Roberta Teta

Genome mining reveals trans-AT polyketide synthase directed antibiotic biosynthesis in the bacterial phylum bacteroidetes.

Complex polyketides such as erythromycin, epothilone, and FK506 greatly contribute to human health. Bacteria synthesize these natural products through large, multifunctional enzymes called modular polyketide synthases (PKSs), which use many catalytic domains in sequence to generate polyketide chains in an assembly-line-like process. Because there is usually a strict correlation between the enzymatic architecture and the structure of the metabolite, it is often possible to predict the biosynthetic product from a PKS gene sequence. This feature has been successfully exploited for natural product discovery through bioinformatic analysis of bacterial DNA sequences. Considering the impressive recent developments in the field of sequencing technologies and the concomitant explosion in genomic data, it is anticipated that genome mining will play a key role in future drug discovery programs. A crucial prerequisite for the success of this strategy is for predictions to be made at a high level of confidence for a wide range of pathways. A recent unexpected discovery by our research group and others is the presence of a novel and evolutionarily distinct group of modular PKSs. These enzymes use free-standing acyltransferases (AT) to select polyketide building blocks and to attach them to the multifunctional PKS in trans, as opposed to cis-acting textbook PKSs that carry integrated AT domains. Such trans-AT PKSs have long been overlooked, as they are rare in actinomycetes that served as initial model organisms to study polyketide biosynthesis. However, a wide range of bioactive polyketides from diverse bacteria, including the clinically used antibiotics of the mupirocin and streptogramin series, are now known to be products of trans-AT PKSs. Trans-AT pathways represent highly interesting targets for natural product discovery for two reasons: they often occur in bacteria that have not been well studied in pharmacological screening programs, and the PKSs exhibit a remarkable array of unusual enzymatic features, such as new catalytic domains, unprecedented module architectures, and non-canonical biosynthetic transformations, which often translate into unusual chemistry. Another consequence of these peculiarities is that textbook collinearity rules cannot be applied to deduce structures from DNA data. However, on the basis of functional and phylogenetic studies, we recently developed a novel predictive approach that allows the assignment of polyketides to transAT PKS sequences with high confidence. This method is based on the observation that the substrate specificity of ketosynthase (KS) domains, which catalyze polyketide chain extensions in a Claisen-condensation-like reaction, correlates with its evolution. Thus, if the position of a particular KS in a phylogenetic tree is known, it can often be predicted whether its substrate carries a b-hydroxy group, an a,b-double bond, an a,breduced moiety, or an aor b-carbon branch. In this way, structural information for an entire polyketide chain can be obtained by combining the information of all KSs present in a PKS. The utility of these trans-AT collinearity rules was demonstrated by the isolation of novel thailandamide polyketides by genome mining of Burkholderia thailandensis. Since then, it was shown that the predictive power can be continuously increased by refining the phylogenetic analysis with KSs of newly sequenced gene clusters. In this work, we demonstrate the utility of trans-AT genome mining by assigning a PKS of Chitinophaga pinensis DSM 2588, a member of the chemically poorly studied bacterial phylum Bacteroidetes, to the biosynthesis of elansolids, antibacterial and cytotoxic agents that arise from an unusual series of post-PKS modifications. The genome of C. pinensis DSM 2588, a heterotrophic gliding bacterium consisting of long unicellular filaments, was found to contain two large gene clusters that encode a hybrid non-ribosomal peptide synthetase-PKS and a trans-AT PKS system (the latter termed els PKS). Intrigued by the fact that only few compounds have been previously reported from the genus Chitinophaga, we were interested in the product of the els pathway. The cluster (Table 1) spans 75.1 kb and contains 18 genes, of which six (elsIJNOPQ) encode multimodular PKS proteins, none of which harbors AT domains. Instead, elsA and elsB exhibit similarities to free-standing AT genes of transAT PKSs. ElsA also contains an additional C-terminal oxidoreductase domain that was recently shown to catalyze enoyl reductions in trans. The els polyketide was therefore expected to contain at least one reduced moiety. Several putative proteins (ElsDEKLM) were identified that are usually involved in the generation of b-carbon branches at the growing polyketide chain. 14] Further gene products resemble 4-hydroxybenzoate synthases (ElsH), cytochromes P450 (ElsF), methyltransferases (ElsR), and proteins with unknown function (ElsC and ElsG). The six large PKS proteins contain a total of 15 modules. The N terminus of ElsI harbors a module that consists of [a] Dr. R. Teta , E. J. N. Helfrich, S. K nne, A. Schneider, Prof. Dr. J. Piel Kekul Institute of Organic Chemistry and Biochemistry University of Bonn, Gerhard-Domagk-Strasse 1, 53121 Bonn (Germany) Fax: (+ 49) 228-739712 E-mail : joern.piel@uni-bonn.de [b] Dr. M. Gurgui, Dr. G. Van Echten-Deckert Kekul Institute LIMES Membrane Biology and Lipid Biochemistry University of Bonn, Gerhard-Domagk-Strasse 1, 53121 Bonn (Germany) [c] Dr. R. Teta , Prof. Dr. A. Mangoni Dipartimento di Chimica delle Sostanze Naturali Universit di Napoli “Federico II” Via D. Montesano 49, 80131 Napoli (Italy) Supporting information for this article is available on the WWW under http ://dx.doi.org/10.1002/cbic.201000542.

Isolation and assessment of the in vitro anti-tumor activity of smenothiazole A and B, chlorinated thiazole-containing peptide/polyketides from the Caribbean sponge, Smenospongia aurea.

The study of the secondary metabolites contained in the organic extract of Caribbean sponge Smenospongia aurea led to the isolation of smenothiazole A (3) and B (4), hybrid peptide/polyketide compounds. Assays performed using four solid tumor cell lines showed that smenothiazoles exert a potent cytotoxic activity at nanomolar levels, with selectivity over ovarian cancer cells and a pro-apoptotic mechanism.

Smenamides A and B, Chlorinated Peptide/Polyketide Hybrids Containing a Dolapyrrolidinone Unit from the Caribbean Sponge Smenospongia aurea. Evaluation of Their Role as Leads in Antitumor Drug Research

An in-depth study of the secondary metabolites contained in the Caribbean sponge Smenospongia aurea led to the isolation of smenamide A (1) and B (2), hybrid peptide/polyketide compounds containing a dolapyrrolidinone unit. Their structures were elucidated using high-resolution ESI-MS/MS and homo- and heteronuclear 2D NMR experiments. Structures of smenamides suggested that they are products of the cyanobacterial metabolism, and 16S rRNA metagenomic analysis detected Synechococcus spongiarum as the only cyanobacterium present in S. aurea. Smenamides showed potent cytotoxic activity at nanomolar levels on lung cancer Calu-1 cells, which for compound 1 is exerted through a clear pro-apoptotic mechanism. This makes smenamides promising leads for antitumor drug design.

Polyketide Synthases in the Microbiome of the Marine Sponge Plakortis halichondrioides: A Metagenomic Update

Sponge-associated microorganisms are able to assemble the complex machinery for the production of secondary metabolites such as polyketides, the most important class of marine natural products from a drug discovery perspective. A comprehensive overview of polyketide biosynthetic genes of the sponge Plakortis halichondrioides and its symbionts was obtained in the present study by massively parallel 454 pyrosequencing of complex and heterogeneous PCR (Polymerase Chain Reaction) products amplified from the metagenomic DNA of a specimen of P. halichondrioides collected in the Caribbean Sea. This was accompanied by a survey of the bacterial diversity within the sponge. In line with previous studies, sequences belonging to supA and swfA, two widespread sponge-specific groups of polyketide synthase (PKS) genes were dominant. While they have been previously reported as belonging to Poribacteria (a novel bacterial phylum found exclusively in sponges), re-examination of current genomic sequencing data showed supA and swfA not to be present in the poribacterial genome. Several non-supA, non-swfA type-I PKS fragments were also identified. A significant portion of these fragments resembled type-I PKSs from protists, suggesting that bacteria may not be the only source of polyketides from P. halichondrioides, and that protistan PKSs should receive further investigation as a source of novel polyketides.

Tedarenes A and B: Structural and Stereochemical Analysis of Two New Strained Cyclic Diarylheptanoids from the Marine Sponge Tedania ignis

Ring strain causes planar chirality in tedarenes A and B, two cyclic diarylheptanoids isolated from the marine sponge Tedania ignis. In both molecules, the chiral plane is an olefinic system, which is very rare among natural products. In tedarene A (1), interconversion is too fast to allow isolation of the enantiomeric atropisomers but still slow enough to cause coalescence of some (1)H and (13)C NMR signals at room temperature. In tedarene B (2), which also shows stable central and axial chirality, the two planar diastereomers are in slow equilibrium. Tedarene B is the smallest natural product with central, axial, and planar chirality in the same simple molecule. The identification of planar chirality as the difference between its conformational isomers allowed the use of theoretical prediction of the CD spectrum to determine the absolute configuration of the stereogenic carbon C-9 as well as of the biphenyl chiral axis.

Manadoperoxides, a new class of potent antitrypanosomal agents of marine origin

Chemical investigation of the marine sponge Plakortis cfr. lita afforded a library of endoperoxyketal polyketides, manadoperoxides B-K (3-5 and 7-13) and peroxyplakoric esters B(3) (6) and C (14). Eight of these metabolites are new compounds and some contain an unprecedented chlorine-bearing THF-type ring in the side chain. The library of endoperoxide derivatives was evaluated for in vitro activity against Trypanosoma brucei rhodesiense and Leishmania donovani. Some compounds, such as manadoperoxide B, exhibited ultrapotent trypanocidal activity (IC(50) = 3 ng mL(-1)) without cytotoxicity. Detailed examination of the antitrypanosomal activity data and comparison with those available in the literature for related dioxane derivatives enabled us to draw a series of structure-activity relationships. Interestingly, it appears that minor structural changes, such as a shift of the methyl group around the dioxane ring, can dramatically affect the antitrypanosomal activity. This information can be valuable to guide the design of optimized antitrypanosomal agents based on the dioxane scaffold.

Polyketide genes in the marine sponge Plakortis simplex: a new group of mono-modular type I polyketide synthases from sponge symbionts.

Summary Sponge symbionts are a largely unexplored source of new and unusual metabolic pathways. Insights into the distribution and function of metabolic genes of sponge symbionts are crucial to dissect and exploit their biotechnological potential. Screening of the metagenome of the marine sponge Plakortis simplex led to the discovery of the swf family, a new group of mono-modular type I polyketide synthase/fatty acid synthase (PKS/FAS) specifically associated with sponge symbionts. Two different examples of the swf cluster were present in the metagenome of P. simplex. A third example of the cluster is present in the previously sequenced genome of a poribacterium from the sponge Aplysina aerophoba but was formerly considered orthologous to the wcb/rkp cluster. The swf cluster was also found in six additional species of sponges. Therefore, the swf cluster represents the second group of mono-modular PKS, after the supA family, to be widespread in marine sponges. The putative swf operon consists of swfA (type I PKS/FAS), swfB (reductase and sulphotransferase domains) and swfC (radical S-adenosylmethionine, or radical SAM). Activation of the acyl carrier protein (ACP) domain of the SwfA protein to its holo-form by co-expression with Svp is the first functional proof of swf type genes in marine sponges. However, the precise biosynthetic role of the swf clusters remains unknown.

Combined LC-MS/MS and Molecular Networking Approach Reveals New Cyanotoxins from the 2014 Cyanobacterial Bloom in Green Lake, Seattle.

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.

Terpioside B, a difucosyl GSL from the marine sponge Terpios sp. is a potent inhibitor of NO release☆

Terpioside B (2a), a unique glycolipid containing two fucose residues in the furanose form in its pentasaccharide chain, was isolated from the marine sponge Terpios sp. Its complete stereostructure was solved by interpretation of mass spectrometric and NMR data along with CD and GG-MS analyses of its degradation products. Terpioside B is a potent inhibitor against LPS-induced NO release, and is considerably more active than simpler glycosphingolipids such as terpioside A and monoglucosylceramide.

A New N -Acyl Homoserine Lactone Synthase in an Uncultured Symbiont of the Red Sea Sponge Theonella swinhoei

ABSTRACT Sponges harbor a remarkable diversity of microbial symbionts in which signal molecules can accumulate and enable cell-cell communication, such as quorum sensing (QS). Bacteria capable of QS were isolated from marine sponges; however, an extremely small fraction of the sponge microbiome is amenable to cultivation. We took advantage of community genome assembly and binning to investigate the uncultured majority of sponge symbionts. We identified a complete N-acyl-homoserine lactone (AHL)-QS system (designated TswIR) and seven partial luxI homologues in the microbiome of Theonella swinhoei. The TswIR system was novel and shown to be associated with an alphaproteobacterium of the order Rhodobacterales, here termed Rhodobacterales bacterium TS309. The tswI gene, when expressed in Escherichia coli, produced three AHLs, two of which were also identified in a T. swinhoei sponge extract. The taxonomic affiliation of the 16S rRNA of Rhodobacterales bacterium TS309 to a sponge-coral specific clade, its enrichment in sponge versus seawater and marine sediment samples, and the presence of sponge-specific features, such as ankyrin-like domains and tetratricopeptide repeats, indicate a likely symbiotic nature of this bacterium.