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2015
Li, YX, Li ZR, Yamanaka K, Xu Y, Zhang WP, Vlamakis H, Kolter R, Moore BS, Qian PY.  2015.  Directed natural product biosynthesis gene cluster capture and expression in the model bacterium Bacillus subtilis. Scientific Reports. 5   10.1038/srep09383   AbstractWebsite

Bacilli are ubiquitous low G+C environmental Gram-positive bacteria that produce a wide assortment of specialized small molecules. Although their natural product biosynthetic potential is high, robust molecular tools to support the heterologous expression of large biosynthetic gene clusters in Bacillus hosts are rare. Herein we adapt transformation-associated recombination (TAR) in yeast to design a single genomic capture and expression vector for antibiotic production in Bacillus subtilis. After validating this direct cloning "plug-and-play'' approach with surfactin, we genetically interrogated amicoumacin biosynthetic gene cluster from the marine isolate Bacillus subtilis 1779. Its heterologous expression allowed us to explore an unusual maturation process involving the N-acyl-asparagine pro-drug intermediates preamicoumacins, which are hydrolyzed by the asparagine-specific peptidase into the active component amicoumacin A. This work represents the first direct cloning based heterologous expression of natural products in the model organism B. subtilis and paves the way to the development of future genome mining efforts in this genus.

Lu, L, Meehan MJ, Gu S, Chen Z, Zhang W, Zhang G, Liu L, Huang X, Dorrestein PC, Xu Y, Moore BS, Qian P-Y.  2015.  Mechanism of action of thalassospiramides, a new class of calpain inhibitors. Scientific Reports. 5   10.1038/srep08783   AbstractWebsite

Thalassospiramides comprise a large family of lipopeptide natural products produced by Thalassospira and Tistrella marine bacteria. Here we provide further evidence of their nanomolar inhibitory activity against the human calpain 1 protease. Analysis of structure-activity relationship data supported our hypothesis that the rigid 12-membered ring containing an alpha,beta-unsaturated carbonyl moiety is the pharmacologically active functional group, in contrast to classic electrophilic "warheads" in known calpain inhibitors. Using a combination of chemical modifications, mass spectrometric techniques, site-directed mutagenesis, and molecular modeling, we show the covalent binding of thalassospiramide's alpha,beta-unsaturated carbonyl moiety to the thiol group of calpain's catalytic Cys115 residue by a Michael 1,4-addition reaction. As nanomolar calpain inhibitors with promising selectivity and low toxicity from natural sources are rare, we consider thalassospiramides as promising drug leads.

Agarwal, V, Li J, Rahman I, Borgen M, Aluwihare LI, Biggs JS, Paul VJ, Moore BS.  2015.  Complexity of naturally produced polybrominated diphenyl ethers revealed via mass spectrometry. Environmental Science & Technology. 49:1339-1346.   10.1021/es505440j   AbstractWebsite

Polybrominated diphenyl ethers (PBDEs) are persistent and bioaccumulative anthropogenic and natural chemicals that are broadly distributed in the marine environment. PBDEs are potentially toxic due to inhibition of various mammalian signaling pathways and enzymatic reactions. PBDE isoforms vary in toxicity in accordance with structural differences, primarily in the number and pattern of hydroxyl moieties afforded upon a conserved core structure. Over four decades of isolation and discovery-based efforts have established an impressive repertoire of natural PBDEs. Based on our recent reports describing the bacterial biosyntheses of PBDEs, we predicted the presence of additional classes of PBDEs to those previously identified from marine sources. Using mass spectrometry and NMR spectroscopy, we now establish the existence of new structural classes of PBDEs in marine sponges. Our findings expand the chemical space explored by naturally produced PBDEs, which may inform future environmental toxicology studies. Furthermore, we provide evidence for iodinated PBDEs and direct attention toward the contribution of promiscuous halogenating enzymes in further expanding the diversity of these polyhalogenated marine natural products.

2014
Diethelm, S, Teufel R, Kaysser L, Moore BS.  2014.  A multitasking vanadium-dependent chloroperoxidase as an inspiration for the chemical synthesis of the merochlorins. Angewandte Chemie-International Edition. 53:11023-11026.   10.1002/anie.201405696   AbstractWebsite

The vanadium-dependent chloroperoxidase Mcl24 was discovered to mediate a complex series of unprecedented transformations in the biosynthesis of the merochlorin meroterpenoid antibiotics. In particular, a site-selective naphthol chlorination is followed by an oxidative dearomatization/terpene cyclization sequence to build up the stereochemically complex carbon framework of the merochlorins in one step. Inspired by the enzyme reactivity, a chemical chlorination protocol paralleling the biocatalytic process was developed. These chemical studies led to the identification of previously overlooked merochlorin natural products.

Teufel, R, Kaysser L, Villaume MT, Diethelm S, Carbullido MK, Baran PS, Moore BS.  2014.  One-pot enzymatic synthesis of merochlorin A and B. Angewandte Chemie-International Edition. 53:11019-11022.   10.1002/anie.201405694   AbstractWebsite

The polycycles merochlorinA and B are complex halogenated meroterpenoid natural products with significant antibacterial activities and are produced by the marine bacterium Streptomyces sp. strain CNH-189. Heterologously produced enzymes and chemical synthesis are employed herein to fully reconstitute the merochlorin biosynthesis invitro. The interplay of a dedicated typeIII polyketide synthase, a prenyl diphosphate synthase, and an aromatic prenyltransferase allow formation of a highly unusual aromatic polyketide-terpene hybrid intermediate which features an unprecedented branched sesquiterpene moiety from isosesquilavandulyl diphosphate. As supported by invivo experiments, this precursor is furthermore chlorinated and cyclized to merochlorinA and isomeric merochlorinB by a single vanadium-dependent haloperoxidase, thus completing the remarkably efficient pathway.

Xu, MM, Hillwig ML, Lane AL, Tiernan MS, Moore BS, Peters RJ.  2014.  Characterization of an orphan diterpenoid biosynthetic operon from Salinispora arenicola. Journal of Natural Products. 77:2144-2147.   10.1021/np500422d   AbstractWebsite

While more commonly associated with plants than microbes, diterpenoid natural products have been reported to have profound effects in marine microbe-microbe interactions. Intriguingly, the genome of the marine bacterium Salinispora arenicola CNS-205 contains a putative diterpenoid biosynthetic operon, terp1. Here recombinant expression studies are reported, indicating that this three-gene operon leads to the production of isopimara-8,15-dien-19-ol (4). Although 4 is not observed in pure cultures of S. arenicola, it is plausible that the terp1 operon is only expressed under certain physiologically relevant conditions such as in the presence of other marine organisms.

Agarwal, V, Moore BS.  2014.  Enzymatic synthesis of polybrominated dioxins from the marine environment. Acs Chemical Biology. 9:1980-1984.   10.1021/cb5004338   AbstractWebsite

Polyhalogenated dibenzo-p-dioxins are arguably among the most toxic molecules known to man. In addition to anthropogenic sources, marine invertebrates also harbor polybrominated dibenzo-p-dioxins of as yet unknown biogenic origin. Here, we report that the bmp gene locus in marine bacteria, a recently characterized source of polybrominated diphenyl ethers, can also synthesize dibenzo-p-dioxins by employing different phenolic initiator molecules. Our findings also diversify the structural classes of diphenyl ethers accessed by the mmp biosynthetic pathway. This report lays the biochemical foundation of a likely biogenetic origin of dibenzo-p-dioxins present in the marine metabolome and greatly expands the toxicity potential of marine derived polyhaloganated natural products.

Agarwal, V, El Gamal AA, Yamanaka K, Poth D, Kersten RD, Schorn M, Allen EE, Moore BS.  2014.  Biosynthesis of polybrominated aromatic organic compounds by marine bacteria. Nature Chemical Biology. 10:640-U182.   10.1038/nchembio.1564   AbstractWebsite

Polybrominated diphenyl ethers (PBDEs) and polybrominated bipyrroles are natural products that bioaccumulate in the marine food chain. PBDEs have attracted widespread attention because of their persistence in the environment and potential toxicity to humans. However, the natural origins of PBDE biosynthesis are not known. Here we report marine bacteria as producers of PBDEs and establish a genetic and molecular foundation for their production that unifies paradigms for the elaboration of bromophenols and bromopyrroles abundant in marine biota. We provide biochemical evidence of marine brominases revealing decarboxylative-halogenation enzymology previously unknown among halogenating enzymes. Biosynthetic motifs discovered in our study were used to mine sequence databases to discover unrealized marine bacterial producers of organobromine compounds.

Mohimani, H, Liu WT, Kersten RD, Moore BS, Dorrestein PC, Pevzner PA.  2014.  NRPquest: Coupling mass spectrometry and genome mining for nonribosomal peptide discovery. Journal of Natural Products. 77:1902-1909.   10.1021/np500370c   AbstractWebsite

Nonribosomal peptides (NRPs) such as vancomycin and daptomycin are among the most effective antibiotics. While NRPs are biomedically important, the computational techniques for sequencing these peptides are still in their infancy. The recent emergence of mass spectrometry techniques for NRP analysis (capable of sequencing an NRP from small amounts of nonpurified material) revealed an enormous diversity of NRPs. However, as many NRPs have nonlinear structure (e.g., cyclic or branched-cyclic peptides), the standard de novo sequencing tools (developed for linear peptides) are not applicable to NRP analysis. Here, we introduce the first NRP identification algorithm, NRPquest, that performs mutation-tolerant and modification-tolerant searches of spectral data sets against a database of putative NRPs. In contrast to previous studies aimed at NRP discovery (that usually report very few NRPs), NRPquest revealed nearly a hundred NRPs (including unknown variants of previously known peptides) in a single study. This result indicates that NRPquest can potentially make MS-based NRP identification as robust as the identification of linear peptides in traditional proteomics.

Niewerth, D, Jansen G, Riethoff LFV, van Meerloo J, Kale AJ, Moore BS, Assaraf YG, Anderl JL, Zweegman S, Kaspers GJL, Cloos J.  2014.  Antileukemic activity and mechanism of drug resistance to the marine Salinispora tropica proteasome inhibitor Salinosporamide A (Marizomib). Molecular Pharmacology. 86:12-19.   10.1124/mol.114.092114   AbstractWebsite

Salinosporamide A (NPI-0052, marizomib) is a naturally occurring proteasome inhibitor derived from the marine actinobacterium Salinispora tropica, and represents a promising clinical agent in the treatment of hematologic malignancies. Recently, these actinobacteria were shown to harbor self-resistance properties to salinosporamide A by expressing redundant catalytically active mutants of the 20S proteasome beta-subunit, reminiscent of PSMB5 mutations identified in cancer cells with acquired resistance to the founding proteasome inhibitor bortezomib (BTZ). Here, we assessed the growth inhibitory potential of salinosporamide A in human acute lymphocytic leukemia CCRF-CEM cells, and its 10-fold (CEM/BTZ7) and 123-fold (CEM/BTZ200) bortezomib-resistant sublines harboring PSMB5 mutations. Parental cells displayed sensitivity to salinosporamide A (IC50=5.1 nM), whereas their bortezomib-resistant sublines were 9-and 17-fold cross-resistant to salinosporamide A, respectively. Notably, combination experiments of salinosporamide A and bortezomib showed synergistic activity in CEM/BTZ200 cells. CEM cells gradually exposed to 20 nM salinosporamide A (CEM/S20) displayed stable 5-fold acquired resistance to salinosporamide A and were 3-fold cross-resistant to bortezomib. Consistent with the acquisition of a PSMB5 point mutation (M45V) in CEM/S20 cells, salinosporamide A displayed a markedly impaired capacity to inhibit beta(5)-associated catalytic activity. Last, compared with parental CEM cells, CEM/S20 cells exhibited up to 2.5-fold upregulation of constitutive proteasome subunits, while retaining unaltered immunoproteasome subunit expression. In conclusion, salinosporamide A displayed potent antileukemic activity against bortezomib-resistant leukemia cells. beta-Subunit point mutations as a common feature of acquired resistance to salinosporamide A and bortezomib in hematologic cells and S. tropica suggest an evolutionarily conservedmechanism of resistance to proteasome inhibitors.

Mohimani, H, Kersten RD, Liu WT, Wang MX, Purvine SO, Wu S, Brewer HM, Pasa-Tolic L, Bandeira N, Moore BS, Pevzner PA, Dorrestein PC.  2014.  Automated genome mining of ribosomal peptide natural products. Acs Chemical Biology. 9:1545-1551.   10.1021/cb500199h   AbstractWebsite

Ribosomally synthesized and posttranslationally modified peptides (RiPPs), especially from microbial sources, are a large group of bioactive natural products that are a promising source of new (bio)chemistry and bioactivity.(1) In light of exponentially increasing microbial genome databases and improved mass spectrometry (MS)-based metabolornic platforms, there is a need for computational tools that connect natural product genotypes predicted from microbial genome sequences with their corresponding chemotypes from metabolomic data sets. Here, we introduce RiPPquest, a tandem mass spectrometry database search tool for identification of microbial RiPPs, and apply it to lanthipeptide discovery. RiPPquest uses genomics to limit search space to the vicinity of RiPP biosynthetic genes and proteomics to analyze extensive peptide modifications and compute p-values of peptide-spectrum matches (PSMs). We highlight RiPPquest by connecting multiple RiPPs from extracts of Streptomyces to their gene clusters and by the discovery of a new class III lanthipeptide, informatipeptin, from Streptomyces viridochromogenes DSM 40736 to reflect that was discovered by mass spectrometry based genome mining using algorithmic tools rather than manual inspection of mass spectrometry data and genetic information. The presented tool is available at cyclo.ucsd.edu.

Trivella, DBB, Pereira AR, Stein ML, Kasai Y, Byrum T, Valeriote FA, Tantillo DJ, Groll M, Gerwick WH, Moore BS.  2014.  Enzyme inhibition by hydroamination: Design and mechanism of a hybrid carmaphycin-syringolin enone proteasome inhibitor. Chemistry & Biology. 21:782-791.   10.1016/j.chembiol.2014.04.010   AbstractWebsite

Hydroamination reactions involving the addition of an amine to an inactivated alkene are entropically prohibited and require strong chemical catalysts. While this synthetic process is efficient at generating substituted amines, there is no equivalent in small molecule-mediated enzyme inhibition. We report an unusual mechanism of proteasome inhibition that involves a hydroamination reaction of alkene derivatives of the epoxyketone natural product carmaphycin. We show that the carmaphycin enone first forms a hemiketal intermediate with the catalytic Thr1 residue of the proteasome before cyclization by an unanticipated intramolecular alkene hydroamination reaction, resulting in a stable six-membered morpholine ring. The carmaphycin enone electrophile, which does not undergo a 1,4-Michael addition as previously observed with vinyl sulfone and alpha,beta-unsaturated amide-based inhibitors, is partially reversible and gives insight into the design of proteasome inhibitors for cancer chemotherapy.

Jensen, PR, Chavarria KL, Fenical W, Moore BS, Ziemert N.  2014.  Challenges and triumphs to genomics-based natural product discovery. Journal of Industrial Microbiology & Biotechnology. 41:203-209.   10.1007/s10295-013-1353-8   AbstractWebsite

Genome sequencing is rapidly changing the field of natural products research by providing opportunities to assess the biosynthetic potential of strains prior to chemical analysis or biological testing. Ready access to sequence data is driving the development of new bioinformatic tools and methods to identify the products of silent or cryptic pathways. While genome mining has fast become a useful approach to natural product discovery, it has also become clear that identifying pathways of interest is much easier than finding the associated products. This has led to bottlenecks in the discovery process that must be overcome for the potential of genomics-based natural product discovery to be fully realized. In this perspective, we address some of these challenges in the context of our work with the marine actinomycete genus Salinispora, which is proving to be a useful model with which to apply genome mining as an approach to natural product discovery.

Yamanaka, K, Reynolds KA, Kersten RD, Ryan KS, Gonzalez DJ, Nizet V, Dorrestein PC, Moore BS.  2014.  Direct cloning and refactoring of a silent lipopeptide biosynthetic gene cluster yields the antibiotic taromycin A. Proceedings of the National Academy of Sciences of the United States of America. 111:1957-1962.   10.1073/pnas.1319584111   AbstractWebsite

Recent developments in next-generation sequencing technologies have brought recognition of microbial genomes as a rich resource for novel natural product discovery. However, owing to the scarcity of efficient procedures to connect genes to molecules, only a small fraction of secondary metabolomes have been investigated to date. Transformation-associated recombination (TAR) cloning takes advantage of the natural in vivo homologous recombination of Saccharomyces cerevisiae to directly capture large genomic loci. Here we report a TAR-based genetic platform that allows us to directly clone, refactor, and heterologously express a silent biosynthetic pathway to yield a new antibiotic. With this method, which involves regulatory gene remodeling, we successfully expressed a 67-kb nonribosomal peptide synthetase biosynthetic gene cluster from the marine actinomycete Saccharomonospora sp. CNQ-490 and produced the dichlorinated lipopeptide antibiotic taromycin A in the model expression host Streptomyces coelicolor. The taromycin gene cluster (tar) is highly similar to the clinically approved antibiotic daptomycin from Streptomyces roseosporus, but has notable structural differences in three amino acid residues and the lipid side chain. With the activation of the tar gene cluster and production of taromycin A, this study highlights a unique "plug-and-play" approach to efficiently gaining access to orphan pathways that may open avenues for novel natural product discoveries and drug development.

Schorn, M, Zettler J, Noel JP, Dorrestein PC, Moore BS, Kaysser L.  2014.  Genetic basis for the biosynthesis of the pharmaceutically important class of epoxyketone proteasome inhibitors. Acs Chemical Biology. 9:301-309.   10.1021/cb400699p   AbstractWebsite

The epoxyketone proteasome inhibitors are an established class of therapeutic agents for the treatment of cancer. Their unique alpha ',beta '-epoxyketone pharmacophore allows binding to the catalytic beta-subunits of the proteasome with extraordinary specificity. Here, we report the characterization of the first gene clusters for the biosynthesis of natural peptidyl-epozyketones. The clusters for epoxomicin, the lead compound for the anticancer drug Kyprolis, and for eponemycin were identified in the actinobacterial producer strains ATCC 53904 and Streptomyces hygroscopicus ATCC 53709, respectively, using a modified protocol for Ion Torrent PGM genome sequencing. Both gene clusters code for a hybrid nonribosomal peptide synthetase/polyketide synthase multifunctional enzyme complex and homologous redox enzymes. Epoxomicin and eponemycin were heterologously produced in Streptomyces albus J1046 via whole pathway expression. Moreover, we employed mass spectral molecular networking for a new comparative metabolomics approach in a heterologous system and discovered a number of putative epoxyketone derivatives. With this study, we have definitively linked epoxyketone proteasome inhibitors and their biosynthesis genes for the first time in any organism, which will now allow for their detailed biochemical investigation.

2013
Lane, AL, Nam SJ, Fukuda T, Yamanaka K, Kauffman CA, Jensen PR, Fenical W, Moore BS.  2013.  Structures and Comparative Characterization of Biosynthetic Gene Clusters for Cyanosporasides, Enediyne-Derived Natural Products from Marine Actinomycetes. Journal of the American Chemical Society. 135:4171-4174.   10.1021/ja311065v   AbstractWebsite

Cyanosporasides are marine bacterial natural products containing a chlorinated cyclopenta[a]indene core of suspected enediyne polyketide biosynthetic origin. Herein, we report the isolation and characterization of novel cyanosporasides C-F (3-6) from the marine actinomycetes Salinispora pacifica CNS-143 and Streptomyces sp. CNT-179, highlighted by the unprecedented C-2' N-acetylcysteamine functionalized hexose group of 6. Cloning, sequencing, and mutagenesis of homologous similar to 50 kb cyanosporaside biosynthetic gene clusters from both bacteria afforded the first genetic evidence supporting cyanosporaside's enediyne, and thereby p-benzyne biradical, biosynthetic origin and revealed the molecular basis for nitrile and glycosyl functionalization. This study provides new opportunities for bioengineering of enediyne derivatives and expands the structural diversity afforded by enediyne gene clusters.

Ross, AC, Xu Y, Lu L, Kersten RD, Shao ZZ, Al-Suwailem AM, Dorrestein PC, Qian PY, Moore BS.  2013.  Biosynthetic Multitasking Facilitates Thalassospiramide Structural Diversity in Marine Bacteria. Journal of the American Chemical Society. 135:1155-1162.   10.1021/ja3119674   AbstractWebsite

Thalassospiramides A and B are immunosuppressant cyclic lipopeptides first reported from the marine alpha-proteobacterium Thalassospira sp. CNJ-328. We describe here the discovery and characterization of an extended family of 14 new analogues from four Tistrella and Thalassospira isolates. These potent calpain 1 protease inhibitors belong to six structure classes in which the length and composition of the acylpeptide side chain varies extensively. Genomic sequence analysis of the thalassospiramide-producing microbes revealed related, genus-specific biosynthetic loci encoding hybrid nonribosomal peptide synthetase/polyketide synthases consistent with thalassospiramide assembly. The bioinformatics analysis of the gene clusters suggests that structural diversity, which ranges from the 803.4 Da thalassospiramide C to the 1291.7 Da thalassospiramide F, results from a complex sequence of reactions involving amino acid substrate channeling and enzymatic multimodule skipping and iteration. Preliminary biochemical analysis of the N-terminal nonribosomal peptide synthetase module from the Thalassospira TtcA megasynthase supports a biosynthetic model in which in cis amino acid activation competes with in trans activation to increase the range of amino acid substrates incorporated at the N terminus.

Horsman, GP, Lechner A, Ohnish Y, Moore BS, Shen B.  2013.  Predictive model for epoxide hydrolase-generated stereochemistry in the biosynthesis of nine-membered enediyne antitumor antibiotics. Biochemistry. 52:5217-5224.   10.1021/bi400572a   AbstractWebsite

Nine-membered enediyne antitumor antibiotics C-1027, neocarzinostatin (NCS), and kedarcidin (KED) possess enediyne cores to which activity-modulating peripheral moieties are attached via (R)- or (S)-vicinal diols. We have previously shown that this stereochemical difference arises from hydrolysis of epoxide precursors by epoxide hydrolases (EHs) with different regioselectivities. The inverting EHs, such as SgcF, hydrolyze an (S)-epoxide substrate to yield an (R)-diol in C-1027 biosynthesis, whereas the retaining EHs, such as NcsF2 and KedF, hydrolyze an (S)-epoxide substrate to yield an (S)-diol in NCS and KED biosynthesis. We now report the characterization of a series of EH mutants and provide a predictive model for EH regioselectivity in the biosynthesis of the nine-membered enediyne antitumor antibiotics. A W236Y mutation in SgcF increased the retaining activity toward (S)-styrene oxide by 3-fold, and a W236Y/Q237M double mutation in SgcF, mimicking NcsF2 and KedF, resulted in a 20-fold increase in the retaining activity. To test the predictive utility of these mutations, two putative enediyne biosynthesis-associated EHs were identified by genome mining and confirmed as inverting enzymes, SpoF from Salinospora tropica CNB-440 and SgrF (SGR_625) from Streptomyces griseus IFO 13350. Finally, phylogenetic analysis of EHs revealed a familial classification according to inverting versus retaining activity. Taken together, these results provide a predictive model for vicinal diol stereochemistry in enediyne biosynthesis and set the stage for further elucidating the origins of EH regioselectivity.

Lechner, A, Wilson MC, Ban YH, Hwang JY, Yoon YJ, Moore BS.  2013.  Designed biosynthesis of 36-methyl-fk506 by polyketide precursor pathway engineering. Acs Synthetic Biology. 2:379-383.   10.1021/sb3001062   AbstractWebsite

The polyketide synthase (PKS) biosynthetic code has recently expanded to include a newly recognized group of extender unit substrates derived from alpha,beta-unsaturated acyl-CoA molecules that deliver diverse side chain chemistry to polyketide backbones. Herein we report the identification of a three-gene operon responsible for the biosynthesis of the PKS building block isobutyrylmalonyl-CoA associated with the macrolide ansalactam A from the marine bacterium Streptomyces sp. CNH189. Using a synthetic biology approach, we engineered the production of unnatural 36-methyl-FK506 in Streptomyces sp. KCTC 11604BP by incorporating the branched extender unit into FK506 biosynthesis in place of its natural C-21 allyl side chain, which has been shown to be critical for FK506's potent immunosuppressant and neurite outgrowth activities.

Nguyen, DD, Wu CH, Moree WJ, Lamsa A, Medema MH, Zhao XL, Gavilan RG, Aparicio M, Atencio L, Jackson C, Ballesteros J, Sanchez J, Watrous JD, Phelan VV, van de Wiel C, Kersten RD, Mehnaz S, De Mot R, Shank EA, Charusanti P, Nagarajan H, Duggan BM, Moore BS, Bandeira N, Palsson BO, Pogliano K, Gutierrez M, Dorrestein PC.  2013.  MS/MS networking guided analysis of molecule and gene cluster families. Proceedings of the National Academy of Sciences of the United States of America. 110:E2611-E2620.   10.1073/pnas.1303471110   AbstractWebsite

The ability to correlate the production of specialized metabolites to the genetic capacity of the organism that produces such molecules has become an invaluable tool in aiding the discovery of biotechnologically applicable molecules. Here, we accomplish this task by matching molecular families with gene cluster families, making these correlations to 60 microbes at one time instead of connecting one molecule to one organism at a time, such as how it is traditionally done. We can correlate these families through the use of nanospray desorption electrospray ionization MS/MS, an ambient pressure MS technique, in conjunction with MS/MS networking and peptidogenomics. We matched the molecular families of peptide natural products produced by 42 bacilli and 18 pseudomonads through the generation of amino acid sequence tags from MS/MS data of specific clusters found in the MS/MS network. These sequence tags were then linked to biosynthetic gene clusters in publicly accessible genomes, providing us with the ability to link particular molecules with the genes that produced them. As an example of its use, this approach was applied to two unsequenced Pseudoalteromonas species, leading to the discovery of the gene cluster for a molecular family, the bromoalterochromides, in the previously sequenced strain P. piscicida JCM 20779(T). The approach itself is not limited to 60 related strains, because spectral networking can be readily adopted to look at molecular family-gene cluster families of hundreds or more diverse organisms in one single MS/MS network.

Mantovani, SM, Moore BS.  2013.  Flavin-linked oxidase catalyzes pyrrolizine formation of dichloropyrrole-containing polyketide extender unit in chlorizidine a. Journal of the American Chemical Society. 135:18032-18035.   10.1021/ja409520v   AbstractWebsite

The marine alkaloid chlorizidine A contains chlorinated pyrroloisoindolone and pyrrolizine rings that are rare chemical features in bacterial natural products. Herein, we report the biosynthetic logic of their construction in Streptomyces sp. CNH-287 based on the identification of the chlorizidine A biosynthetic gene cluster. Using whole pathway heterologous expression and genetic manipulations, we show that chlorizidine A is assembled by a polyketide synthase that uniquely incorporates a fatty acid synthase-derived dichloropyrrolyl extender unit into the pyrroloisoindolone enzymatic product. We further provide the first biochemical characterization of a flavoenzyme associated with the oxidative formation of chlorizidine A's distinctive pyrrolizine ring. This work illuminates new enzymatic assembly line processes leading to rare nitrogen-containing rings in nature.

Teufel, R, Miyanaga A, Michaudel Q, Stull F, Louie G, Noel JP, Baran PS, Palfey B, Moore BS.  2013.  Flavin-mediated dual oxidation controls an enzymatic Favorskii-type rearrangement. Nature. 503:552-+.   10.1038/nature12643   AbstractWebsite

Flavoproteins catalyse a diversity of fundamental redox reactions and are one of the most studied enzyme families(1,2). As monooxygenases, they are universally thought to control oxygenation by means of a peroxyflavin species that transfers a single atom of molecular oxygen to an organic substrate(1,3,4). Here we report that the bacterial flavoenzyme EncM(5,6) catalyses the peroxyflavin-independent oxygenation-dehydrogenation dual oxidation of a highly reactive poly(beta-carbonyl). The crystal structure of EncM with bound substrate mimics and isotope labelling studies reveal previously unknown flavin redox biochemistry. We show that EncM maintains an unexpected stable flavin-oxygenating species, proposed to be a flavin-N5-oxide, to promote substrate oxidation and trigger a rare Favorskii-type rearrangement that is central to the biosynthesis of the antibiotic enterocin. This work provides new insight into the fine-tuning of the flavin cofactor in offsetting the innate reactivity of a polyketide substrate to direct its efficient electrocyclization.

Kersten, RD, Ziemert N, Gonzalez DJ, Duggan BM, Nizet V, Dorrestein PC, Moore BS.  2013.  Glycogenomics as a mass spectrometry-guided genome-mining method for microbial glycosylated molecules. Proceedings of the National Academy of Sciences of the United States of America. 110:E4407-E4416.   10.1073/pnas.1315492110   AbstractWebsite

Glycosyl groups are an essential mediator of molecular interactions in cells and on cellular surfaces. There are very few methods that directly relate sugar-containing molecules to their biosynthetic machineries. Here, we introduce glycogenomics as an experiment-guided genome-mining approach for fast characterization of glycosylated natural products (GNPs) and their biosynthetic pathways from genome-sequenced microbes by targeting glycosyl groups in microbial metabolomes. Microbial GNPs consist of aglycone and glycosyl structure groups in which the sugar unit(s) are often critical for the GNP's bioactivity, e.g., by promoting binding to a target biomolecule. GNPs are a structurally diverse class of molecules with important pharmaceutical and agrochemical applications. Herein, O- and N- glycosyl groups are characterized in their sugar monomers by tandem mass spectrometry (MS) and matched to corresponding glycosylation genes in secondary metabolic pathways by a MS-glycogenetic code. The associated aglycone biosynthetic genes of the GNP genotype then classify the natural product to further guide structure elucidation. We highlight the glycogenomic strategy by the characterization of several bioactive glycosylated molecules and their gene clusters, including the anticancer agent cinerubin B from Streptomyces sp. SPB74 and an antibiotic, arenimycin B, from Salinispora arenicola CNB-527.

Kersten, RD, Lane AL, Nett M, Richter TKS, Duggan BM, Dorrestein PC, Moore BS.  2013.  Bioactivity-Guided Genome Mining Reveals the Lomaiviticin Biosynthetic Gene Cluster in Salinispora tropica. ChemBioChem. 14:955-962.: WILEY-VCH Verlag   10.1002/cbic.201300147   AbstractWebsite

The use of genome sequences has become routine in guiding the discovery and identification of microbial natural products and their biosynthetic pathways. In silico prediction of molecular features, such as metabolic building blocks, physico-chemical properties or biological functions, from orphan gene clusters has opened up the characterization of many new chemo- and genotypes in genome mining approaches. Here, we guided our genome mining of two predicted enediyne pathways in Salinispora tropica CNB-440 by a DNA interference bioassay to isolate DNA-targeting enediyne polyketides. An organic extract of S. tropica showed DNA-interference activity that surprisingly was not abolished in genetic mutants of the targeted enediyne pathways, ST_pks1 and spo. Instead we showed that the product of the orphan type II polyketide synthase pathway, ST_pks2, is solely responsible for the DNA-interfering activity of the parent strain. Subsequent comparative metabolic profiling revealed the lomaiviticins, glycosylated diazofluorene polyketides, as the ST_pks2 products. This study marks the first report of the 59 open reading frame lomaiviticin gene cluster (lom) and supports the biochemical logic of their dimeric construction through a pathway related to the kinamycin monomer.

Arnison, PG, Bibb MJ, Bierbaum G, Bowers AA, Bugni TS, Bulaj G, Camarero JA, Campopiano DJ, Challis GL, Clardy J, Cotter PD, Craik DJ, Dawson M, Dittmann E, Donadio S, Dorrestein PC, Entian KD, Fischbach MA, Garavelli JS, Goransson U, Gruber CW, Haft DH, Hemscheidt TK, Hertweck C, Hill C, Horswill AR, Jaspars M, Kelly WL, Klinman JP, Kuipers OP, Link AJ, Liu W, Marahiel MA, Mitchell DA, Moll GN, Moore BS, Muller R, Nair SK, Nes IF, Norris GE, Olivera BM, Onaka H, Patchett ML, Piel J, Reaney MJT, Rebuffat S, Ross RP, Sahl HG, Schmidt EW, Selsted ME, Severinov K, Shen B, Sivonen K, Smith L, Stein T, Sussmuth RD, Tagg JR, Tang GL, Truman AW, Vederas JC, Walsh CT, Walton JD, Wenzel SC, Willey JM, van der Donk WA.  2013.  Ribosomally synthesized and post-translationally modified peptide natural products: overview and recommendations for a universal nomenclature. Natural Product Reports. 30:108-160.   10.1039/c2np20085f   AbstractWebsite

This review presents recommended nomenclature for the biosynthesis of ribosomally synthesized and post-translationally modified peptides (RiPPs), a rapidly growing class of natural products. The current knowledge regarding the biosynthesis of the >20 distinct compound classes is also reviewed, and commonalities are discussed.