Recent innovations in peptide natural product biosynthesis reveal a surprising wealth of previously uncharacterized biochemical reactions that have potential applications in synthetic biology. Among these, the cyanobactins are noteworthy because these peptides are protected at their N- and C-termini by macrocyclization. Here, we use a novel bifunctional enzyme AgeMTPT to protect linear peptides by attaching prenyl and methyl groups at their free N- and C-termini. Using this peptide protectase in combination with other modular biosynthetic enzymes, we describe the total synthesis of the natural product aeruginosamide B and the biosynthesis of linear cyanobactin natural products. Our studies help to define the enzymatic mechanism of macrocyclization, providing evidence against the water exclusion hypothesis of transpeptidation and favoring the kinetic lability hypothesis.

Serotonin (5-HT) receptors are important in health and disease, but the existence of 14 subtypes necessitates selective ligands. Previously, the pulicatins were identified as ligands that specifically bound to the subtype 5-HT2B in the 500 nM to 10 μM range and that exhibited in vitro effects on cultured mouse neurons. Here, we examined the structure–activity relationship of 30 synthetic and natural pulicatin derivatives using binding, receptor functionality, and in vivo assays. The results reveal the 2-arylthiazoline scaffold as a tunable serotonin receptor-targeting pharmacophore. Tests in mice show potential antiseizure and antinociceptive activities at high doses without motor impairment.

•Observations from marine RiPP biosynthesis teaches us lessons about peptide combinatorial libraries.•Marine RiPP substrates contain mutable hypervariable cores, which are coupled with promiscuous posttranslational modification enzymes.•Marine RiPPs led to the discovery of conserved enzyme recognition sequences outside the core sequence, which serve as docking sites for the promiscuous biosynthetic enzymes.•Portability of recognition sequences allow modularity and enables hybridization of RiPP pathways.•Several unique posttranslational modifications were discovered in marine RiPPs.Ribosomally synthesized natural products are found in all forms of life. Their biosynthesis uses simple ribosomally synthesized peptides as starting materials that are transformed into complex structures via posttranslational modifications, enriched with elaborate chemical scaffolds that make them desirable as pharmacological tools. In addition, these natural products often exhibit combinatorial biosynthesis, making them attractive targets for engineering. An increasing knowledge of their biosynthetic machinery has provided key insights into their fascinating chemistry. Marine organisms have been a rich source of this class of natural products and here we review the lessons learned from marine life that enables exploitation of their potential for combinatorial engineering, opening up new routes for peptide-based drug discovery.

The cyanobactin prenyltransferases catalyze a series of known or unprecedented reactions on millions of different substrates, with no easily observable recognition motif and exquisite regioselectivity. Here we define the basis of broad substrate tolerance for the otherwise uncharacterized TruF family. We determined the structures of the Tyr-prenylating enzyme PagF, in complex with an isoprenoid donor analog and a panel of linear and macrocyclic peptide substrates. Unexpectedly, the structures reveal a truncated barrel fold, wherein binding of large peptide substrates is necessary to complete a solvent-exposed hydrophobic pocket to form the catalytically competent active site. Kinetic, mutational, chemical, and computational analyses revealed the structural basis of selectivity, showing a small motif within peptide substrates that is sufficient for recognition by the enzyme. Attaching this 2-residue motif to two random peptides results in their isoprenylation by PagF, demonstrating utility as a general biocatalytic platform for modifications on any peptide substrate.

A conventional metabolic pathway leads to a specific product. In stark contrast, there are diversity-generating metabolic pathways that naturally produce different chemicals, sometimes of great diversity. We demonstrate that for one such pathway, tru, each ensuing metabolic step is slower, in parallel with the increasing potential chemical divergence generated as the pathway proceeds. Intermediates are long lived and accumulate progressively, in contrast with conventional metabolic pathways, in which the first step is rate-limiting and metabolic intermediates are short-lived. Understanding these fundamental differences enables several different practical applications, such as combinatorial biosynthesis, some of which we demonstrate here. We propose that these principles may provide a unifying framework underlying diversity-generating metabolism in many different biosynthetic pathways.

A biosynthetic pathway to fungal polyketide–nonribosomal peptide natural products, Sch210971 (1a) and Sch210972 (1b) from Hapsidospora irregularis, was characterized by reconstitution and heterologous expression in Fusarium heterosporum. Using genetic, biochemical, and feeding experiments, we show that the incorporated amino acid 4-hydroxyl-4-methyl glutamate (HMG) is synthesized by an aldolase, probably using pyruvate as the precursor.

Cone snails are renowned for producing peptide-based venom, containing conopeptides and conotoxins, to capture their prey. A novel small-molecule guanine derivative with unprecedented features, genuanine, was isolated from the venom of two cone snail species. Genuanine causes paralysis in mice, indicating that small molecules and not just polypeptides may contribute to the activity of cone snail venom.

Ribosomally synthesized and posttranslationally modified peptide (RiPP) natural products are of broad interest because of their intrinsic bioactivities and potential for synthetic biology. The RiPP cyanobactin pathways pat and tru have been experimentally shown to be extremely tolerant of mutations. In nature, the pathways exhibit “substrate evolution”, where enzymes remain constant while the substrates of those enzymes are hypervariable and readily evolvable. Here, we sought to determine the mechanism behind this promiscuity. Analysis of a series of different enzyme–substrate combinations from five different cyanobactin gene clusters, in addition to engineered substrates, led us to define short discrete recognition elements within substrates that are responsible for directing enzymes. We show that these recognition sequences (RSs) are portable and can be interchanged to control which functional groups are added to the final natural product. In addition to the previously assigned N- and C-terminal proteolysis RSs, here we assign the RS for heterocyclization modification. We show that substrate elements can be swapped in vivo leading to successful production of natural products in E. coli. The exchangeability of these elements holds promise in synthetic biology approaches to tailor peptide products in vivo and in vitro.Keywords: cyanobactins; posttranslational modifications; recognition sequences; ribosomally synthesized and posttranslationally modified peptides (RiPP); substrate evolution

Strategies are needed for the robust production of cryptic, silenced, or engineered secondary metabolites in fungi. The filamentous fungus Fusarium heterosporum natively synthesizes the polyketide equisetin at >2 g L–1 in a controllable manner. We hypothesized that this production level was achieved by regulatory elements in the equisetin pathway, leading to the prediction that the same regulatory elements would be useful in producing other secondary metabolites. This was tested by using the native eqxS promoter and eqxR regulator in F. heterosporum, synthesizing heterologous natural products in yields of ∼1 g L–1. As proof of concept for the practical application, we resurrected an extinct pathway from an endophytic fungus with an initial yield of >800 mg L–1, leading to the practical synthesis of a selective antituberculosis agent. Finally, the method enabled new insights into the function of polyketide synthases in filamentous fungi. These results demonstrate a strategy for optimally employing native regulators for the robust synthesis of secondary metabolites.

•The use of cyanobactin enzymes in vitro to synthesize modified cyclic peptides•Manipulation of cyanobactin enzyme requirements for one-pot synthesis•In vitro synthesis of cyanobactins inaccessible through expression in E. coli•TruF1 was shown to be a Ser/Thr prenyltransferase in vitroMacrocyclases 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.Figure optionsDownload full-size imageDownload high-quality image (138 K)Download as PowerPoint slide

Ascidians contain abundant, diverse secondary metabolites, which are thought to serve a defensive role and which have been applied to drug discovery. It is known that bacteria in symbiosis with ascidians produce several of these metabolites, but very little is known about factors governing these ‘chemical symbioses’. To examine this phenomenon across a wide geographical and species scale, we performed bacterial and chemical analyses of 32 different ascidians, mostly from the didemnid family from Florida, Southern California and a broad expanse of the tropical Pacific Ocean. Bacterial diversity analysis showed that ascidian microbiomes are highly diverse, and this diversity does not correlate with geographical location or latitude. Within a subset of species, ascidian microbiomes are also stable over time (R=−0.037, P-value=0.499). Ascidian microbiomes and metabolomes contain species-specific and location-specific components. Location-specific bacteria are found in low abundance in the ascidians and mostly represent strains that are widespread. Location-specific metabolites consist largely of lipids, which may reflect differences in water temperature. By contrast, species-specific bacteria are mostly abundant sequenced components of the microbiomes and include secondary metabolite producers as major components. Species-specific chemicals are dominated by secondary metabolites. Together with previous analyses that focused on single ascidian species or symbiont type, these results reveal fundamental properties of secondary metabolic symbiosis. Different ascidian species have established associations with many different bacterial symbionts, including those known to produce toxic chemicals. This implies a strong selection for this property and the independent origin of secondary metabolite-based associations in different ascidian species. The analysis here streamlines the connection of secondary metabolite to producing bacterium, enabling further biological and biotechnological studies.

The programming of the fungal polyketide synthase (PKS) is quite complex, with a simple domain architecture leading to elaborate products. An additional level of complexity has been found within PKS-based pathways where the PKS is fused to a single module nonribosomal peptide synthetase (NRPS) to synthesize polyketides conjugated to amino acids. Here, we sought to understand the communication between these modules that enable correct formation of polyketide-peptide hybrid products. To do so, we fused together the genes that are responsible for forming five highly chemically diverse fungal natural products in a total of 57 different combinations, comprising 34 distinct module swaps. Gene fusions were formed with the idea of testing the connection and compatibility of the PKS and NRPS modules mediated by the acyl carrier protein (ACP), condensation (C) and ketoreductase (KR) domains. The resulting recombinant gene fusions were analyzed in a high-yielding expression platform to avail six new compounds, including the first successful fusion between a PKS and NRPS that make highly divergent products, and four previously reported molecules. Our results show that C domains are highly selective for a subset of substrates. We discovered that within the highly reducing (hr) PKS class, noncognate ACPs of closely related members complement PKS function. We intercepted a pre-Diels–Alder intermediate in lovastatin synthesis for the first time, shedding light on this canonical fungal biochemical reaction. The results of these experiments provide a set of ground rules for the successful engineering of hr-PKS and PKS-NRPS products in fungi.

A racemic, prenylated polyketide dimer, oxazinin A (1), was isolated from a novel filamentous fungus in the class Eurotiomycetes, and its structure was elucidated spectroscopically. The pentacyclic structure of oxazinin A (1) is a unique combination of benzoxazine, isoquinoline, and a pyran ring. Oxazinin A (1) exhibited antimycobacterial activity and modestly antagonized transient receptor potential (TRP) channels.

The griseorhodins belong to a family of extensively modified aromatic polyketides that exhibit activities such as inhibition of HIV reverse transcriptase and human telomerase. The vast structural diversity of this group of polyketides is largely introduced by enzymatic oxidations, which can significantly influence the bioactivity profile. Four new compounds, griseorhodins D–F, were isolated from a griseorhodin producer, Streptomyces sp. CN48+, based upon their enhancement of calcium uptake in a mouse dorsal root ganglion primary cell culture assay. Two of these compounds, griseorhodins D1 and D2, were shown to be identical to the major, previously uncharacterized products of a grhM mutant in an earlier griseorhodin biosynthesis study. Their structures enabled the establishment of a more complete hypothesis for the biosynthesis of griseorhodins and related compounds. The other two compounds, griseorhodins E and F, represent new products of post-polyketide synthase tailoring in griseorhodin biosynthesis and showed significant binding activity in a human dopamine active transporter assay.

A novel lumun-lumun sampling methodology was used to obtain a large diversity of micromollusks, including the new species Lienardia totopotens. In turn, from L. totopotens we cultivated a Streptomyces sp. strain that contained new and known spirotetronate polyketides, lobophorins (1–5). The structures were elucidated using spectroscopy, and the compounds were evaluated for cytotoxicity to human cells and activity against Mycobacterium tuberculosis, Bacillus subtilis, Pseudomonas aeruginosa and Burkholderia cepacia. Compounds 2–5 showed varying degrees of activity against human cells, M. tuberculosis and B. subtilis in the low μM to mid nM range but were inactive against the other strains, while 1 lacking digitoxose was inactive. Very slight structural changes in 2–5 led to varying antibacterial:cytotoxicity ratios, providing a possible basis to synthesize more selective derivatives.

In the oceans, secondary metabolites often protect otherwise poorly defended invertebrates, such as shell-less mollusks, from predation. The origins of these metabolites are largely unknown, but many of them are thought to be made by symbiotic bacteria. In contrast, mollusks with thick shells and toxic venoms are thought to lack these secondary metabolites because of reduced defensive needs. Here, we show that heavily defended cone snails also occasionally contain abundant secondary metabolites, γ-pyrones known as nocapyrones, which are synthesized by symbiotic bacteria. The bacteria, Nocardiopsis alba CR167, are related to widespread actinomycetes that we propose to be casual symbionts of invertebrates on land and in the sea. The natural roles of nocapyrones are unknown, but they are active in neurological assays, revealing that mollusks with external shells are an overlooked source of secondary metabolite diversity.Graphical AbstractFigure optionsDownload full-size imageDownload high-quality image (280 K)Download as PowerPoint slideHighlights► Symbiotic bacteria synthesize compounds found in mollusks ► Physically defended mollusks provide a source for bioactive metabolite discovery ► A type of pyrone polyketide pathway described ► Nocardiopsis strains may be widespread, casually associated symbionts of inveterbrates

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.

We report 12 cyanobactin cyclic peptides, the aestuaramides, from the cultivated cyanobacterium Lyngbya aestuarii. We show that aestuaramides are synthesized enzymatically as reverse O-prenylated tyrosine ethers that subsequently undergo a Claisen rearrangement to produce forward C-prenylated tyrosine. These results reveal that a nonenzymatic Claisen rearrangement dictates isoprene regiochemistry in a natural system. They also reveal one of the mechanisms that organisms use to generate structurally diverse compound libraries starting from simple ribosomal peptide pathways (RiPPs).

The bacterium Gordonia sp. 647 W.R.1a.05 was cultivated from the venom duct of the cone snail, Conus circumcisus. The Gordonia sp. organic extract modulated the action potential of mouse dorsal root ganglion neurons. Assay-guided fractionation led to the identification of the new compound circumcin A (1) and 11 known analogs (2–12). Two of these compounds, kurasoin B (7) and soraphinol A (8), were active in a human norepinephrine transporter assay with Ki values of 2575 and 867 nM, respectively. No neuroactivity had previously been reported for compounds in this structural class. Gordonia species have been reproducibly isolated from four different cone snail species, indicating a consistent association between these organisms.

Ribosomal peptide natural products (RiPPs) offer a new frontier for discovering and engineering bioactive small molecules. In this issue of Chemistry & Biology, Young and colleagues overcome hurdles that have slowed the development of RiPPs, leading to in vivo synthesis of new thiopeptide antibiotics.

Cyanobactins, a class of ribosomally encoded macrocylic natural products, are biosynthesized through the proteolytic processing and subsequent N-C macrocylization of ribosomal peptide precursors. Macrocylization occurs through a two-step process in which the first protease (PatA) removes the amino terminal flanking sequence from the precursor to yield a free N terminus of the precursor peptide, and the second protease (PatG) removes the C-terminal flanking sequence and then catalyzes the transamidation reaction to yield an N-C cyclized product. Here, we present the crystal structures of the protease domains of PatA and PatG from the patellamide cluster and of PagA from the prenylagaramide cluster. A comparative structural and biochemical analysis of the transamidating PatG protease reveals the presence of a unique structural element distinct from canonical subtilisin proteases, which may facilitate the N-C macrocylization of the peptide substrate.Graphical AbstractFigure optionsDownload full-size imageDownload high-quality image (488 K)Download as PowerPoint slideHighlights► Boundaries for cyanobactin maturation protease domains determined ► Structures of protease domains for cyanobactin maturation enzymes determined ► Additional structural motif in PatG assists in cyclizing transamidation ► Rational mutagenesis of active site leads to altered catalytic profiles for PatG

Complex biosynthetic enzymes such as polyketide synthases make mistakes. In this issue of Chemistry & Biology, Jensen et al. report that a discrete family of acyltransferases is responsible for error correction, hydrolyzing key biosynthetic intermediates from a multi-enzyme complex. This activity might find use in understanding polyketide biosynthesis, particularly in uncultivated organisms and in tailoring the synthesis of small molecules.

Ascidians (tunicates) are rich sources of structurally elegant, pharmaceutically potent secondary metabolites and, more recently, potential biofuels. It has been demonstrated that some of these compounds are made by symbiotic bacteria and not by the animals themselves, and for a few other compounds evidence exists supporting a symbiotic origin. In didemnid ascidians, compounds are highly variable even in apparently identical animals. Recently, we have explained this variation at the genomic and metagenomic levels and have applied the basic scientific findings to drug discovery and development. This review discusses what is currently known about the origin and variation of symbiotically derived metabolites in ascidians, focusing on the family Didemnidae, where most research has occurred. Applications of our basic studies are also described.

Two new compounds, the peptide–polyketide glycoside totopotensamide A (1) and its aglycone totopotensamide B (2), were isolated from a Streptomyces sp. cultivated from the gastropod mollusk Lienardia totopotens collected in the Philippines. The compounds contain a previously undescribed polyketide component, a novel 2,3-diaminobutyric acid-containing macrolactam, and a new amino acid, 4-chloro-5,7-dihydroxy-6-methylphenylglycine. The application of Marfey’s method to phenylglycine derivatives was explored using quantum mechanical calculations and NMR.

Burkholdines are cyclic lipopeptides with unusual antifungal potency, making them promising leads as a new class of antifungal agents. However, a recent report using knockout mutagenesis indicates that these and related compounds, such as occidiofungins, xylocandins, and cepacidines, may also be synonymous with the long-known hemolytic virulence factors found in diverse Burkholderia isolates. Because of their possible roles in causing Burkholderia infections or curing fungal infections, it is important to fully define their structures and biological activities using pure compounds. Here, we report the structures of three further burkholdines, Bk-1119, Bk-1213, and Bk-1215, which were elucidated using spectroscopic methods. The absolute configuration of this compound class was determined for the first time using a combination of spectroscopy and chemical degradation techniques. Antifungal and hemolytic activities were assessed for five pure burkholdines, representative of the structural diversity of this lipopeptide class. All of the burkholdines were potent antifungal and hemolytic agents, validating their probable role in virulence. However, one of the burkholdines (Bk-1119) exhibited a >30-fold selectivity for fungi versus sheep erythrocytes and was more than 25-fold more potent than amphotericin against some fungal strains. Therefore, burkholdines have potential to selectively target fungal infections.

Secondary metabolites are ubiquitous in bacteria, but by definition, they are thought to be nonessential. Highly toxic secondary metabolites such as patellazoles have been isolated from marine tunicates, where their exceptional potency and abundance implies a role in chemical defense, but their biological source is unknown. Here, we describe the association of the tunicate Lissoclinum patella with a symbiotic α-proteobacterium, Candidatus Endolissoclinum faulkneri, and present chemical and biological evidence that the bacterium synthesizes patellazoles. We sequenced and assembled the complete Ca. E. faulkneri genome, directly from metagenomic DNA obtained from the tunicate, where it accounted for 0.6% of sequence data. We show that the large patellazoles biosynthetic pathway is maintained, whereas the remainder of the genome is undergoing extensive streamlining to eliminate unneeded genes. The preservation of this pathway in streamlined bacteria demonstrates that secondary metabolism is an essential component of the symbiotic interaction.

The cyanobactin ribosomal peptide (RP) natural product pathway was manipulated to incorporate multiple tandem mutations and non-proteinogenic amino acids, using eight heterologous components simultaneously expressed in Escherichia coli. These studies reveal the potential of RPs for the rational synthesis of complex, new small molecules over multiple-step biosynthetic pathways using simple genetic engineering.

The enzymatic basis of ribosomal peptide natural product prenylation has not been reported. Here, we characterize a prenyltransferase, LynF, from the TruF enzyme family. LynF is the first characterized representative of the TruF protein family, which is responsible for both reverse- and forward-O-prenylation of tyrosine, serine, and threonine in cyclic peptides known as cyanobactins. We show that LynF reverse O-prenylates tyrosine in macrocyclic peptides. Based upon these results, we propose that the TruF family prenylates mature cyclic peptides, from which the leader sequence and other enzyme recognition elements have been excised. This differs from the common model of ribosomal peptide biosynthesis, in which a leader sequence is required to direct post-translational modifications. In addition, we find that reverse O-prenylated tyrosine derivatives undergo a facile Claisen rearrangement at ‘physiological’ temperature in aqueous buffers, leading to forward C-prenylated products. Although the Claisen rearrangement route to natural products has been chemically anticipated for at least 40 years, it has not been demonstrated as a route to prenylated natural products. Here, we show that the Claisen rearrangement drives phenolic C-prenylation in at least one case, suggesting that this route should be reconsidered as a mechanism for the biosynthesis of prenylated phenolic compounds.

New compounds nobilamides A–H and related known compounds A-3302-A and A-3302-B were isolated based upon their suppression of capsaicin-induced calcium uptake in a mouse dorsal root ganglion primary cell culture assay. Two of these compounds, nobilamide B and A-3302-A, were shown to be long-acting antagonists of mouse and human TRPV1 channels, abolishing activity for >1 h after removal of drug presumably via a covalent attachment. Other derivatives also inhibited the TRPV1 channel, albeit with low potency, affording a structure–activity profile to support the proposed mechanism of action. While the activities were modest, we propose a new mechanism of action and a new site of binding for these inhibitors that may spur development of related analogues for treatment of pain.

Ribosomal peptide natural products are ubiquitous, yet relatively few tools exist to predict structures and clone new pathways. Cyanobactin ribosomal peptides are found in ∼30% of all cyanobacteria, but the connection between gene sequence and structure was not defined, limiting the rapid identification of new compounds and pathways. Here, we report discovery of four orphan cyanobactin gene clusters by genome mining and an additional pathway by targeted cloning, which represented a tyrosine O-prenylating biosynthetic pathway. Genome mining enabled discovery of five cyanobactins, including peptide natural products from Spirulina supplements. A phylogenetic model defined four cyanobactin genotypes, which explain the synthesis of multiple cyanobactin structural classes and help direct pathway cloning and structure prediction efforts. These strategies were applied to DNA isolated from a mixed cyanobacterial bloom containing cyanobactins.Graphical AbstractFigure optionsDownload full-size imageDownload high-quality image (403 K)Download as PowerPoint slideHighlights► A tyrosine-prenylating gene cluster and a previously uncharacterized compound were found ► Cyanobactin natural products were identified in Spirulina health food supplements ► Cyanobactin chemical structure-gene sequence relationships were defined ► These relationships enabled gene cloning from complex environmental samples

The relationship between tunicates and the uncultivated cyanobacterium Prochloron didemni has long provided a model symbiosis. P. didemni is required for survival of animals such as Lissoclinum patella and also makes secondary metabolites of pharmaceutical interest. Here, we present the metagenomes, chemistry, and microbiomes of four related L. patella tunicate samples from a wide geographical range of the tropical Pacific. The remarkably similar P. didemni genomes are the most complex so far assembled from uncultivated organisms. Although P. didemni has not been stably cultivated and comprises a single strain in each sample, a complete set of metabolic genes indicates that the bacteria are likely capable of reproducing outside the host. The sequences reveal notable peculiarities of the photosynthetic apparatus and explain the basis of nutrient exchange underlying the symbiosis. P. didemni likely profoundly influences the lipid composition of the animals by synthesizing sterols and an unusual lipid with biofuel potential. In addition, L. patella also harbors a great variety of other bacterial groups that contribute nutritional and secondary metabolic products to the symbiosis. These bacteria possess an enormous genetic potential to synthesize new secondary metabolites. For example, an antitumor candidate molecule, patellazole, is not encoded in the genome of Prochloron and was linked to other bacteria from the microbiome. This study unveils the complex L. patella microbiome and its impact on primary and secondary metabolism, revealing a remarkable versatility in creating and exchanging small molecules.

A protease from ribosomal peptide biosynthesis macrocyclizes diverse substrates, including those resembling nonribosomal peptide and hybrid polyketide−peptide products. The proposed mechanism is analogous to thioesterase-catalyzed chemistry, but the substrates are amide bonds rather than thioesters.

The cyanobactin biosynthetic pathways pat and tru, isolated from metagenomes of marine animals, lead to diverse natural products containing heterocycles derived from Cys, Ser, and Thr. Previous work has shown that PatD and TruD are extremely broad-substrate heterocyclase enzymes. These enzymes are virtually identical in their N-terminal putative catalytic domains, but only ∼77% identical in their C-terminal putative substrate-binding domains. Here, we show that these differences allow the enzymes to control regioselectivity of posttranslational modifications, helping to control product chemistry in this hypervariable family of marine natural products.

Potent antifungal cyclic lipopeptides, burkholdines (Bk), were isolated from a culture of Burkholderia ambifaria 2.2N. Bk-1229 (1) and Bk-1097 (2) are octapeptides comprised of nonproteinogenic amino acids, including β-hydroxytyrosine, β-hydroxyasparagine, and a new fatty acyl amino acid. 1 and 2 are fungicidal against a panel of fungi with potencies 2−60-fold better than amphotericin B control.

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 Ca2+ 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-HT2B receptor. Here, we report the structure elucidation of the new compounds and results of the neurological assays.

Covering: up to the end of January 2009

Covering: up to July 2008

Fungal reduced polyketides possess diverse structures exploring a broad region of chemical space despite their synthesis by very similar enzymes. Many fungal polyketides are capped by diverse amino acid-derived five-membered rings, the tetramic acids and related pyrrolidine-2-ones. The known tetramic acid synthetase enzymes in fungi contain C-terminal reductive (R) domains that were proposed to release reduced pyrrolidine-2-one intermediates en route to the tetramic acids. To determine the enzymatic basis of pyrrolidine-2-one diversity, we overexpressed equisetin synthetase (EqiS) R domains and analyzed their reactivity with synthetic substrate analogs. We show that the EqiS R domain does not perform a reducing function and does not bind reducing cofactors. Instead, the EqiS R catalyzes a Dieckmann condensation, with an estimated kcat ≈ 15 s−1. This role differs from the redox reactions normally catalyzed by short chain dehydrogenase/reductase superfamily enzymes.

An iterative polyketide synthase–peptide synthetase hybrid assembles the HIV-1 integrase inhibitory tetramic acid, equisetin, in the filamentous fungus Fusarium heterosporum.

Ascidians (tunicates; sea squirts) are sources of diverse, bioactive natural products, one of which is an approved drug and many of which are potent drug leads. It has been shown that symbiotic bacteria living with ascidians produce some of the bioactive compounds isolated from whole animals, and indirect evidence strongly implicates symbiotic bacteria in the synthesis of many others. However, for the majority the producing organism has not been identified. In cases where a symbiotic origin has been definitively assigned, the resulting data lead to improved paths to drug discovery and development from marine animals. This review traces evidence for symbiotic production where such evidence exists and describes the strengths and limitations of that evidence.