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Saleem-Batcha, R, Stull F, Sanders JN, Moore BS, Palfey BA, Houk KN, Teufel R.  2018.  Enzymatic control of dioxygen binding and functionalization of the flavin cofactor. Proceedings of the National Academy of Sciences of the United States of America. 115:4909-4914.   10.1073/pnas.1801189115   AbstractWebsite

The reactions of enzymes and cofactors with gaseous molecules such as dioxygen (O-2) are challenging to study and remain among the most contentious subjects in biochemistry. To date, it is largely enigmatic how enzymes control and fine-tune their reactions with O-2, as exemplified by the ubiquitous flavin-dependent enzymes that commonly facilitate redox chemistry such as the oxygenation of organic substrates. Here we employ O-2-pressurized X-ray crystallography and quantum mechanical calculations to reveal how the precise positioning of O-2 within a flavoenzyme's active site enables the regio-specific formation of a covalent flavin-oxygen adduct and oxygenating species (i.e., the flavin-N5-oxide) by mimicking a critical transition state. This study unambiguously demonstrates how enzymes may control the O-2 functionalization of an organic cofactor as prerequisite for oxidative catalysis. Our work thus illustrates how O-2 reactivity can be harnessed in an enzymatic environment and provides crucial knowledge for future rational design of O-2-reactive enzymes.

Zheng, J, McKinnie SMK, El Gama A, Fengit W, Dong Y, Agarwal V, Fenical W, Kumar A, Cao ZY, Moore BS, Pessah IN.  2018.  Organohalogens naturally biosynthesized in marine environments and produced as disinfection byproducts alter sarco/endoplasmic reticulum ca2+ dynamics. Environmental Science & Technology. 52:5469-5478.   10.1021/acs.est.8b00512   AbstractWebsite

Contemporary sources of organohalogens produced as disinfection byproducts (DBPs) are receiving considerable attention as emerging pollutants because of their abundance, persistence, and potential to structurally mimic natural organohalogens produced by bacteria that serve signaling or toxicological functions in marine environments. Here, we tested 34 organohalogens from anthropogenic and marine sources to identify compounds active toward ryanodine receptor (RyR1), known toxicological targets of non-dioxin like polychlorinated biphenyls (PCBs) and polybrominated diphenyl ethers (PBDEs). [H-3]Ryanodine ([H-3]Ry) binding screening (<= 2 mu M) identified 10 highly active organohalogens. Further analysis indicated that 2,3-dibromoindole (14), tetrabromopyrrole (31), and 2,3,S-tribromopyrrole (34) at 10 mu M were the most efficacious at enhancing [H-3]Ry binding. Interestingly, these congeners also inhibited microsomal sarcoplasmic/endoplasmic reticulum (SR/ER) Ca2+ ATPase (SERCA1a). Dual SERCAla inhibition and RyR1 activation triggered Ca2+ efflux from microsomal vesicles with initial rates rank ordered 31 > 34 > 14. Hexabromobipyrroles (25) enhanced [H-3]Ry binding moderately with strong SERCAla inhibition, whereas pyrrole (24), 2,3,4-tribromopyrrole (26), and ethyl-4-bromopyrrole-2-carboxylate (27) were inactive. Of three PBDE derivatives of marine origin active in the [H-3]Ry assay, 4'-hydroxy-2,3',4,5',6-pentabromodiphenyl ether (18) was also a highly potent SERCAla inhibitor. Molecular targets of marine organohalogens that are also DBPs of emerging environmental concern are likely to contribute to their toxicity.

Moore, BS.  2018.  Asymmetric Alkene and Arene Halofunctionalization Reactions in Meroterpenoid Biosynthesis. Synlett. 29:401-409.   10.1055/s-0036-1590919   AbstractWebsite

Meroterpenoid natural products are important bioactive molecules with broad distribution throughout nature. In Streptomyces bacteria, naphthoquinone-based meroterpenoids comprise a simple yet structurally fascinating group of natural product antibiotics that are enzymatically constructed through a series of asymmetric alkene and arene halofunctionalization reactions. This account article highlights our discovery and characterization of a group of vanadium-dependent chloroperoxidase enzymes that catalyze halogen-assisted cyclization and rearrangement reactions and have inspired biomimetic syntheses of numerous meroterpenoid natural products. 1 Introduction 2 Early Biosynthetic Insights and the Characterization of Alkene Halofunctionalization in Napyradiomycin Biosynthesis 3 Discovery of the Merochlorin Natural Products and Enzymatic Aryl Halofunctionalization 4 Discovery and Development of Unifying THN-Based Meroterpenoid Biosynthesis and Synthesis Approaches 5 Insights into Naphterpin and Marinone Biosynthesis Involving Cryptic Aryl Halofunctionalization Reactions 6 Closing Thoughts

Bruns, H, Crusemann M, Letzel AC, Alanjary M, McInerney JO, Jensen PR, Schulz S, Moore BS, Ziemert N.  2018.  Function-related replacement of bacterial siderophore pathways. Isme Journal. 12:320-329.   10.1038/ismej.2017.137   AbstractWebsite

Bacterial genomes are rife with orphan biosynthetic gene clusters (BGCs) associated with secondary metabolism of unrealized natural product molecules. Often up to a tenth of the genome is predicted to code for the biosynthesis of diverse metabolites with mostly unknown structures and functions. This phenomenal diversity of BGCs coupled with their high rates of horizontal transfer raise questions about whether they are really active and beneficial, whether they are neutral and confer no advantage, or whether they are carried in genomes because they are parasitic or addictive. We previously reported that Salinispora bacteria broadly use the desferrioxamine family of siderophores for iron acquisition. Herein we describe a new and unrelated group of peptidic siderophores called salinichelins from a restricted number of Salinispora strains in which the desferrioxamine biosynthesis genes have been lost. We have reconstructed the evolutionary history of these two different siderophore families and show that the acquisition and retention of the new salinichelin siderophores co- occurs with the loss of the more ancient desferrioxamine pathway. This identical event occurred at least three times independently during the evolution of the genus. We surmise that certain BGCs may be extraneous because of their functional redundancy and demonstrate that the relative evolutionary pace of natural pathway replacement shows high selective pressure against retention of functionally superfluous gene clusters.

Reynolds, KA, Luhavaya H, Li J, Dahesh S, Nizet V, Yamanaka K, Moore BS.  2018.  Isolation and structure elucidation of lipopeptide antibiotic taromycin B from the activated taromycin biosynthetic gene cluster. Journal of Antibiotics. 71:333-338.   10.1038/ja.2017.146   AbstractWebsite

In the ongoing effort to unlock the chemical potential of marine bacteria, genetic engineering of biosynthetic gene clusters (BGCs) is increasingly used to awake or improve expression of biosynthetic genes that may lead to discovery of novel bioactive natural products. Previously, we reported the successful capture, engineering and heterologous expression of an orphan BGC from the marine actinomycete Saccharomonospora sp. CNQ-490, which resulted in the isolation of the novel lipopeptide antibiotic taromycin A. Herein we report the isolation and structure elucidation of taromycin B, the second most abundant product of the taromycin biosynthetic series, and show that taromycins A and B exhibit complex chromatographic properties indicative of interconverting conformations. Taromycins A and B display potent activity against methicillin-resistant Staphylococcus aureus and vancomycin-resistant Enterococcus faecium clinical isolates, suggestive that the taromycin molecular scaffold is a promising starting point for further derivatization to produce compounds with promising antibiotic characteristics.

Amos, GCA, Awakawa T, Tuttle RN, Letzel AC, Kim MC, Kudo Y, Fenical W, Moore BS, Jensen PR.  2017.  Comparative transcriptomics as a guide to natural product discovery and biosynthetic gene cluster functionality. Proceedings of the National Academy of Sciences of the United States of America. 114:E11121-E11130.   10.1073/pnas.1714381115   AbstractWebsite

Bacterial natural products remain an important source of new medicines. DNA sequencing has revealed that a majority of natural product biosynthetic gene clusters (BGCs) maintained in bacterial genomes have yet to be linked to the small molecules whose biosynthesis they encode. Efforts to discover the products of these orphan BGCs are driving the development of genome mining techniques based on the premise that many are transcriptionally silent during normal laboratory cultivation. Here, we employ comparative transcriptomics to assess BGC expression among four closely related strains of marine bacteria belonging to the genus Salinispora. The results reveal that slightly more than half of the BGCs are expressed at levels that should facilitate product detection. By comparing the expression profiles of similar gene clusters in different strains, we identified regulatory genes whose inactivation appears linked to cluster silencing. The significance of these subtle differences between expressed and silent BGCs could not have been predicted a priori and was only revealed by comparative transcriptomics. Evidence for the conservation of silent clusters among a larger number of strains for which genome sequences are available suggests they may be under different regulatory control from the expressed forms or that silencing may represent an underappreciated mechanism of gene cluster evolution. Coupling gene expression and metabolomics data established a bioinformatic link between the salinipostins and their associated BGC, while genetic manipulation established the genetic basis for this series of compounds, which were previously unknown from Salinispora pacifica.

Miles, ZD, Diethelm S, Pepper HP, Huang DM, George JH, Moore BS.  2017.  A unifying paradigm for naphthoquinone-based meroterpenoid (bio)synthesis. Nature Chemistry. 9:1235-1242.   10.1038/nchem.2829   AbstractWebsite

Bacterial meroterpenoids constitute an important class of natural products with diverse biological properties and therapeutic potential. The biosynthetic logic for their production is unknown and defies explanation via classical biochemical paradigms. A large subgroup of naphthoquinone-based meroterpenoids exhibits a substitution pattern of the polyketide-derived aromatic core that seemingly contradicts the established reactivity pattern of polyketide phenol nucleophiles and terpene diphosphate electrophiles. We report the discovery of a hitherto unprecedented enzyme-promoted alpha-hydroxyketone rearrangement catalysed by vanadium-dependent haloperoxidases to account for these discrepancies in the merochlorin and napyradiomycin class of meroterpenoid antibiotics, and we demonstrate that the alpha-hydroxyketone rearrangement is potentially a conserved biosynthetic reaction in this molecular class. The biosynthetic alpha-hydroxyketone rearrangement was applied in a concise total synthesis of naphthomevalin, a prominent member of the napyradiomycin meroterpenes, and sheds further light on the mechanism of this unifying enzymatic transformation.

Zhang, JJ, Tang XY, Zhang M, Nguyen D, Moore BS.  2017.  Broad-host-range expression reveals native and host regulatory elements that influence heterologous antibiotic production in gram-negative bacteria. Mbio. 8   10.1128/mBio.01291-17   AbstractWebsite

Heterologous expression has become a powerful tool for studying microbial biosynthetic gene clusters (BGCs). Here, we extend the transformation-associated recombination cloning and heterologous expression platform for microbial BGCs to include Gram-negative proteobacterial expression hosts. Using a broad-hostrange expression platform, we test the implicit assumption that biosynthetic pathways are more successfully expressed in more closely related heterologous hosts. Cloning and expression of the violacein BGC from Pseudoalteromonas luteoviolacea 2ta16 revealed robust production in two proteobacterial hosts, Pseudomonas putida KT2440 and Agrobacterium tumefaciens LBA4404, but very little production of the antibiotic in various laboratory strains of Escherichia coli, despite their closer phylogenetic relationship. We identified a nonclustered LuxR-type quorum-sensing receptor from P. luteoviolacea 2ta16, PviR, that increases pathway transcription and violacein production in E. coli by similar to 60-fold independently of acyl-homoserine lactone autoinducers. Although E. coli harbors the most similar homolog of PviR identified from all of the hosts tested, overexpression of various E. coli transcription factors did not result in a statistically significant increase in violacein production, while overexpression of two A. tumefaciens PviR homologs significantly increased production. Thus, this work not only introduces a new genetic platform for the heterologous expression of microbial BGCs, it also challenges the assumption that host phylogeny is an accurate predictor of host compatibility. IMPORTANCE Although Gram-positive heterologous hosts such as Streptomyces have been developed and optimized to support diverse secondary metabolic reactions, there has been comparatively less work on Gram-negative hosts, some of which grow faster and are easier to work with. This work presents a new genetic platform for direct cloning and broad-host-range heterologous expression of BGCs in Gram-negative proteobacterial expression hosts, and we leverage this platform to uncover regulatory elements that influence violacein expression from Pseudoalteromonas. Although it is often assumed that BGCs will be more successfully expressed in more closely related hosts, our work suggests that this may not be a general rule of thumb, as heterologous production of natural products can be influenced by specific host regulatory and/or biosynthetic elements, and the identity and effectiveness of those elements are difficult to predict. We argue for the use of a diverse set of heterologous hosts, which may also provide insights into BGC mechanism and function.

Li, J, Tang XY, Awakawa T, Moore BS.  2017.  Enzymatic C-H Oxidation-Amidation Cascade in the Production of Natural and Unnatural Thiotetronate Antibiotics with Potentiated Bioactivity. Angewandte Chemie-International Edition. 56:12234-12239.   10.1002/anie.201705239   AbstractWebsite

The selective activation of unreactive hydrocarbons by biosynthetic enzymes has inspired new synthetic methods in C-H bond activation. Herein, we report the unprecedented two-step biosynthetic conversion of thiotetromycin to thiotetroamideC involving the tandem oxidation and amidation of an unreactive ethyl group. We detail the genetic and biochemical basis for the terminal amidation in thiotetroamideC biosynthesis, which involves a uniquely adapted cytochrome P450-amidotransferase enzyme pair and highlights the first oxidation-amidation enzymatic cascade reaction leading to the selective formation of a primary amide group from a chemically inert alkyl group. Motivated by the ten-fold increase in antibiotic potency of thiotetroamideC ascribed to the acetamide group and the unusual enzymology involved, we enzymatically interrogated diverse thiolactomycin analogues and prepared an unnatural thiotetroamideC analogue with potentiated bioactivity compared to the parent molecule.

Letzel, AC, Li J, Amos GCA, Millan-Aguinaga N, Ginigini J, Abdelmohsen UR, Gaudencio SP, Ziemert N, Moore BS, Jensen PR.  2017.  Genomic insights into specialized metabolism in the marine actinomycete Salinispora. Environmental Microbiology. 19:3660-3673.   10.1111/1462-2920.13867   AbstractWebsite

Comparative genomics is providing new opportunities to address the diversity and distributions of genes encoding the biosynthesis of specialized metabolites. An analysis of 119 genome sequences representing three closely related species of the marine actinomycete genus Salinispora reveals extraordinary biosynthetic diversity in the form of 176 distinct biosynthetic gene clusters (BGCs) of which only 24 have been linked to their products. Remarkably, more than half of the BGCs were observed in only one or two strains, suggesting they were acquired relatively recently in the evolutionary history of the genus. These acquired gene clusters are concentrated in specific genomic islands, which represent hot spots for BGC acquisition. While most BGCs are stable in terms of their chromosomal position, others migrated to different locations or were exchanged with unrelated gene clusters suggesting a plug and play type model of evolution that provides a mechanism to test the relative fitness effects of specialized metabolites. Transcriptome analyses were used to address the relationships between BGC abundance, chromosomal position and product discovery. The results indicate that recently acquired BGCs can be functional and that complex evolutionary processes shape the micro-diversity of specialized metabolism observed in closely related environmental bacteria.

Tang, XY, Li J, Moore BS.  2017.  Minimization of the thiolactomycin biosynthetic pathway reveals that the cytochrome p450 enzyme tlmf is required for five-membered thiolactone ring formation. Chembiochem. 18:1072-1076.   10.1002/cbic.201700090   AbstractWebsite

Thiolactomycin (TLM) belongs to a class of rare and unique thiotetronate antibiotics that inhibit bacterial fatty acid synthesis. Although this group of natural product antibiotics was first discovered over 30 years ago, the study of TLM biosynthesis remains in its infancy. We recently discovered the biosynthetic gene cluster (BGC) for TLM from the marine bacterium Salinispora pacifica CNS-863. Here, we report the investigation of TLM biosynthetic logic through mutagenesis and comparative metabolic analyses. Our results revealed that only four genes (tlmF, tlmG, tlmH, and tlmI) are required for the construction of the characteristic -thiolactone skeleton of this class of antibiotics. We further showed that the cytochrome P450 TlmF does not directly participate in sulfur insertion and C-S bond formation chemistry but rather in the construction of the five-membered thiolactone ring as, upon its deletion, we observed the alternative production of the six-membered -thiolactomycin. Our findings pave the way for future biochemical investigation of the biosynthesis of this structurally unique group of thiotetronic acid natural products.

Agarwal, V, Miles ZD, Winter JM, Eustaquio AS, El Gamal AA, Moore BS.  2017.  Enzymatic halogenation and dehalogenation reactions: Pervasive and mechanistically diverse. Chemical Reviews. 117:5619-5674.   10.1021/acs.chemrev.6b00571   AbstractWebsite

Naturally produced halogenated compounds are ubiquitous across all domains of life where they perform a multitude of biological functions and adopt a diversity of chemical structures. Accordingly, a diverse collection of enzyme catalysts to install and remove halogens from organic scaffolds has evolved in nature. Accounting for the different chemical properties of the four halogen atoms (fluorine, chlorine, bromine, and iodine) and the diversity and chemical reactivity of their organic substrates, enzymes performing biosynthetic and degradative halogenation chemistry utilize numerous mechanistic strategies involving oxidation, reduction, and substitution. Biosynthetic halogenation reactions range from simple aromatic substitutions to stereoselective C-H functionalizations on remote carbon centers and can initiate the formation of simple to complex ring structures. Dehalogenating enzymes, on the other hand, are best known for removing halogen atoms from man-made organohalogens, yet also function naturally, albeit rarely, in metabolic pathways. This review details the scope and mechanism of nature's halogenation and dehalogenation enzymatic strategies, highlights gaps in our understanding, and posits where new advances in the field might arise in the near future.

Larson, CB, Crusemann M, Moore BS.  2017.  PCR-independent method of transformation-associated recombination reveals the cosmomycin biosynthetic gene cluster in an ocean streptomycete. Journal of Natural Products. 80:1200-1204.   10.1021/acs.jnatprod.6b01121   AbstractWebsite

The transformation-associated recombination cloning methodology facilitates the genomic capture and heterologous expression of natural product biosynthetic gene clusters (BGCs). We have streamlined this procedure by introduction of synthetic DNA gene blocks for the efficient capture of BGCs. We show the successful capture and expression of the aromatic polyketide antitumor agent cosmomycin from streptomycete bacteria and the discovery of new cosmomycin analogues by mass spectral molecular networking.

Agarwal, V, Blanton JM, Podell S, Taton A, Schorn MA, Busch J, Lin Z, Schmidt EW, Jensen PR, Paul VJ, Biggs JS, Golden JW, Allen EE, Moore BS.  2017.  Metagenomic discovery of polybrominated diphenyl ether biosynthesis by marine sponges. Nat Chem Biol. advance online publication: Nature Publishing Group, a division of Macmillan Publishers Limited. All Rights Reserved.   10.1038/nchembio.2330   Abstract

Naturally produced polybrominated diphenyl ethers (PBDEs) pervade the marine environment and structurally resemble toxic man-made brominated flame retardants. PBDEs bioaccumulate in marine animals and are likely transferred to the human food chain. However, the biogenic basis for PBDE production in one of their most prolific sources, marine sponges of the order Dysideidae, remains unidentified. Here, we report the discovery of PBDE biosynthetic gene clusters within sponge-microbiome-associated cyanobacterial endosymbionts through the use of an unbiased metagenome-mining approach. Using expression of PBDE biosynthetic genes in heterologous cyanobacterial hosts, we correlate the structural diversity of naturally produced PBDEs to modifications within PBDE biosynthetic gene clusters in multiple sponge holobionts. Our results establish the genetic and molecular foundation for the production of PBDEs in one of the most abundant natural sources of these molecules, further setting the stage for a metagenomic-based inventory of other PBDE sources in the marine environment.

Crusemann, M, O'Neill EC, Larson CB, Melnik AV, Floros DJ, da Silva RR, Jensen PR, Dorrestein PC, Moore BS.  2017.  Prioritizing natural product diversity in a collection of 146 bacterial strains based on growth and extraction protocols. Journal of Natural Products. 80:588-597.   10.1021/acsjnatprod.6b00722   AbstractWebsite

In order to expedite the rapid and efficient discovery and isolation of novel specialized metabolites, while minimizing the waste of resources on rediscovery of known compounds, it is crucial to develop efficient approaches for strain prioritization, rapid dereplication, and the assessment of favored cultivation and extraction conditions. Herein we interrogated bacterial strains by systematically evaluating cultivation and extraction parameters with LC-MS/MS analysis and subsequent dereplication through the Global Natural Product Social Molecular Networking (GNPS) platform. The developed method is fast, requiring minimal time and sample material, and is compatible with high throughput extract analysis, thereby streamlining strain prioritization and evaluation of culturing parameters. With this approach, we analyzed 146 marine Salinispora and Streptomyces strains that were grown and extracted using multiple different protocols. In total, 603 samples were analyzed, generating approximately 1.8 million mass spectra. We constructed a comprehensive molecular network and identified 15 molecular families of diverse natural products and their analogues. The size and breadth of this network shows statistically supported trends in molecular diversity when comparing growth and extraction conditions. The network provides an extensive survey of the biosynthetic capacity of the strain collection and a method to compare strains based on the variety and novelty of their metabolites. This approach allows us to quickly identify patterns in metabolite production that can be linked to taxonomy, culture conditions, and extraction methods, as well as informing the most valuable growth and extraction conditions.

Patin, NV, Schorn M, Aguinaldo K, Lincecum T, Moore BS, Jensen PR.  2017.  Effects of actinomycete secondary metabolites on sediment microbial communities. Applied and Environmental Microbiology. 83   10.1128/aem.02676-16   Abstract

Marine sediments harbor complex microbial communities that remain poorly studied relative to other biomes such as seawater. Moreover, bacteria in these communities produce antibiotics and other bioactive secondary metabolites, yet little is known about how these compounds affect microbial community structure. In this study, we used next-generation amplicon sequencing to assess native microbial community composition in shallow tropical marine sediments. The results revealed complex communities comprised of largely uncultured taxa, with considerable spatial heterogeneity and known antibiotic producers comprising only a small fraction of the total diversity. Organic extracts from cultured strains of the sedimentdwelling actinomycete genus Salinispora were then used in mesocosm studies to address how secondary metabolites shape sediment community composition. We identified predatory bacteria and other taxa that were consistently reduced in the extract-treated mesocosms, suggesting that they may be the targets of allelopathic interactions. We tested related taxa for extract sensitivity and found general agreement with the culture-independent results. Conversely, several taxa were enriched in the extract-treated mesocosms, suggesting that some bacteria benefited from the interactions. The results provide evidence that bacterial secondary metabolites can have complex and significant effects on sediment microbial communities. IMPORTANCE Ocean sediments represent one of Earth's largest and most poorly studied biomes. These habitats are characterized by complex microbial communities where competition for space and nutrients can be intense. This study addressed the hypothesis that secondary metabolites produced by the sediment-inhabiting actinomycete Salinispora arenicola affect community composition and thus mediate interactions among competing microbes. Next-generation amplicon sequencing of mesocosm experiments revealed complex communities that shifted following exposure to S. arenicola extracts. The results reveal that certain predatory bacteria were consistently less abundant following exposure to extracts, suggesting that microbial metabolites mediate competitive interactions. Other taxa increased in relative abundance, suggesting a benefit from the extracts themselves or the resulting changes in the community. This study takes a first step toward assessing the impacts of bacterial metabolites on sediment microbial communities. The results provide insight into how low-abundance organisms may help structure microbial communities in ocean sediments.

Nguyen, DD, Melnik AV, Koyama N, Lu XW, Schorn M, Fang JS, Aguinaldo K, Lincecum TL, Ghequire MGK, Carrion VJ, Cheng TL, Duggan BM, Malone JG, Mauchline TH, Sanchez LM, Kilpatrick AM, Raaijmakers JM, De Mot R, Moore BS, Medema MH, Dorrestein PC.  2017.  Indexing the Pseudomonas specialized metabolome enabled the discovery of poaeamide B and the bananamides. Nature Microbiology. 2   10.1038/nmicrobiol.2016.197   AbstractWebsite

Pseudomonads are cosmopolitan microorganisms able to produce a wide array of specialized metabolites. These molecules allow Pseudomonas to scavenge nutrients, sense population density and enhance or inhibit growth of competing microorganisms. However, these valuable metabolites are typically characterized one-molecule-one-microbe at a time, instead of being inventoried in large numbers. To index and map the diversity of molecules detected from these organisms, 260 strains of ecologically diverse origins were subjected to mass-spectrometry-based molecular networking. Molecular networking not only enables dereplication of molecules, but also sheds light on their structural relationships. Moreover, it accelerates the discovery of new molecules. Here, by indexing the Pseudomonas specialized metabolome, we report the molecular-networking-based discovery of four molecules and their evolutionary relationships: a poaeamide analogue and a molecular subfamily of cyclic lipopeptides, bananamides 1, 2 and 3. Analysis of their biosynthetic gene cluster shows that it constitutes a distinct evolutionary branch of the Pseudomonas cyclic lipopeptides. Through analysis of an additional 370 extracts of wheat-associated Pseudomonas, we demonstrate how the detailed knowledge from our reference index can be efficiently propagated to annotate complex metabolomic data from other studies, akin to the way in which newly generated genomic information can be compared to data from public databases.

Jordan, PA, Moore BS.  2016.  Biosynthetic pathway connects cryptic ribosomally synthesized posttranslationally modified peptide genes with pyrroloquinoline alkaloids. Cell Chemical Biology. 23:1504-1514.   10.1016/j.chembiol.2016.10.009   AbstractWebsite

In an era where natural product biosynthetic gene clusters can be rapidly identified from sequenced genomes, it is unusual for the biosynthesis of an entire natural product class to remain unknown. Yet, the genetic determinates for pyrroloquinoline alkaloid biosynthesis have remained obscure despite their abundance and deceptive structural simplicity. In this work, we have identified the biosynthetic gene cluster for ammosamides A-C, pyrroloquinoline alkaloids from Streptomyces sp. CNR-698. Through direct cloning, heterologous expression and gene deletions we have validated the ammosamide biosynthetic gene cluster and demonstrated that these seemingly simple molecules are derived from a surprisingly complex set of biosynthetic genes that are also found in the biosynthesis of lymphostin, a structurally related pyrroloquinoline alkaloid from Salinispora and Streptomyces. Our results implicate a conserved set of genes driving pyrroloquinoline biosynthesis that consist of genes frequently associated with ribosomal peptide natural product biosynthesis, and whose exact biochemical role remains enigmatic.

Zhang, WP, Lu L, Lai QL, Zhu BK, Li ZR, Xu Y, Shao ZZ, Herrup K, Moore BS, Ross AC, Qian PY.  2016.  Family-wide structural characterization and genomic comparisons decode the diversity-oriented biosynthesis of thalassospiramides by marine proteobacteria. Journal of Biological Chemistry. 291:27228-+.   10.1074/jbc.M116.756858   AbstractWebsite

The thalassospiramide lipopeptides have great potential for therapeutic applications; however, their structural and functional diversity and biosynthesis are poorly understood. Here, by cultivating 130 Rhodospirillaceae strains sampled from oceans worldwide, we discovered 21 new thalassospiramide analogues and demonstrated their neuroprotective effects. To investigate the diversity of biosynthetic gene cluster (BGC) architectures, we sequenced the draft genomes of 28 Rhodospirillaceae strains. Our family-wide genomic analysis revealed three types of dysfunctional BGCs and four functional BGCs whose architectures correspond to four production patterns. This correlation allowed us to reassess the "diversity-oriented biosynthesis" proposed for the microbial production of thalassospiramides, which involves iteration of several key modules. Preliminary evolutionary investigation suggested that the functional BGCs could have arisen through module/domain loss, whereas the dysfunctional BGCs arose through horizontal gene transfer. Further comparative genomics indicated that thalassospiramide production is likely to be attendant on particular genes/pathways for amino acid metabolism, signaling transduction, and compound efflux. Our findings provide a systematic understanding of thalassospiramide production and new insights into the underlying mechanism.

Schorn, MA, Alanjary MM, Aguinaldo K, Korobeynikov A, Podell S, Patin N, Lincecum T, Jensen PR, Ziemert N, Moore BS.  2016.  Sequencing rare marine actinomycete genomes reveals high density of unique natural product biosynthetic gene clusters. Microbiology-Sgm. 162:2075-2086.   10.1099/mic.0.000386   AbstractWebsite

Traditional natural product discovery methods have nearly exhausted the accessible diversity of microbial chemicals, making new sources and techniques paramount in the search for new molecules. Marine actinomycete bacteria have recently come into the spotlight as fruitful producers of structurally diverse secondary metabolites, and remain relatively untapped. In this study, we sequenced 21 marine-derived actinomycete strains, rarely studied for their secondary metabolite potential and under-represented in current genomic databases. We found that genome size and phylogeny were good predictors of biosynthetic gene cluster diversity, with larger genomes rivalling the well-known marine producers in the Streptomyces and Salinispora genera. Genomes in the Micrococcineae suborder, however, had consistently the lowest number of biosynthetic gene clusters. By networking individual gene clusters into gene cluster families, we were able to computationally estimate the degree of novelty each genus contributed to the current sequence databases. Based on the similarity measures between all actinobacteria in the Joint Genome Institute's Atlas of Biosynthetic gene Clusters database, rare marine genera show a high degree of novelty and diversity, with Corynebacterium, Gordonia, Nocardiopsis, Saccharomonospora and Pseudonocardia genera representing the highest gene cluster diversity. This research validates that rare marine actinomycetes are important candidates for exploration, as they are relatively unstudied, and their relatives are historically rich in secondary metabolites.

Groenhagen, U, De Oliveira ALL, Fielding E, Moore BS, Schulz S.  2016.  Coupled biosynthesis of volatiles and salinosporamideA in Salinispora tropica. Chembiochem. 17:1978-1985.   10.1002/cbic.201600388   AbstractWebsite

Terrestrial bacteria, especially actinomycetes, are known to be prolific producers of volatile compounds. We show here that bacteria from ocean sediments can also release complex bouquets of volatiles. The actinomycete Salinispora tropica produces cyclohexenyl compounds not previously known in nature, such as methyl cyclohex-2-ene-1-carboxylate (9), methyl 2-(cyclohex-2-en-1-yl)acetate (10), methyl (E/Z)-2-(cyclohex-2-en-1-ylidene)acetate (11/12), and related alcohols 8 and 13. These compounds were identified by GC/MS and confirmed by synthesis. In addition, rare spiroacetals, aromatic compounds, short-chain acids and esters, alcohols, and various cyclic compounds were produced by the bacteria. The biosynthesis of the cyclohexenyl compounds is closely coupled to that of cyclohexenylalanine (4), a building block of salinosporamideA, a proteasome inhibitor produced by S.tropica. Analysis of S.tropica strains that harbor knockouts of the salinosporamide biosynthetic genes salX and salD, coupled with feeding experiments, revealed that 3-(cyclohex-2-en-1-yl)-2-oxopropanoic acid (60) and 3-(cyclohex-2-en-1-ylidene)-2-oxopropanoic acid (isomers 61 and 62) are important intermediates in the biosynthesis of salinosporamideA, 4, and 8-13.

Li, ZR, Li J, Gu JP, Lai JYH, Duggan BM, Zhang WP, Li ZL, Li YX, Tong RB, Xu Y, Lin DH, Moore BS, Qian PY.  2016.  Divergent biosynthesis yields a cytotoxic aminomalonate-containing precolibactin. Nature Chemical Biology. 12:773-+.   10.1038/nchembio.2157   AbstractWebsite

Colibactin is an as-yet-uncharacterized genotoxic secondary metabolite produced by human gut bacteria. Here we report the biosynthetic discovery of two new precolibactin molecules from Escherichia coli, including precolibactin-886, which uniquely incorporates the highly sought genotoxicity-associated aminomalonate building block into its unprecedented macrocyclic structure. This work provides new insights into the biosynthetic logic and mode of action of this colorectal-cancer-linked microbial chemical.

Elgamal, A, Agarwal V, Rahman I, Moore BS.  2016.  Enzymatic reductive dehalogenation controls the biosynthesis of marine bacterial pyrroles. Journal of the American Chemical Society. 138:13167-13170.   10.1021/jacs.6b08512   AbstractWebsite

Enzymes capable of performing dehalogenating reactions have attracted tremendous contemporary attention due to their potential application in the bioremediation of anthropogenic polyhalogenated persistent organic pollutants. Nature, in particular the marine environment, is also a prolific source of polyhalogenated organic natural products. The study of the biosynthesis of these natural products has furnished a diverse array of halogenation biocatalysts, but thus far no examples of dehalogenating enzymes have been reported from a secondary metabolic pathway. Here we show that the penultimate step in the biosynthesis of the highly brominated marine bacterial product pentabromopseudilin is catalyzed by an unusual debrominase Bmp8 that utilizes a redox thiol mechanism to remove the C-2 bromine atom of 2,3,4,5-tetrabromopyrrole to facilitate oxidative coupling to 2,4-dibromophenol. To the best of our knowledge, Bmp8 is first example of a dehalogenating enzyme from the established genetic and biochemical context of a natural product biosynthetic pathway.

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.

Elgamal, A, Agarwal V, Diethelm S, Rahman I, Schorn MA, Sneed JM, Louie GV, Whalen KE, Mincer TJ, Noel JP, Paul VJ, Moore BS.  2016.  Biosynthesis of coral settlement cue tetrabromopyrrole in marine bacteria by a uniquely adapted brominase-thioesterase enzyme pair. Proceedings of the National Academy of Sciences of the United States of America. 113:3797-3802.   10.1073/pnas.1519695113   AbstractWebsite

Halogenated pyrroles (halopyrroles) are common chemical moieties found in bioactive bacterial natural products. The halopyrrole moieties of mono-and dihalopyrrole-containing compounds arise from a conserved mechanism in which a proline-derived pyrrolyl group bound to a carrier protein is first halogenated and then elaborated by peptidic or polyketide extensions. This paradigm is broken during the marine pseudoalteromonad bacterial biosynthesis of the coral larval settlement cue tetrabromopyrrole (1), which arises from the substitution of the proline-derived carboxylate by a bromine atom. To understand the molecular basis for decarboxylative bromination in the biosynthesis of 1, we sequenced two Pseudoalteromonas genomes and identified a conserved four-gene locus encoding the enzymes involved in its complete biosynthesis. Through total in vitro reconstitution of the biosynthesis of 1 using purified enzymes and biochemical interrogation of individual biochemical steps, we show that all four bromine atoms in 1 are installed by the action of a single flavin-dependent halogenase: Bmp2. Tetrabromination of the pyrrole induces a thioesterase-mediated offloading reaction from the carrier protein and activates the biosynthetic intermediate for decarboxylation. Insights into the tetrabrominating activity of Bmp2 were obtained from the high-resolution crystal structure of the halogenase contrasted against structurally homologous halogenase Mpy16 that forms only a dihalogenated pyrrole in marinopyrrole biosynthesis. Structure-guided mutagenesis of the proposed substrate- binding pocket of Bmp2 led to a reduction in the degree of halogenation catalyzed. Our study provides a biogenetic basis for the biosynthesis of 1 and sets a firm foundation for querying the biosynthetic potential for the production of 1 in marine (meta) genomes.