Towards Prediction of Metabolic Products of Polyketide Synthases: An In Silico Analysis

Sequence data arising from an increasing number of partial and complete genome projects is revealing the presence of the polyketide synthase (PKS) family of genes not only in microbes and fungi but also in plants and other eukaryotes. PKSs are huge multifunctional megasynthases that use a variety of biosynthetic paradigms to generate enormously diverse arrays of polyketide products that posses several pharmaceutically important properties. The remarkable conservation of these gene clusters across organisms offers abundant scope for obtaining novel insights into PKS biosynthetic code by computational analysis. We have carried out a comprehensive in silico analysis of modular and iterative gene clusters to test whether chemical structures of the secondary metabolites can be predicted from PKS protein sequences. Here, we report the success of our method and demonstrate the feasibility of deciphering the putative metabolic products of uncharacterized PKS clusters found in newly sequenced genomes. Profile Hidden Markov Model analysis has revealed distinct sequence features that can distinguish modular PKS proteins from their iterative counterparts. For iterative PKS proteins, structural models of iterative ketosynthase (KS) domains have revealed novel correlations between the size of the polyketide products and volume of the active site pocket. Furthermore, we have identified key residues in the substrate binding pocket that control the number of chain extensions in iterative PKSs. For modular PKS proteins, we describe for the first time an automated method based on crucial intermolecular contacts that can distinguish the correct biosynthetic order of substrate channeling from a large number of non-cognate combinatorial possibilities. Taken together, our in silico analysis provides valuable clues for formulating rules for predicting polyketide products of iterative as well as modular PKS clusters. These results have promising potential for discovery of novel natural products by genome mining and rational design of novel natural products.     

Non-modular polyketide synthases in myxobacteria

Myxobacteria are prolific producers of a wide variety of secondary metabolites. The vast majority of these compounds are complex polyketides which are biosynthesised by multimodular polyketide synthases (PKSs). In contrast, few myxobacterial metabolites isolated to date are derived from non-modular PKSs, in particular type III PKSs. This review reports our progress on the characterisation of type III PKSs in myxobacteria. We also summarize current knowledge on bacterial type III PKSs, with a special focus on the evolutionary relationship between plant and bacterial enzymes. The biosynthesis of a quinoline alkaloid in Stigmatella aurantiaca by a non-modular PKS is also discussed.     

Chimeric 6-methylsalicylic acid synthase with domains of acyl carrier protein and methyltransferase from Pseudallescheria boydii shows novel biosynthetic activity

Polyketides are important secondary metabolites, many of which exhibit potent pharmacological applications. Biosynthesis of polyketides is carried out by a single polyketide synthase (PKS) or multiple PKSs in successive elongations of enzyme-bound intermediates related to fatty acid biosynthesis. The polyketide gene PKS306 from Pseudallescheria boydii NTOU2362 containing domains of ketosynthase (KS), acyltransferase (AT), dehydratase (DH), acyl carrier protein (ACP) and methyltransferase (MT) was cloned in an attempt to produce novel chemical compounds, and this PKS harbouring green fluorescent protein (GFP) was expressed in Saccharomyces cerevisiae. Although fluorescence of GFP and fusion protein analysed by anti-GFP antibody were observed, no novel compound was detected. 6-methylsalicylic acid synthase (6MSAS) was then used as a template and engineered with PKS306 by combinatorial fusion. The chimeric PKS containing domains of KS, AT, DH and ketoreductase (KR) from 6MSAS with ACP and MT from PKS306 demonstrated biosynthesis of a novel compound. The compound was identified with a deduced chemical formula of C₇H₁₀O₃, and the chemical structure was named as 2-hydroxy-2-(propan-2-yl) cyclobutane-1,3-dione. The novel compound synthesized by the chimeric PKS in this study demonstrates the feasibility of combinatorial fusion of PKS genes to produce novel polyketides.     

The biosynthesis of the aromatic myxobacterial electron transport inhibitor stigmatellin is directed by a novel type of modular polyketide synthase.

Deductions from the molecular analysis of the 65,000-bp stigmatellin biosynthetic gene cluster are reported. The biosynthetic genes (stiA-J) encode an unusual bacterial modular type I polyketide synthase (PKS) responsible for the formation of this aromatic electron transport inhibitor produced by the myxobacterium Stigmatella aurantiaca. Involvement of the PKS gene cluster in stigmatellin biosynthesis is shown using site-directed mutagenesis. One module of the PKS is assumed to be used iteratively during the biosynthetic process, which seems to involve an unusual transacylation of the biosynthetic intermediate from an acyl carrier protein domain back to the preceding ketosynthase domain. Finally, the polyketide chain which is presumably catalyzed by a novel C-terminal domain in StiJ that does not resemble thioesterases, is cyclized and aromatized. The presented results of feeding experiments are in good agreement with the proposed biosynthetic scheme. In contrast to all other PKS type I systems reported to date, each module of StiA-J is encoded on a separate gene. The gene cluster contains a "stand alone" O-methyltransferase and two unusual O-methyltransferase domains embedded in the PKS. In addition, inactivation of a cytochrome P450 monooxygenase-encoding gene involved in post-PKS hydroxylation of the aromatic ring leads to the formation of two novel stigmatellin derivatives.     

A gene cluster involved in nogalamycin biosynthesis fromStreptomyces nogalater: sequence analysis and complementation of early-block mutations in the anthracycline pathway

We have analyzed an anthracycline biosynthesis gene cluster fromStreptomyces nogalater. Based on sequence analysis, a contiguous region of 11 kb is deduced to include genes for the early steps in anthracycline biosynthesis, a regulatory gene (snoA) promoting the expression of the biosynthetic genes, and at least one gene whose product might have a role in modification of the glycoside moiety. The three ORFs encoding a minimal polyketide synthase (PKS) are separated from the regulatory gene (snoA) by a comparatively AT-rich region (GC content 60%). Subfragments of the DNA region were transferred toStreptomyces galilaeus mutants blocked in aclacinomycin biosynthesis, and to a regulatory mutant ofS. nogalater. TheS. galilaeus mutants carrying theS. nogalater minimal PKS genes produced auramycinone glycosides, demonstrating replacement of the starter unit for polyketide biosynthesis. The product ofsnoA seems to be needed for expression of at least the genes for the minimal PKS.     

Natural biocombinatorics in the polyketide synthase genes of the actinobacterium Streptomyces avermitilis

Modular polyketide synthases (PKSs) of bacteria provide an enormous reservoir of natural chemical diversity. Studying natural biocombinatorics may aid in the development of concepts for experimental design of genes for the biosynthesis of new bioactive compounds. Here we address the question of how the modularity of biosynthetic enzymes and the prevalence of multiple gene clusters in Streptomyces drive the evolution of metabolic diversity. The phylogeny of ketosynthase (KS) domains of Streptomyces PKSs revealed that the majority of modules involved in the biosynthesis of a single compound evolved by duplication of a single ancestor module. Using Streptomyces avermitilis as a model organism, we have reconstructed the evolutionary relationships of different domain types. This analysis suggests that 65% of the modules were altered by recombinational replacements that occurred within and between biosynthetic gene clusters. The natural reprogramming of the biosynthetic pathways was unambiguously confined to domains that account for the structural diversity of the polyketide products and never observed for the KS domains. We provide examples for natural acyltransferase (AT), ketoreductase (KR), and dehydratase (DH)–KR domain replacements. Potential sites of homologous recombination could be identified in interdomain regions and within domains. Our results indicate that homologous recombination facilitated by the modularity of PKS architecture is the most important mechanism underlying polyketide diversity in bacteria.     

Identification of functionally clustered nystatin-like biosynthetic genes in a rare actinomycetes, <Emphasis Type="Italic">Pseudonocardia autotrophica</Emphasis>

The polyene antibiotics, including nystatin, pimaricin, amphotericin, and candicidin, comprise a family of very valuable antifungal polyketide compounds, and they are typically produced by soil actinomycetes. Previously, using a polyene cytochrome P450 hydroxylase-specific genome screening strategy, Pseudonocardia autotrophica KCTC9441 was determined to contain genes potentially encoding polyene biosynthesis. Here, sequence information of an approximately 125.7-kb contiguous DNA region in five overlapping cosmids isolated from the P. autotrophica KCTC9441 genomic library revealed a total of 23 open reading frames, which are presumably involved in the biosynthesis of a nystatin-like compound tentatively named NPP. The deduced roles for six multi-modular polyketide synthase (PKS) catalytic domains were found to be highly homologous to those of previously identified nystatin biosynthetic genes. Low NPP productivity suggests that the functionally clustered NPP biosynthetic pathway genes are tightly regulated in P. autotrophica. Disruption of a NPP PKS gene completely abolished both NPP biosynthesis and antifungal activity against Candida albicans, suggesting that polyene-specific genome screening may constitute an efficient method for isolation of potentially valuable previously identified polyene genes and compounds from various rare actinomycetes widespread in nature.     

Functional replacement of the ketosynthase domain of FUM1 for the biosynthesis of fumonisins, a group of fungal reduced polyketides

The genetic manipulation of the biosynthesis of fungal reduced polyketides has been challenging due to the lack of knowledge on the biosynthetic mechanism, the difficulties in the detection of the acyclic, non-aromatic metabolites, and the complexity in genetically manipulating filamentous fungi. Fumonisins are a group of economically important mycotoxins that contaminate maize-based food and feed products worldwide. Fumonisins contain a linear dimethylated C₁₈ chain that is synthesized by Fum1p, which is a single module polyketide synthase (PKS). Using a genetic system that allows the specific manipulation of PKS domains in filamentous fungus Fusarium verticillioides, we replaced the KS domain of fumonisin FUM1 with the KS domain of T-toxin PKS1 from Cochliobolus heterostrophus. Although PKS1 synthesizes different polyketides, the F. verticillioides strain carrying the chimeric PKS produced fumonisins. This represents the first successful domain swapping in PKSs for fungal reduced polyketides and suggests that KS domain alone may not be sufficient to control the product’s structure. To further test if the whole fumonisin PKS could be functionally replaced by a PKS that has a similar domain architecture, we replaced entire FUM1 with PKS1. This strain did not produce any fumonisin or new metabolites, suggesting that the intrinsic interactions between the intact PKS and downstream enzymes in the biosynthetic pathway may play a role in the control of fungal reduced polyketides.     

A Single Sfp-Type Phosphopantetheinyl Transferase Plays a Major Role in the Biosynthesis of PKS and NRPS Derived Metabolites in Streptomyces ambofaciens ATCC23877

The phosphopantetheinyl transferases (PPTases) are responsible for the activation of the carrier protein domains of the polyketide synthases (PKS), non ribosomal peptide synthases (NRPS) and fatty acid synthases (FAS). The analysis of the Streptomyces ambofaciens ATCC23877 genome has revealed the presence of four putative PPTase encoding genes. One of these genes appears to be essential and is likely involved in fatty acid biosynthesis. Two other PPTase genes, samT0172 (alpN) and samL0372, are located within a type II PKS gene cluster responsible for the kinamycin production and an hybrid NRPS-PKS cluster involved in antimycin production, respectively, and their products were shown to be specifically involved in the biosynthesis of these secondary metabolites. Surprisingly, the fourth PPTase gene, which is not located within a secondary metabolite gene cluster, appears to play a pleiotropic role. Its product is likely involved in the activation of the acyl- and peptidyl-carrier protein domains within all the other PKS and NRPS complexes encoded by S. ambofaciens. Indeed, the deletion of this gene affects the production of the spiramycin and stambomycin macrolide antibiotics and of the grey spore pigment, all three being PKS-derived metabolites, as well as the production of the nonribosomally produced compounds, the hydroxamate siderophore coelichelin and the pyrrolamide antibiotic congocidine. In addition, this PPTase seems to act in concert with the product of samL0372 to activate the ACP and/or PCP domains of the antimycin biosynthesis cluster which is also responsible for the production of volatile lactones.     

Designing and Implementing an Assay for the Detection of Rare and Divergent NRPS and PKS Clones in European,Antarctic and Cuban Soils

The ever increasing microbial resistome means there is an urgent need for new antibiotics. Metagenomics is an underexploited tool in the field of drug discovery. In this study we aimed to produce a new updated assay for the discovery of biosynthetic gene clusters encoding bioactive secondary metabolites. PCR assays targeting the polyketide synthases (PKS) and non-ribosomal peptide synthetases (NRPS) were developed. A range of European soils were tested for their biosynthetic potential using clone libraries developed from metagenomic DNA. Results revealed a surprising number of NRPS and PKS clones with similarity to rare Actinomycetes. Many of the clones tested were phylogenetically divergent suggesting they were fragments from novel NRPS and PKS gene clusters. Soils did not appear to cluster by location but did represent NRPS and PKS clones of diverse taxonomic origin. Fosmid libraries were constructed from Cuban and Antarctic soil samples; 17 fosmids were positive for NRPS domains suggesting a hit rate of less than 1 in 10 genomes. NRPS hits had low similarities to both rare Actinobacteria and Proteobacteria; they also clustered with known antibiotic producers suggesting they may encode for pathways producing novel bioactive compounds. In conclusion we designed an assay capable of detecting divergent NRPS and PKS gene clusters from the rare biosphere; when tested on soil samples results suggest the majority of NRPS and PKS pathways and hence bioactive metabolites are yet to be discovered.     

Characterization of the biosynthetic gene cluster for the antifungal polyketide soraphen A from Sorangium cellulosum So ce26

A genomic DNA region of over 80 kb that contains the complete biosynthetic gene cluster for the synthesis of the antifungal polyketide metabolite soraphen A was cloned from Sorangium cellulosum So ce26. The nucleotide sequence of the soraphen A gene region, including 67,523 bp was determined. Examination of this sequence led to the identification of two adjacent type I polyketide synthase (PKS) genes that encode the soraphen synthase. One of the soraphen A PKS genes includes three biosynthetic modules and the second contains five additional modules for a total of eight. The predicted substrate specificities of the acyltransferase (AT) domains, as well as the reductive loop domains identified within each module, are consistent with expectations from the structure of soraphen A. Genes were identified in the regions flanking the two soraphen synthase genes that are proposed to have roles in the biosynthesis of soraphen A. Downstream of the soraphen PKS genes is an O-methyltransferase (OMT) gene. Upstream of the soraphen PKS genes there is a gene encoding a reductase and a group of genes that are postulated to have roles in the synthesis of methoxymalonyl-acyl carrier protein (ACP). This unusual extender unit is proposed to be incorporated in two positions of the soraphen polyketide chain. One of the genes in this group contains distinct domains for an AT, an ACP, and an OMT.     

Characterization of two novel non-reducing polyketide synthase genes from the lichen-forming fungus Hypogymnia physodes

Lichens are known to produce a variety of secondary metabolites including polyketides, which have valuable biological activities. Some polyketides are produced solely by lichens. The biosynthesis of these compounds is primarily governed by iterative type I polyketide synthases. Hypogymnia physodes synthesize polyketides such as physodic, physodalic and hydroxyphysodic acid and atranorin, which are non-reducing polyketides. Two novel non-reducing polyketide synthase (PKS) genes were isolated from a fosmid genomic library of a mycobiont of H. physodes using a 409bp fragment corresponding to part of the reductase (R) domain as a probe. H. physodes PKS1 (Hyopks1) and PKS2 (Hypopks2) contain keto synthase (KS), acyl transferase (AT), acyl carrier protein (ACP), methyl transferase (ME) and R domains. Classification based on phylogeny analysis using the translated KS and AT domains demonstrated that Hypopks1 and Hypopks2 are members of the fungal non-reducing PKSs clade III. This is the first report of non-reducing PKSs containing the R domain-mediated release mechanisms in lichens, which are also rare fungal type I PKS in non-lichenized filamentous fungi.     

Diversity and evolution of polyketide biosynthesis gene clusters in the Ceratocystidaceae

Polyketides are secondary metabolites with diverse biological activities. Polyketide synthases (PKS) are often encoded from genes clustered in the same genomic region. Functional analyses and genomic studies show that most fungi are capable of producing a repertoire of polyketides. We considered the potential of Ceratocystidaceae for producing polyketides using a comparative genomics approach. Our aims were to identify the putative polyketide biosynthesis gene clusters, to characterize them and predict the types of polyketide compounds they might produce. We used sequences from nineteen species in the genera, Ceratocystis, Endoconidiophora, Davidsoniella, Huntiella, Thielaviopsis and Bretziella, to identify and characterize PKS gene clusters, by employing a range of bioinformatics and phylogenetic tools. We showed that the genomes contained putative clusters containing a non-reducing type I PKS and a type III PKS. Phylogenetic analyses suggested that these genes were already present in the ancestor of the Ceratocystidaceae. By contrast, the various reducing type I PKS-containing clusters identified in these genomes appeared to have distinct evolutionary origins. Although one of the identified clusters potentially allows for the production of melanin, their functional characterization will undoubtedly reveal many novel and important compounds implicated in the biology of the Ceratocystidaceae.     

Polyketide synthases of bacterial symbionts in sponges – Evolution-based applications in natural products research

Marine sponges are an unusually rich source of bioactive natural products with clinical potential. They also often harbor rich communities of symbiotic bacteria that have often been suspected as the true producers of sponge-derived compounds. To date, these bacteria can in most cases not be cultivated, but culture-independent methods, such as isolating and analyzing biosynthetic gene clusters using metagenomic strategies, have recently provided first insights into their chemical potential. This review summarizes recent work of our laboratory on the study of polyketide synthases (PKSs). These studies revealed two evolutionarily distinct, unusual PKS types that are commonly found in sponge metagenomes and were shown to be of bacterial origin. One, the sup PKS, dominates sponge metagenomic DNA libraries, occurs widespread in bacteriosponges and is to date exclusively known from such animals. Data suggest that it is a type of synthase that generates methyl-branched fatty acids, which are commonly present in sponges. The other PKS type, termed trans-acyltransferase (AT) PKS, is responsible for the biosynthesis of complex, bioactive polyketides, such as the onnamides, and also occurs in free-living bacteria. The diversity of PKS genes present in a single sponge metagenome can be enormous. However, the phylogenetic approaches outlined in this review can provide valuable insights into the PKS function and structures of polyketides and can assist in the targeted isolation of gene clusters.     

Evolution of ketosynthase domains of polyketide synthase genes in the Cladonia chlorophaea species complex (Cladoniaceae)

Lichen-forming fungi synthesize a diversity of polyketides, but only a few non-reducing polyketide synthase (PKS) genes from a lichen-forming fungus have been linked with a specific polyketide. While it is a challenge to link the large number of PKS paralogs in fungi with specific products, it might be expected that the PKS paralogs from closely related species would be similar because of recent evolutionary divergence. The objectives of this study were to reconstruct a PKS gene phylogeny of the Cladonia chlorophaea species complex based on the ketosynthase domain, a species phylogeny of the complex, and to explore the presence of PKS gene paralogs among members of the species complex. DNA was isolated from 51 individuals of C. chlorophaea and allies to screen for the presence of 13 PKS paralogs. A 128 sequence PKS gene phylogeny using deduced amino acid sequences estimated from the 13 PKS paralogs and sequences subjected to BLASTx comparisons showed losses of each of two PKS domains (reducing and methylation). This research provided insight into the evolution of PKS genes in the C. chlorophaea group, species evolution in the group, and it identified potential directions for further investigation of polyketide synthesis in the C. chlorophaea species complex.     

Novel Polyketide Synthase from Nectria haematococca

We identified a polyketide synthase (PKS) gene, pksN, from a strain of Nectria haematococca by complementing a mutant unable to synthesize a red perithecial pigment. pksN encodes a 2,106-amino-acid polypeptide with conserved motifs characteristic of type I PKS enzymatic domains: β-ketoacyl synthase, acyltransferase, duplicated acyl carrier proteins, and thioesterase. The pksN product groups with the Aspergillus nidulans WA-type PKSs involved in conidial pigmentation and melanin, bikaverin, and aflatoxin biosynthetic pathways. Inactivation of pksN did not cause any visible change in fungal growth, asexual sporulation, or ascospore formation, suggesting that it is involved in a specific developmental function. We propose that pksN encodes a novel PKS required for the perithecial red pigment biosynthesis.     

A Single Module Type I Polyketide Synthase Directs de Novo Macrolactone Biogenesis during Galbonolide Biosynthesis in Streptomyces galbus

Galbonolide (GAL) A and B are antifungal macrolactone polyketides produced by Streptomyces galbus. During their polyketide chain assembly, GAL-A and -B incorporate methoxymalonate and methylmalonate, respectively, in the fourth chain extension step. The methoxymalonyl-acyl carrier protein biosynthesis locus (galG to K) is specifically involved in GAL-A biosynthesis, and this locus is neighbored by a gene cluster composed of galA-E. GalA-C constitute a single module, highly reducing type I polyketide synthase (PKS). GalD and GalE are cytochrome P450 and Rieske domain protein, respectively. Gene knock-out experiments verified that galB, -C, and -D are essential for GAL biosynthesis. A galD mutant accumulated a GAL-C that lacked two hydroxyl groups and a double bond when compared with GAL-B. A [U-¹³C]propionate feeding experiment indicated that no rare precursor other than methoxymalonate was incorporated during GAL biogenesis. A search of the S. galbus genome for a modular type I PKS system, the type that was expected to direct GAL biosynthesis, resulted in the identification of only one modular type I PKS gene cluster. Homology analysis indicated that this PKS gene cluster is the locus for vicenistatin biosynthesis. This cluster was previously reported in Streptomyces halstedii. A gene deletion of the vinP2 ortholog clearly demonstrated that this modular type I PKS system is not involved in GAL biosynthesis. Therefore, we propose that GalA-C direct macrolactone polyketide formation for GAL. Our studies provide a glimpse into a novel biochemical strategy used for polyketide synthesis; that is, the iterative assembly of propionates with highly programmed β-keto group modifications.     

Organization of the biosynthetic gene cluster for the polyketide macrolide mycinamicin in Micromonospora griseorubida

Mycinamicin, composed of a branched lactone and two sugars, desosamine and mycinose, at the C-5 and C-21 positions, is a 16-membered macrolide antibiotic produced by Micromonospora griseorubida A11725, which shows strong antimicrobial activity against Gram-positive bacteria. The nucleotide sequence (62 kb) of the mycinamicin biosynthetic gene cluster, in which there were 22 open reading frames (ORFs), was completely determined. All of the products from the 22 ORFs are responsible for the biosynthesis of mycinamicin II and self-protection against the compounds synthesized. Central to the cluster is a polyketide synthase locus (mycA), which encodes a seven-module system comprised of five multifunctional proteins. Immediately downstream of mycA, there is a set of genes for desosamine biosynthesis (mydA-G and mycB). Moreover, mydH, whose product is responsible for the biosynthesis of mycinose, lies between mydA and B. On the other hand, eight ORFs were detected upstream of the mycinamicin PKS gene. The myrB, mycG, and mycF genes had already been characterized by Inouye et al. The other five ORFs (mycCI, mycCII, mydI, mycE, and mycD) lie between mycA1 and mycF, and these five genes and mycF are responsible for the biosynthesis of mycinose. In the PKS gene, four regions of KS and AT domains in modules 1, 4, 5, and 6 indicated that it does not show the high GC content typical for Streptomyces genes, nor the unusual frame plot patterns for Streptomyces genes. Methylmalonyl-CoA was used as substrate in the functional units of those four modules. The relationship between the substrate and the unusual frame plot pattern of the KS and AT domains was observed in the other PKS genes, and it is suggested that the KS-AT original region was horizontally transferred into the PKS genes on the chromosomal DNA of several actinomycetes strains.     

Metagenomic Analysis Reveals Diverse Polyketide Synthase Gene Clusters in Microorganisms Associated with the Marine Sponge Discodermia dissoluta

Sponge-associated bacteria are thought to produce many novel bioactive compounds, including polyketides. PCR amplification of ketosynthase domains of type I modular polyketide synthases (PKS) from the microbial community of the marine sponge Discodermia dissoluta revealed great diversity and a novel group of sponge-specific PKS ketosynthase domains. Metagenomic libraries totaling more than four gigabases of bacterial genomes associated with this sponge were screened for type I modular PKS gene clusters. More than 90% of the clones in total sponge DNA libraries represented bacterial DNA inserts, and 0.7% harbored PKS genes. The majority of the PKS hybridizing clones carried small PKS clusters of one to three modules, although some clones encoded large multimodular PKSs (more than five modules). The most abundant large modular PKS appeared to be encoded by a bacterial symbiont that made up <1% of the sponge community. Sequencing of this PKS revealed 14 modules that, if expressed and active, is predicted to produce a multimethyl-branched fatty acid reminiscent of mycobacterial lipid components. Metagenomic libraries made from fractions enriched for unicellular or filamentous bacteria differed significantly, with the latter containing numerous nonribosomal peptide synthetase (NRPS) and mixed NRPS-PKS gene clusters. The filamentous bacterial community of D. dissoluta consists mainly of Entotheonella spp., an unculturable sponge-specific taxon previously implicated in the biosynthesis of bioactive peptides.