetic pathway analysis concerning filamentous cyanobacteria.

Skiba, MA, Sikkema AP, Moss NA, Tran CL, Sturgis RM, Gerwick L, Gerwick WH, Sherman DH, Smith JL.  2017.  A mononuclear iron-dependent methyltransferase catalyzes initial steps in assembly of the apratoxin A polyketide starter unit. Acs Chemical Biology. 12:3039-3048.   10.1021/acschembio.7b00746   AbstractWebsite

Natural product biosynthetic pathways contain a plethora of enzymatic tools to carry out difficult biosynthetic transformations. Here, we discover an unusual mononuclear iron dependent methyltransferase that acts in the initiation steps of apratoxin A biosynthesis (AprA MT1). Fe3+-replete AprA MT1 catalyzes one or two methyl transfer reactions on the substrate malonyl-ACP (acyl carrier protein), whereas Co2+, Fe2+, Mn2+, and Ni2+ support only a single methyl transfer. MT1 homologues' exist;Within the "GNAT" (GCNS-related N-acetyltransferase) loading modules of several modular biosynthetic pathways with propionyl, isobutyryt or pivaloyl starter units. GNAT domains are thought to catalyze decarboXylation of malonyl-CoA and acetyl transfer to a carrier protein. In AprA, the GNAT domain lacks both decarboxylation and acyl transfer activity. A crystal structure of the AprA MT1-GNAT di-domain with bound Mn2+, malonate, and the methyl donor S-adenosylmethionine (SAM) reveals that the malonyl substrate is a bidentate metal ligand, indicating that the metal acts as a Lewis acid to promote methylation of the malonyl alpha-carbon. The GNAT domain is truncated relative to functional homologues. These results afford an expanded understanding of MT1-GNAT structure and activity arid permit the functional annotation of homologous GNAT loading modules both with and without methyltransferases, additionally revealing their rapid evolutionary adaptation in different biosynthetic contexts.

Luzzatto-Knaan, T, Garg N, Wang MX, Glukhov E, Peng Y, Ackermann G, Amir A, Duggan BM, Ryazanov S, Gerwick L, Knight R, Alexandov T, Bandeira N, Gerwick WH, Dorrestein PC.  2017.  Digitizing mass spectrometry data to explore the chemical diversity and distribution of marine cyanobacteria and algae. Elife. 6   10.7554/eLife.24214   AbstractWebsite

Natural product screening programs have uncovered molecules from diverse natural sources with various biological activities and unique structures. However, much is yet underexplored and additional information is hidden in these exceptional collections. We applied untargeted mass spectrometry approaches to capture the chemical space and dispersal patterns of metabolites from an in-house library of marine cyanobacterial and algal collections. Remarkably, 86% of the metabolomics signals detected were not found in other available datasets of similar nature, supporting the hypothesis that marine cyanobacteria and algae possess distinctive metabolomes. The data were plotted onto a world map representing eight major sampling sites, and revealed potential geographic locations with high chemical diversity. We demonstrate the use of these inventories as a tool to explore the diversity and distribution of natural products. Finally, we utilized this tool to guide the isolation of a new cyclic lipopeptide, yuvalamide A, from a marine cyanobacterium.

Kinnel, RB, Esquenazi E, Leao T, Moss N, Mevers E, Pereira AR, Monroe EA, Korobeynikov A, Murray TF, Sherman D, Gerwick L, Dorrestein PC, Gerwick WH.  2017.  A maldiisotopic approach to discover natural products: Cryptomaldamide, a hybrid tripeptide from the marine cyanobacterium Moorea producens. Journal of Natural Products. 80:1514-1521.   10.1021/acs.jnatprod.7b00019   AbstractWebsite

Genome sequencing of microorganisms has revealed a greatly increased capacity for natural products biosynthesis than was previously recognized from compound isolation efforts alone. Hence, new methods are needed for the discovery and description of this hidden secondary metabolite potential. Here we show that provision of heavy nitrogen N-15-nitrate to marine cyanobacterial cultures followed by single-filament MALDI analysis over a period of days was highly effective in identifying a new natural product with an exceptionally high nitrogen content. The compound, named cryptomaldamide, was subsequently isolated using MS to guide the purification process, and its structure determined by 2D NMR and other spectroscopic and chromatographic methods. Bioinformatic analysis of the draft genome sequence identified a 28.7 kB gene cluster that putatively encodes for cryptomaldamide biosynthesis. Notably, an amidinotransferase is proposed to initiate the biosynthetic process by transferring an amidino group from arginine to serine to produce the first residue to be incorporated by the hybrid NRPS-PKS pathway. The maldiisotopic approach presented here is thus demonstrated to provide an orthogonal method by which to discover novel chemical diversity from Nature.

Leao, T, Castelao G, Korobeynikov A, Monroe EA, Podell S, Glukhov E, Allen EE, Gerwick WH, Gerwick L.  2017.  Comparative genomics uncovers the prolific and distinctive metabolic potential of the cyanobacterial genus Moorea. Proceedings of the National Academy of Sciences of the United States of America. 114:3198-3203.   10.1073/pnas.1618556114   AbstractWebsite

Cyanobacteria are major sources of oxygen, nitrogen, and carbon in nature. In addition to the importance of their primary metabolism, some cyanobacteria are prolific producers of unique and bioactive secondary metabolites. Chemical investigations of the cyanobacterial genus Moorea have resulted in the isolation of over 190 compounds in the last two decades. However, preliminary genomic analysis has suggested that genome-guided approaches can enable the discovery of novel compounds from even well-studied Moorea strains, highlighting the importance of obtaining complete genomes. We report a complete genome of a filamentous tropical marine cyanobacterium, Moorea producens PAL, which reveals that about one-fifth of its genome is devoted to production of secondary metabolites, an impressive four times the cyanobacterial average. Moreover, possession of the complete PAL genome has allowed improvement to the assembly of three other Moorea draft genomes. Comparative genomics revealed that they are remarkably similar to one another, despite their differences in geography, morphology, and secondary metabolite profiles. Gene cluster networking highlights that this genus is distinctive among cyanobacteria, not only in the number of secondary metabolite pathways but also in the content of many pathways, which are potentially distinct from all other bacterial gene clusters to date. These findings portend that future genome-guided secondary metabolite discovery and isolation efforts should be highly productive.

Maloney, FP, Gerwick L, Gerwick WH, Sherman DH, Smith JL.  2016.  Anatomy of the beta-branching enzyme of polyketide biosynthesis and its interaction with an acyl-ACP substrate. Proceedings of the National Academy of Sciences of the United States of America. 113:10316-10321.   10.1073/pnas.1607210113   AbstractWebsite

Alkyl branching at the beta position of a polyketide intermediate is an important variation on canonical polyketide natural product biosynthesis. The branching enzyme, 3-hydroxy-3-methylglutaryl synthase (HMGS), catalyzes the aldol addition of an acyl donor to a beta-keto-polyketide intermediate acceptor. HMGS is highly selective for two specialized acyl carrier proteins (ACPs) that deliver the donor and acceptor substrates. The HMGS from the curacin A biosynthetic pathway (CurD) was examined to establish the basis for ACP selectivity. The donor ACP (CurB) had high affinity for the enzyme (K-d = 0.5 mu M) and could not be substituted by the acceptor ACP. High-resolution crystal structures of HMGS alone and in complex with its donor ACP reveal a tight interaction that depends on exquisite surface shape and charge complementarity between the proteins. Selectivity is explained by HMGS binding to an unusual surface cleft on the donor ACP, in a manner that would exclude the acceptor ACP. Within the active site, HMGS discriminates between pre-and postreaction states of the donor ACP. The free phosphopantetheine (Ppant) cofactor of ACP occupies a conserved pocket that excludes the acetyl-Ppant substrate. In comparison with HMG-CoA (CoA) synthase, the homologous enzyme from primary metabolism, HMGS has several differences at the active site entrance, including a flexible-loop insertion, which may account for the specificity of one enzyme for substrates delivered by ACP and the other by CoA.

Cummings, SL, Barbe D, Leao TF, Korobeynikov A, Engene N, Glukhov E, Gerwick WH, Gerwick L.  2016.  A novel uncultured heterotrophic bacterial associate of the cyanobacterium Moorea producens JHB. Bmc Microbiology. 16   10.1186/s12866-016-0817-1   AbstractWebsite

Background: Filamentous tropical marine cyanobacteria such as Moorea producens strain JHB possess a rich community of heterotrophic bacteria on their polysaccharide sheaths; however, these bacterial communities have not yet been adequately studied or characterized. Results and discussion: Through efforts to sequence the genome of this cyanobacterial strain, the 5.99 MB genome of an unknown bacterium emerged from the metagenomic information, named here as Mor1. Analysis of its genome revealed that the bacterium is heterotrophic and belongs to the phylum Acidobacteria, subgroup 22; however, it is only 85 % identical to the nearest cultured representative. Comparative genomics further revealed that Mor1 has a large number of genes involved in transcriptional regulation, is completely devoid of transposases, is not able to synthesize the full complement of proteogenic amino acids and appears to lack genes for nitrate uptake. Mor1 was found to be present in lab cultures of M. producens collected from various locations, but not other cyanobacterial species. Diverse efforts failed to culture the bacterium separately from filaments of M. producens JHB. Additionally, a co-culturing experiment between M. producens JHB possessing Mor1 and cultures of other genera of cyanobacteria indicated that the bacterium was not transferable. Conclusion: The data presented support a specific relationship between this novel uncultured bacterium and M. producens, however, verification of this proposed relationship cannot be done until the "uncultured" bacterium can be cultured.

Wang, MX, Carver JJ, Phelan VV, Sanchez LM, Garg N, Peng Y, Nguyen DD, Watrous J, Kapono CA, Luzzatto-Knaan T et al..  2016.  Sharing and community curation of mass spectrometry data with Global Natural Products Social Molecular Networking. Nature Biotechnology. 34:828-837.   10.1038/nbt.3597   AbstractWebsite

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 (MS) techniques are well-suited to high-throughput characterization of NP, there is a pressing need for an infrastructure to enable sharing and curation of data. We present Global Natural Products Social Molecular Networking (GNPS;, 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.

Moss, NA, Bertin MJ, Kleigrewe K, Leao TF, Gerwick L, Gerwick WH.  2016.  Integrating mass spectrometry and genomics for cyanobacterial metabolite discovery. Journal of Industrial Microbiology & Biotechnology. 43:313-324.   10.1007/s10295-015-1705-7   AbstractWebsite

Filamentous marine cyanobacteria produce bioactive natural products with both potential therapeutic value and capacity to be harmful to human health. Genome sequencing has revealed that cyanobacteria have the capacity to produce many more secondary metabolites than have been characterized. The biosynthetic pathways that encode cyanobacterial natural products are mostly uncharacterized, and lack of cyanobacterial genetic tools has largely prevented their heterologous expression. Hence, a combination of cutting edge and traditional techniques has been required to elucidate their secondary metabolite biosynthetic pathways. Here, we review the discovery and refined biochemical understanding of the olefin synthase and fatty acid ACP reductase/aldehyde deformylating oxygenase pathways to hydrocarbons, and the curacin A, jamaicamide A, lyngbyabellin, columbamide, and a trans-acyltransferase macrolactone pathway encoding phormidolide. We integrate into this discussion the use of genomics, mass spectrometric networking, biochemical characterization, and isolation and structure elucidation techniques.

Bertin, MJ, Vulpanovici A, Monroe EA, Korobeynikov A, Sherman DH, Gerwick L, Gerwick WH.  2016.  The phormidolide biosynthetic gene cluster: A trans-at pks pathway encoding a toxic macrocyclic polyketide. Chembiochem. 17:164-173.   10.1002/cbic.201500467   AbstractWebsite

Phormidolide is a polyketide produced by a cultured filamentous marine cyanobacterium and incorporates a 16-membered macrolactone. Its complex structure is recognizably derived from a polyketide synthase pathway, but possesses unique and intriguing structural features that prompted interest in investigating its biosynthetic origin. Stable isotope incorporation experiments confirmed the polyketide nature of this compound. We further characterized the phormidolide gene cluster (phm) through genome sequencing followed by bioinformatic analysis. Two discrete trans-type acyltransferase (trans-AT) ORFs along with KS-AT adaptor regions (ATd) within the polyketide synthase (PKS) megasynthases, suggest that the phormidolide gene cluster is a trans-AT PKS. Insights gained from analysis of the mode of acetate incorporation and ensuing keto reduction prompted our reevaluation of the stereochemistry of phormidolide hydroxy groups located along the linear polyketide chain.

Gerwick, L, Mascuch SJ, Navarro G, Boudreau P, Carland TM, Gaasterland T, Gerwick WH.  2015.  Honaucin A, mechanism of action and role as a potential cancer prevention agent. Planta Medica. 81:927-927. AbstractWebsite
Teta, R, Della Sala G, Glukhov E, Gerwick L, Gerwick WH, Mangoni A, Costantino V.  2015.  Combined LC-MS/MS and molecular networking approach reveals new cyanotoxins from the 2014 cyanobacterial bloom in Green Lake, Seattle. Environmental Science & Technology. 49:14301-14310.   10.1021/acs.est.5b04415   AbstractWebsite

Cyanotoxins obtained from a freshwater cyanobacterial collection at Green Lake, Seattle during a cyanobacterial harmful algal bloom in the summer of 2014 were studied using a new approach based on molecular networking analysis of liquid chromatography tandem mass spectrometry (LC-MS/MS) data. This MS networking approach is particularly well-suited for the detection of new cyanotoxin variants and resulted in the discovery of three new cyclic peptides, namely microcystin-MhtyR (6), which comprised about half of the total microcystin content in the bloom, and ferintoic acids C (12) and D (13). Structure elucidation of 6 was aided by a new microscale methylation procedure. Metagenomic analysis of the bloom using the 16S-ITS rRNA region identified Microcystis aeruginosa as the predominant cyanobacterium in the sample. Fragments of the putative biosynthetic genes for the new cyanotoxins were also identified, and their sequences correlated to the structure of the isolated cyanotoxins.

Shao, CL, Linington RG, Balunas MJ, Centeno A, Boudreau P, Zhang C, Engene N, Spadafora C, Mutka TS, Kyle DE, Gerwick L, Wang CY, Gerwick WH.  2015.  Bastimolide A, a potent antimalarial polyhydroxy macrolide from the marine cyanobacterium Okeania hirsuta. Journal of Organic Chemistry. 80:7849-7855.   10.1021/acs.joc.5b01264   AbstractWebsite

Bastimolide A (1), a polyhydroxy macrolide with a 40-membered ring, was isolated from a new genus of the tropical marine cyanobacterium Okeania hirsuta. This novel macrolide was defined by spectroscopy and chemical reactions to possess one 1,3-diol, one 1,3,5-triol, six 1,5-diols, and one tert-butyl group; however, the relationships of these moieties to one another were obscured by a highly degenerate H-1 NMR spectrum. Its complete structure and absolute configuration were therefore unambiguously determined by X-ray diffraction analysis of the nona-p-nitrobenzoate derivative (1d). Pure bastimolide A (1) showed potent antimalarial activity against four resistant strains of Plasmodium falciparum with IC50 values between 80 and 270 nM, although with some toxicity to the control Vero cells (IC50 = 2.1 mu M), and thus represents a potentially promising lead for antimalarial drug discovery. Moreover, rigorous establishment of its molecular arrangement gives fresh insight into the structures and biosynthesis of cyanobacterial polyhydroxymacrolides.

Kleigrewe, K, Almaliti J, Tian IY, Kinnel RB, Korobeynikov A, Monroe EA, Duggan BM, Di Marzo V, Sherman DH, Dorrestein PC, Gerwick L, Gerwick WH.  2015.  Combining mass spectrometric metabolic profiling with genomic analysis: A powerful approach for discovering natural products from cyanobacteria. Journal of Natural Products. 78:1671-1682.   10.1021/acs.jnatprod.5b00301   AbstractWebsite

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 trichlorinated 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. On the basis of their distinctive acyl amide structure, the most prevalent metabolite was evaluated for cannabinomimetic properties and found to be moderate affinity ligands for CB1.

Boudreau, PD, Monroe EA, Mehrotra S, Desfor S, Korobeynikov A, Sherman DH, Murray TF, Gerwick L, Dorrestein PC, Gerwick WH.  2015.  Expanding the described metabolome of the marine cyanobacterium Moorea producens JHB through orthogonal natural products workflows. Plos One. 10   10.1371/journal.pone.0133297   AbstractWebsite

Moorea producens JHB, a Jamaican strain of tropical filamentous marine cyanobacteria, has been extensively studied by traditional natural products techniques. These previous bioassay and structure guided isolations led to the discovery of two exciting classes of natural products, hectochlorin (1) and jamaicamides A (2) and B (3). In the current study, mass spectrometry-based 'molecular networking' was used to visualize the metabolome of Moorea producens JHB, and both guided and enhanced the isolation workflow, revealing additional metabolites in these compound classes. Further, we developed additional insight into the metabolic capabilities of this strain by genome sequencing analysis, which subsequently led to the isolation of a compound unrelated to the jamaicamide and hectochlorin families. Another approach involved stimulation of the biosynthesis of a minor jamaicamide metabolite by cultivation in modified media, and provided insights about the underlying biosynthetic machinery as well as preliminary structure-activity information within this structure class. This study demonstrated that these orthogonal approaches are complementary and enrich secondary metabolomic coverage even in an extensively studied bacterial strain.

Navarro, G, Cummings S, Lee J, Moss N, Glukhov E, Valeriote FA, Gerwick L, Gerwick WH.  2015.  Isolation of polycavernoside D from a marine cyanobacterium. Environmental Science & Technology Letters. 2:166-170.   10.1021/acs.estlett.5b00116   AbstractWebsite

The polycavernosides make up a unique class of marine-derived macrolides that were implicated in the poisoning of 49 people in the South Western Pacific resulting in 11 deaths. The original source ascribed to these environmental toxins was from the edible red alga Polycavernosa tsudai (also known as Gracilaria edulis); however, the inability to reisolate these metabolites from the alga, along with structural resemblance to several marine cyanobacterial natural products, suggested that these compounds derive from these latter photosynthetic prokaryotes. In this study, we identified a new analogue "polycavernoside D" from an environmental sample of the marine cyanobacterium Okeania sp., thus providing the first experimental evidence that these lethal toxins are in fact cyanobacterial secondary metabolites. Moreover, the new metabolite was obtained from a Caribbean cyanobacterial collection, thus suggesting this toxin family to be of broader environmental occurrence than previously realized, and raising concerns about unrecognized human exposure in diverse tropical marine environments.

Mascuch, SJ, Moree WJ, Hsu CC, Turner GG, Cheng TL, Blehert DS, Kilpatrick AM, Frick WF, Meehan MJ, Dorrestein PC, Gerwick L.  2015.  Direct detection of fungal siderophores on bats with white-nose syndrome via fluorescence microscopy-guided ambient ionization mass spectrometry. Plos One. 10   10.1371/journal.pone.0119668   AbstractWebsite

White-nose syndrome (WNS) caused by the pathogenic fungus Pseudogymnoascus destructans is decimating the populations of several hibernating North American bat species. Little is known about the molecular interplay between pathogen and host in this disease. Fluorescence microscopy ambient ionization mass spectrometry was used to generate metabolic profiles from the wings of both healthy and diseased bats of the genus Myotis. Fungal siderophores, molecules that scavenge iron from the environment, were detected on the wings of bats with WNS, but not on healthy bats. This work is among the first examples in which microbial molecules are directly detected from an infected host and highlights the ability of atmospheric ionization methodologies to provide direct molecular insight into infection.

Bertin, MJ, Schwartz SL, Lee J, Korobeynikov A, Dorrestein PC, Gerwick L, Gerwick WH.  2015.  Spongosine production by a Vibrio harveyi strain sssociated with the sponge Tectitethya crypta. Journal of Natural Products. 78:493-499.   10.1021/np5009762   AbstractWebsite

Spongosine (1), deoxyspongosine (2), spongothymidine (Ara T) (3), and spongouridine (Ara U) were isolated from the Caribbean sponge Tectitethya crypta and given the general name "spongonucleosides". Spongosine, a methoxyadenosine derivative, has demonstrated a diverse bioactivity profile including anti-inflammatory activity and analgesic and vasodilation properties. Investigations into unusual nucleoside production by T. crypta-associated microorganisms using mass spectrometric techniques have identified a spongosine-producing strain of Vibrio harveyi and several structurally related compounds from multiple strains.

Micallef, ML, Sharma D, Bunn BM, Gerwick L, Viswanathan R, Moffitt MC.  2014.  Comparative analysis of hapalindole, ambiguine and welwitindolinone gene clusters and reconstitution of indole-isonitrile biosynthesis from cyanobacteria. Bmc Microbiology. 14   10.1186/s12866-014-0213-7   AbstractWebsite

Background: The hapalindole-type family of natural products is a group of hybrid isoprenoid-indole alkaloids, produced solely by members of the Subsection V cyanobacterial strains. This family broadly includes the hapalindoles, welwitindolinones, fisherindoles and ambiguines amongst others, all of which have an isonitrile-or isothiocyanate-containing indole alkaloid skeleton, with a cyclized isoprene unit. The hapalindoles are diversified into the welwitindolinones, fischerindoles and ambiguines through the employment of tailoring oxygenase, methyltransferase and prenyltransferase enzymes. We compare the genetic basis for the biosynthesis of this diverse group of natural products and identify key early biosynthetic intermediates. Results: Whole genome sequencing of freshwater and terrestrial cyanobacteria Westiella intricata UH strain HT-29-1, Hapalosiphon welwitschii UH strain IC-52-3, Fischerella ambigua UTEX 1903 and Fischerella sp. ATCC 43239 led to the identification of a candidate hapalindole-type gene cluster in each strain. These were compared with the recently published ambiguine and welwitindolinone gene clusters and four unpublished clusters identified within publicly available genomes. We present detailed comparative bioinformatic analysis of the gene clusters and the biosynthesis of a pivotal indole-isonitrile intermediate resulting in both cis and trans geometrical isomers. Enzyme analyses and metabolite extractions from two hapalindole-producing Fischerella strains indicate the presence of cis and trans indole-isonitriles as biosynthetic intermediates in the early steps of the pathway. Conclusions: Interestingly, the organization of the welwitindolinone gene cluster is conserved in all producing strains but distinct from the hapalindole and ambiguine clusters. Enzymatic assays using WelI1 and WelI3 from Westiella intricata UH strain HT-29-1 demonstrated the ability to catalyze the formation of both cis and trans geometrical isomers when using a cell lysate. The enzymatic and metabolic characterization of both cis and trans indole-isonitrile intermediates implies conservation of their stereochemical integrity towards members of the ambiguine and welwitindolinone products. In summary, we present data that supports a unified biosynthetic pathway towards hapalindoles in nine individual species of cyanobacteria. Diversification of the pathway occurs later through the employment of specialized enzymatic steps towards fischerindoles, ambiguines and welwitindolinones.

Coates, CR, Podell S, Korobeynikov A, Lapidus A, Pevzner P, Sherman DH, Allen EE, Gerwick L, Gerwick WH.  2014.  Characterization of cyanobacterial hydrocarbon composition and distribution of biosynthetic pathways. PLoS ONE. 9:e85140.: Public Library of Science   10.1371/journal.pone.0085140   AbstractWebsite

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.

Gerwick, L, Boudreau P, Choi H, Mascuch S, Villa FA, Balunas MJ, Malloy KL, Teasdale ME, Rowley DC, Gerwick WH.  2013.  Interkingdom signaling by structurally related cyanobacterial and algal secondary metabolites. Phytochemistry Reviews. 12:459-465.   10.1007/s11101-012-9237-5   AbstractWebsite

Several groups of structurally-related compounds, comprised of either five or six-membered ring structures with attached lipophilic carbon chains and in some cases possessing halogen atoms, have been isolated from various marine algae and filamentous cyanobacteria. The related compounds considered in the present work include the coibacins, laurenciones, honaucins, malyngamides and the tumonoic acids. Members of all of these compound families were assayed and found to inhibit the production of nitric oxide in lipopolysaccharides-stimulated macrophages, indicating their anti-inflammatory potential. In addition, several of these same marine natural products were found to inhibit quorum sensing mediated phenotypes in Vibrio harveyi BB120 and/or Escherichia coli JB525. The mechanism and evolutionary significance for inhibition of these cellular processes in prokaryotic and eukaryotic systems are speculated on and discussed.

Hsu, CC, ElNaggar MS, Peng Y, Fang JS, Sanchez LM, Mascuch SJ, Moller KA, Alazzeh EK, Pikula J, Quinn RA, Zeng Y, Wolfe BE, Dutton RJ, Gerwick L, Zhang LX, Liu XT, Mansson M, Dorrestein PC.  2013.  Real-time metabolomics on living microorganisms using ambient electrospray ionization flow-probe. Analytical Chemistry. 85:7014-7018.   10.1021/ac401613x   AbstractWebsite

Microorganisms such as bacteria and fungi produce a variety of specialized metabolites that are invaluable for agriculture, biological research, and drug discovery. However, the screening of microbial metabolic output is usually a time-intensive task. Here, we utilize a liquid microjunction surface sampling probe for electrospray ionization-mass spectrometry to extract and ionize metabolite mixtures directly from living microbial colonies grown on soft nutrient agar in Petri-dishes without any sample pretreatment. To demonstrate the robustness of the method, this technique was applied to observe the metabolic output of more than 30 microorganisms, including yeast, filamentous fungi, pathogens, and marine-derived bacteria, that were collected worldwide. Diverse natural products produced from different microbes, including Streptomyces coelicolor, Bacillus subtilis, and Pseudomonas aeruginosa are further characterized.

Sanchez, LM, Knudsen GM, Helbig C, De Muylder G, Mascuch SM, Mackey ZB, Gerwick L, Clayton C, McKerrow JH, Linington RG.  2013.  Examination of the Mode of Action of the Almiramide Family of Natural Products against the Kinetoplastid Parasite Trypanosoma brucei. Journal of Natural Products. 76:630-641.   10.1021/np300834q   AbstractWebsite

Almiramide C is a marine natural product with low micromolar activity against Leishmania donovani, the causative agent of leishmaniasis. We have now shown that almiramide C is also active against the related parasite Ttypanosoma brucei, the causative agent of human African trypanosomiasis. A series of activity-based probes have been synthesized to explore both the molecular target of this compound series in T. brucei lysates and site localization through epifluorescence microscopy. These target identification studies indicate that the almiramides likely perturb glycosomal function through disruption of membrane assembly machinery. Glycosomes, which are organelles specific to kinetoplastid parasites, house the first seven steps of glycolysis and have been shown to be essential for parasite survival in the bloodstream stage. There are currently no reported smallmolecule disruptors of glycosome function, making the almiramides unique molecular probes for this understudied parasitespecific organelle. Additionally, examination of toxicity in an in vivo zebrafish model has shown that these compounds have little effect on organism development, even at high concentrations, and has uncovered a potential side effect through localization of fluorescent derivatives to zebrafish neuromast cells. Combined, these results further our understanding of the potential value of this lead series as development candidates against T. brucei.