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 by employing an unbiased metagenome mining approach. By 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.

SUMMARY Macrocyclases and other posttranslational enzymes afford derived peptides with improved properties for pharmaceutical and biotechnological applications. Here, we asked whether multiple posttranslational modifications could be simultaneously controlled and matched to rationally generate new peptide derivatives. We reconstituted the cyanobactin peptide natural products in vitro with up to five different posttranslational enzymes in a single tube. By manipulating the order of addition and identity of enzymes and exploiting their broad-substrate tolerance, we engineered the production of highly unnatural derivatives, including an N-C peptide macrocycle of 22 amino acids in length. In addition to engineering, this work better defines the macrocyclization mechanism, provides the first biochemical demonstration of Ser/Thr posttranslational prenylation, and is the first example of reconstitution of a native, multistep RiPP pathway with multiple enzymes in one pot. Overall, this work demonstrates how the modularity of posttranslational modification enzymes can be used to design and synthesize desirable peptide motifs.

Equisetin synthetase (EqiS), from the filamentous fungus Fusarium heterosporum ATCC 74349, was initially assigned on the basis of genetic knockout and expression analysis. Increasing inconsistencies in experimental results led us to question this assignment. Here, we sequenced the F. heterosporum genome, revealing two hybrid polyketide-peptide proteins that were candidates for the equisetin synthetase. The surrounding genes in both clusters had the needed auxiliary genes that might be responsible for producing equisetin. Genetic mutation, biochemical analysis, and recombinant expression in the fungus enabled us to show that the initially assigned EqiS does not produce equisetin but instead produces a related 2,4-pyrrolidinedione, fusaridione A, that was previously unknown. Fusaridione A is methylated in the 3-position of the pyrrolidinedione, which has not otherwise been found in natural products, leading to spontaneous reverse-Dieckmann reactions. A newly described gene cluster, eqx, is responsible for producing equisetin.

The cone snail Conus pulicarius from the Philippines provides a specific habitat for actinomycetes and other bacteria. A phenotypic screen using primary cultures of mouse dorsal root ganglion neurons revealed that one C. pulicarius associate, Streptomyces sp. CP32, produces a series of natural products that enhance or diminish whole-cell Ca 2+ flux. These compounds include known thiazoline compounds and a series of new derivatives, pulicatins A-E (6-10). Individual compounds were shown to bind to a series of human receptors, with selective binding to the human serotonin 5-HT 2B receptor. Here, we report the structure elucidation of the new compounds and results of the neurological assays.Symbiotic bacteria living with animals have been implicated in the synthesis of defensive and other natural products of importance to drug discovery.1 -5 In the marine environment, most studies of symbiont-derived natural products have focused on soft-bodied, benthic invertebrates that are otherwise vulnerable to predation. By contrast, cone snail mollusks are well defended by their shells and their arsenal of extremely diverse, snail-derived peptide toxins that act on channels and receptors.6 We sought to investigate whether this type of well-defended organism would also house symbiotic bacteria involved in the synthesis of allelochemicals, potentially including venom components, antibacterial defenses, and other small-molecule natural products. In addition to investigating this basic question, we reasoned that exploration of an untapped bacterial niche could lead to the discovery of new bioactive natural products.We recently reported that at least some cone snails are associated with diverse actinomycetes and other bacteria, and that extracts of some of these bacteria exhibit neurological activity.7 In order to maximize the discovery of new active bacterial metabolites from these new niches, we applied a broad-scope assay involving primary * Corresponding author. Mailing address: College of Pharmacy, Medicinal Chemistry, 307 Skaggs Hall, 30 S. 2000 E., Rm. 201, Salt Lake City, Utah 84112-5820. Tel: (801) 585-9119. ews1@utah.edu. Supporting Information Available: NMR data for pulicatins A-E (6-10), DRG assay results, application of LIS, 1 H NMR of MTPA esters for compounds 1, 6 and 7, HRESIMS data of compounds 1 and 6-10. This material is available free of charge via the Internet at http://pubs.acs.org. NIH Public Access Results and DiscussionExtracts of pilot-scale cultures of strain CP32 were strongly active in the DRG assay, resulting in K + -stimulated Ca 2+ influx. CP32 was fermented in 2.8 L Fernbach flasks for eight days. In an assay-guided procedure, the culture broth was centrifuged and subjected to HP20 adsorption chromatography, followed by C 18 flash chromatography and HPLC to yield compounds 1-10.A known compound, aerugine (1) was isolated as a pale yellow solid.10 , 11 The NMR and low resolution MS data also exactly matched a siderophore compound (1a) isolated from a bacterium associated with a marin...