RESUMO
Coral terpenes are important molecules with numerous applications. Here, we describe a robust and simple method to produce coral terpene scaffolds at scale. As an example of the approach, here we discover, express, and characterize further klysimplexin R synthases, expanding the known enzymology of soft coral terpene cyclases. We hope that the underlying method described will enable widespread basic research into the functions of coral terpenes and their biosynthetic genes, as well as the commercial development of biomedically and technologically important molecules.
Assuntos
Antozoários , Terpenos , Antozoários/enzimologia , Antozoários/metabolismo , Antozoários/genética , Terpenos/metabolismo , Terpenos/química , Animais , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Alquil e Aril Transferases/metabolismo , Alquil e Aril Transferases/genéticaRESUMO
Sponges (Porifera) contain many peptide-specialized metabolites with potent biological activities and significant roles in shaping marine ecology. It is well established that symbiotic bacteria produce bioactive "sponge" peptides, both on the ribosome (RiPPs) and nonribosomally. Here, we demonstrate that sponges themselves also produce many bioactive macrocyclic peptides, such as phakellistatins and related proline-rich macrocyclic peptides (PRMPs). Using the Stylissa carteri sponge transcriptome, methods were developed to find sequences encoding 46 distinct RiPP-type core peptides, of which ten encoded previously identified PRMP sequences. With this basis set, the genome and transcriptome of the sponge Axinella corrugata was interrogated to find 35 PRMP precursor peptides encoding 31 unique core peptide sequences. At least 11 of these produced cyclic peptides that were present in the sponge and could be characterized by mass spectrometry, including stylissamides A-D and seven previously undescribed compounds. Precursor peptides were encoded in the A. corrugata genome, confirming their animal origin. The peptides contained signal peptide sequences and highly repetitive recognition sequence-core peptide elements with up to 25 PRMP copies in a single precursor. In comparison to sponges without PRMPs, PRMP sponges are incredibly enriched in potentially secreted polypeptides, with >23,000 individual signal peptide encoding genes found in a single transcriptome. The similarities between PRMP biosynthetic genes and neuropeptides in terms of their biosynthetic logic suggest a fundamental biology linked to circular peptides, possibly indicating a widespread and underappreciated diversity of signaling peptide post-translational modifications across the animal kingdom.
Assuntos
Peptídeos Cíclicos , Peptídeos , Animais , Peptídeos/genética , Peptídeos Cíclicos/genética , Sequência de Aminoácidos , Bandagens , Sinais Direcionadores de ProteínasRESUMO
Peptide cyclization improves conformational rigidity, providing favorable pharmacological properties, such as proteolytic resistance, target specificity, and membrane permeability. Thus, many synthetic and biosynthetic peptide circularization strategies have been developed. PatG and related natural macrocyclases process diverse peptide sequences, generating millions of cyclic derivatives. However, the application of these cyclases is limited by low yields and the potential presence of unwanted intermediates. Here, we designed a covalently fused G macrocyclase with substrates that efficiently and spontaneously release cyclic peptides. To increase the fidelity of synthesis, we developed an orthogonal control mechanism enabling precision synthesis in Escherichia coli. As a result, a library comprising 4.8 million cyclic derivatives was constructed, producing an estimated 2.6 million distinct cyclic peptides with an improved yield and fidelity.
Assuntos
Peptídeos Cíclicos , Peptídeos , Peptídeos Cíclicos/metabolismo , Peptídeos/genética , Peptídeos/química , Sequência de Aminoácidos , Peptídeo Hidrolases/química , CiclizaçãoRESUMO
Animals synthesize simple lipids using a distinct fatty acid synthase (FAS) related to the type I polyketide synthase (PKS) enzymes that produce complex specialized metabolites. The evolutionary origin of the animal FAS and its relationship to the diversity of PKSs remain unclear despite the critical role of lipid synthesis in cellular metabolism. Recently, an animal FAS-like PKS (AFPK) was identified in sacoglossan molluscs. Here, we explore the phylogenetic distribution of AFPKs and other PKS and FAS enzymes across the tree of life. We found AFPKs widely distributed in arthropods and molluscs (>6300 newly described AFPK sequences). The AFPKs form a clade with the animal FAS, providing an evolutionary link bridging the type I PKSs and the animal FAS. We found molluscan AFPK diversification correlated with shell loss, suggesting AFPKs provide a chemical defense. Arthropods have few or no PKSs, but our results indicate AFPKs contributed to their ecological and evolutionary success by facilitating branched hydrocarbon and pheromone biosynthesis. Although animal metabolism is well studied, surprising new metabolic enzyme classes such as AFPKs await discovery.
Assuntos
Policetídeos , Animais , Policetídeos/metabolismo , Ácidos Graxos , Metabolismo dos Lipídeos/genética , Filogenia , Policetídeo Sintases/genética , Policetídeo Sintases/metabolismo , Ácido Graxo Sintases/genética , Ácido Graxo Sintases/metabolismoRESUMO
IMPORTANCE: Bacterial resistance to antibiotics is a crisis. Acinetobacter baumannii is among the CDC urgent threat pathogens in part for this reason. Lipopeptides known as turnercyclamycins are produced by symbiotic bacteria that normally live in marine mollusks, where they may be involved in shaping their symbiotic niche. Turnercyclamycins killed Gram-negative pathogens including drug-resistant Acinetobacter, but how do the mechanisms of resistance compare to other lipopeptide drugs? Here, we define resistance from a truncation of MlaA, a protein involved in regulating bacterial membrane phospholipids. Intriguingly, this resistance mechanism only affected one turnercyclamycin variant, which differed only in two atoms in the lipid tail of the compounds. We could not obtain significant resistance to the second turnercyclamycin variant, which was also effective in an infection model. This study reveals an unexpected subtlety in resistance to lipopeptide antibiotics, which may be useful in the design and development of antibiotics to combat drug resistance.
Assuntos
Colistina , Lipopeptídeos , Colistina/farmacologia , Lipopeptídeos/farmacologia , Antibacterianos/farmacologia , Bactérias Gram-Negativas , Bactérias , Testes de Sensibilidade Microbiana , Farmacorresistência Bacteriana MúltiplaRESUMO
Animal cytoplasmic fatty acid synthase (FAS) represents a unique family of enzymes that are classically thought to be most closely related to fungal polyketide synthase (PKS). Recently, a widespread family of animal lipid metabolic enzymes has been described that bridges the gap between these two ubiquitous and important enzyme classes: the animal FAS-like PKSs (AFPKs). Although very similar in sequence to FAS enzymes that produce saturated lipids widely found in animals, AFPKs instead produce structurally diverse compounds that resemble bioactive polyketides. Little is known about the factors that bridge lipid and polyketide synthesis in the animals. Here, we describe the function of EcPKS2 from Elysia chlorotica, which synthesizes a complex polypropionate natural product found in this mollusc. EcPKS2 starter unit promiscuity potentially explains the high diversity of polyketides found in and among molluscan species. Biochemical comparison of EcPKS2 with the previously described EcPKS1 reveals molecular principles governing substrate selectivity that should apply to related enzymes encoded within the genomes of photosynthetic gastropods. Hybridization experiments combining EcPKS1 and EcPKS2 demonstrate the interactions between the ketoreductase and ketosynthase domains in governing the product outcomes. Overall, these findings enable an understanding of the molecular principles of structural diversity underlying the many molluscan polyketides likely produced by the diverse AFPK enzyme family.
Assuntos
Produtos Biológicos , Gastrópodes , Policetídeos , Animais , Policetídeo Sintases/genética , Ácido Graxo Sintases , LipídeosRESUMO
Thousands of coral terpenes originate from simple scaffolds that undergo oxidative tailoring. While corals are excellent sources of drug leads, the challenge of supplying structurally complex drug leads from marine organisms has sometimes slowed their development. Making this even more challenging, in comparison to other organisms, such as plants and microbes, for which the terpene literature is substantial, very little is known about how the unique coral terpenes are biosynthesized and elaborated in nature. In this study, we used a semisynthetic strategy to produce at gram scale in yeast the eunicellane scaffold that underlies >200 coral compounds. Synthetic oxidation reactions were explored, generating key scaffolds that reflect three of the four structural classes derived from eunicellane and enabling the first asymmetric syntheses of the natural products solenopodin C and klysimplexin Q. Biomimetic methods and detailed mechanistic studies of synthetic reactions shed light on potential enzymological reactivity, including the role of epoxide rearrangement in eunicellane biosynthesis.
Assuntos
Antozoários , Produtos Biológicos , Diterpenos , Animais , Biomimética , Diterpenos/química , Terpenos/química , Saccharomyces cerevisiae , Produtos Biológicos/químicaRESUMO
Pain is often debilitating, and current treatments are neither universally efficacious nor without risks. Transient receptor potential (TRP) ion channels offer alternative targets for pain relief, but little is known about the regulation or identities of endogenous TRP ligands that affect inflammation and pain. Here, transcriptomic and targeted lipidomic analysis of damaged tissue from the mouse spinal nerve ligation (SNL)-induced chronic pain model revealed a time-dependent increase in Cyp1b1 mRNA and a concurrent accumulation of 8,9-epoxyeicosatrienoic acid (EET) and 19,20-EpDPA post injury. Production of 8,9-EET and 19,20-EpDPA by human/mouse CYP1B1 was confirmed in vitro, and 8,9-EET and 19,20-EpDPA selectively and dose-dependently sensitized and activated TRPA1 in overexpressing HEK-293 cells and Trpa1-expressing/AITC-responsive cultured mouse peptidergic dorsal root ganglia (DRG) neurons. TRPA1 activation by 8,9-EET and 19,20-EpDPA was attenuated by the antagonist A967079, and mouse TRPA1 was more responsive to 8,9-EET and 19,20-EpDPA than human TRPA1. This latter effect mapped to residues Y933, G939, and S921 of TRPA1. Intra-plantar injection of 19,20-EpDPA induced acute mechanical, but not thermal hypersensitivity in mice, which was also blocked by A967079. Similarly, Cyp1b1-knockout mice displayed a reduced chronic pain phenotype following SNL injury. These data suggest that manipulation of the CYP1B1-oxylipin-TRPA1 axis might have therapeutic benefit.
RESUMO
Chemists have studied marine animals for the better part of a century because they contain a diverse array of bioactive compounds. Tens of thousands of compounds have been reported, many with elaborate structural motifs and biological mechanisms of action found nowhere else. The challenge holding back the field has long been that of supply. Compounds are sometimes obtained by cultivating marine animals or by wild harvest, but this often presents logistical and environmental challenges. Some of the most medically important marine animal compounds are supplied by synthesis, often through multistep procedures that delay drug development. A relatively small number of such agents have been approved by the U.S. Food and Drug Administration, often after a heroic effort. In a recent mBio paper, Uppal and coworkers (https://doi.org/10.1128/mBio.01524-22) address key hurdles underlying the supply issue, discovering an uncultivated new bacterial genus from a marine sponge and reconstituting the biosynthetic pathway for expression.
Assuntos
Produtos Biológicos , Poríferos , Animais , Poríferos/microbiologia , Bactérias/metabolismo , Desenvolvimento de Medicamentos , Descoberta de Drogas , Produtos Biológicos/metabolismoRESUMO
A key goal of synthetic biology is to enable designed modification of peptides and proteins, both in vivo and in vitro. N- and C-Terminal modification enzymes are crucial in this regard, but there are a few enzymatic options to protect peptide termini. AgeMTPT protects the N-terminus of short peptides with isoprene and the C-terminus as a methyl ester, but its substrate scope is unknown, limiting its application. Here, we investigate the substrate selectivity of the prenyltransferase domain, revealing a requirement for N-terminal aromatic amino acids, but with tolerance for diverse uncharged amino acids in the remaining positions. To demonstrate the potential of the enzyme, substrate selectivity data were used in the enzymatic modification of leu-enkephalin at the critical N-terminal residue. AgeMTPT active site mutagenesis led to an enzyme with expanded substrate scope, including the reverse geranylation of the N-termini of peptides. These data reveal potential applications of enzymatic peptide protection in synthetic biology.
Assuntos
Peptídeos , Transferases , Transferases/metabolismo , Peptídeos/metabolismo , Metiltransferases , ProteínasRESUMO
The methylation of peptide backbone amides is a hallmark of bioactive natural products, and it also greatly modifies the pharmacology of synthetic peptides. Usually, bioactive N-methylated peptides are cyclic. However, there is very limited knowledge about how post-translational enzymes can be applied to the synthesis of designed N-methylated peptides or peptide libraries. Here, driven by the established ability of some RiPP enzymes to process diverse substrates, we sought to define catalysts for the in vivo and in vitro macrocyclization of backbone-methylated peptides. We developed efficient methods in which short, synthetic N-methylated peptides could be modified using side chain and mainchain macrocyclases, PsnB and PCY1 from plesiocin and orbitide biosynthetic pathways, respectively. Most significantly, a strategy for PsnB cyclase was designed enabling simple in vitro methods compatible with solid-phase peptide synthesis. We show that cyanobactin N-terminal protease PatA is a broadly useful catalyst that is also compatible with N-methylation chemistry, but that cyanobactin macrocyclase PatG is strongly biased against N-methylated substrates. Finally, we sought to marry these macrocyclase tools with an enzyme that N-methylates its core peptide: OphMA from the omphalotin pathway. However, instead, we reveal some limitations of OphMA and demonstrate that it unexpectedly and extensively modified the enzyme itself in vivo. Together, these results demonstrate proof-of-concept for enzymatic synthesis of N-methylated peptide macrocycles.
Assuntos
Peptídeos Cíclicos , Peptídeos , Vias Biossintéticas , Metilação , Peptídeo Hidrolases/metabolismo , Peptídeos/química , Peptídeos Cíclicos/química , Processamento de Proteína Pós-TraducionalRESUMO
Natural product-encoding biosynthetic gene clusters (BGCs) within microbial genomes far outnumber the known natural products; chemical products from such BGCs remain cryptic. These silent BGCs hold promise not only for the elaboration of new natural products but also for the discovery of useful biosynthetic enzymes. Here, we describe a genome mining strategy targeted toward the discovery of substrate promiscuous natural product biosynthetic enzymes. In the genome of the methanotrophic bacterium Methylovulum psychrotolerans Sph1T, we discover a transcriptionally silent natural product BGC that encoded numerous ribosomally synthesized and post-translationally modified peptide (RiPP) natural products. These cryptic RiPP natural products were accessed using heterologous expression of the substrate peptide and biosynthetic enzyme-encoded genes. In line with our genome mining strategy, the RiPP biosynthetic enzymes in this BGC were found to be substrate promiscuous, which allowed us to use them in a combinatorial fashion with a similarly substrate-tolerant cyanobactin biosynthetic enzyme to introduce head-to-tail macrocyclization in the proteusin family of RiPP natural products.
Assuntos
Produtos Biológicos , Família Multigênica , Peptídeos/metabolismoRESUMO
Diterpenes are major defensive small molecules that enable soft corals to survive without a tough exterior skeleton, and, until now, their biosynthetic origin has remained intractable. Furthermore, biomedical application of these molecules has been hampered by lack of supply. Here, we identify and characterize coral-encoded terpene cyclase genes that produce the eunicellane precursor of eleutherobin and cembrene, representative precursors for the >2,500 terpenes found in octocorals. Related genes are found in all sequenced octocorals and form their own clade, indicating a potential ancient origin concomitant with the split between the hard and soft corals. Eleutherobin biosynthetic genes are colocalized in a single chromosomal region. This demonstrates that, like plants and microbes, animals also harbor defensive biosynthetic gene clusters, supporting a recombinational model to explain why specialized or defensive metabolites are adjacently encoded in the genome.
Assuntos
Antozoários , Terpenos , Animais , Antozoários/genética , Antozoários/metabolismo , Cromossomos , Genoma , Família Multigênica , Terpenos/metabolismoRESUMO
Nearly every animal species on Earth contains a unique polyketide synthase (PKS) encoded in its genome, yet no animal-clade PKS has been biochemically characterized, and even the chemical products of these ubiquitous enzymes are known in only a few cases. The earliest animal genome-encoded PKS gene to be identified was SpPks1 from sea urchins. Previous genetic knockdown experiments implicated SpPks1 in synthesis of the sea urchin pigment echinochrome. Here, we express and purify SpPks1, performing biochemical experiments to demonstrate that the sea urchin protein is responsible for the synthesis of 2-acetyl-1,3,6,8-tetrahydroxynaphthalene (ATHN). Since ATHN is a plausible precursor of echinochromes, this result defines a biosynthetic pathway to the ubiquitous echinoderm pigments and rewrites the previous hypothesis for echinochrome biosynthesis. Truncation experiments showed that, unlike other type I iterative PKSs so far characterized, SpPks1 produces the naphthalene core using solely ketoacylsynthase (KS), acyltransferase, and acyl carrier protein domains, delineating a unique class of animal nonreducing aromatic PKSs (aPKSs). A series of amino acids in the KS domain define the family and are likely crucial in cyclization activity. Phylogenetic analyses indicate that SpPks1 and its homologs are widespread in echinoderms and their closest relatives, the acorn worms, reinforcing their fundamental importance to echinoderm biology. While the animal microbiome is known to produce aromatic polyketides, this work provides biochemical evidence that animals themselves also harbor ancient, convergent, dedicated pathways to carbocyclic aromatic polyketides. More fundamentally, biochemical analysis of SpPks1 begins to define the vast and unexplored biosynthetic space of the ubiquitous animal PKS family.
Assuntos
Policetídeo Sintases , Policetídeos , Animais , Naftalenos , Filogenia , Policetídeo Sintases/metabolismo , Policetídeos/metabolismo , Ouriços-do-Mar/metabolismoRESUMO
Biologically active peptides are a major growing class of drugs, but their therapeutic potential is constrained by several limitations including bioavailability and poor pharmacokinetics. The attachment of functional groups like lipids has proven to be a robust and effective strategy for improving their therapeutic potential. Biochemical and bioactivity-guided screening efforts have identified the cyanobactins as a large class of ribosomally synthesized and post-translationally modified peptides (RiPPs) that are modified with lipids. These lipids are attached by the F superfamily of peptide prenyltransferase enzymes that utilize 5-carbon (prenylation) or 10-carbon (geranylation) donors. The chemical structures of various cyanobactins initially showed isoprenoid attachments on Ser, Thr, or Tyr. Biochemical characterization of the F prenyltransferases from the corresponding clusters shows that the different enzymes have different acceptor residue specificities but are otherwise remarkably sequence tolerant. Hence, these enzymes are well suited for biotechnological applications. The crystal structure of the Tyr O-prenyltransferase PagF reveals that the F enzyme shares a domain architecture reminiscent of a canonical ABBA prenyltransferase fold but lacks secondary structural elements necessary to form an enclosed active site. Binding of either cyclic or linear peptides is sufficient to close the active site to allow for productive catalysis, explaining why these enzymes cannot use isolated amino acids as substrates.Almost all characterized isoprenylated cyanobactins are modified with 5-carbon isoprenoids. However, chemical characterization demonstrates that the piricyclamides are modified with a 10-carbon geranyl moiety, and in vitro reconstitution of the corresponding PirF shows that the enzyme is a geranyltransferase. Structural analysis of PirF shows an active site nearly identical with that of the PagF prenyltransferase but with a single amino acid substitution. Of note, mutation at this residue in PagF or PirF can completely switch the isoprenoid donor specificity of these enzymes. Recent efforts have resulted in significant expansion of the F family with enzymes identified that can carry out C-prenylations of Trp, N-prenylations of Trp, and bis-N-prenylations of Arg. Additional genome-guided efforts based on the sequence of F enzymes identify linear cyanobactins that are α-N-prenylated and α-C-methylated by a bifunctional prenyltransferase/methyltransferase fusion and a bis-α-N- and α-C-prenylated linear peptide. The discovery of these different classes of prenyltransferases with diverse acceptor residue specificities expands the biosynthetic toolkit for enzymatic prenylation of peptide substrates.In this Account, we review the current knowledge scope of the F family of peptide prenyltransferases, focusing on the biochemical, structure-function, and chemical characterization studies that have been carried out in our laboratories. These enzymes are easily amenable for diversity-oriented synthetic efforts as they can accommodate substrate peptides of diverse sequences and are thus attractive catalysts for use in synthetic biology approaches to generate high-value peptidic therapeutics.
Assuntos
Dimetilaliltranstransferase , Carbono , Catálise , Dimetilaliltranstransferase/química , Dimetilaliltranstransferase/genética , Dimetilaliltranstransferase/metabolismo , Lipídeos , Peptídeos/química , TerpenosRESUMO
Members of the YcaO superfamily are among the most common post-translational modification enzymes in natural product biosynthesis, with wide usage in biotechnology and synthetic biology applications. Here, we use domain-swapped chimeras and discovered unstructured regions in cyanobactin YcaOs that guide interactions with the substrates, governing access to interior amino acids in the substrates and explaining the chemoselectivity between PatD and TruD. These results define how the cyanobactin heterocyclases modify exceptionally sequence diverse substrates, yet with a high degree of positional and nucleophile selectivity.
Assuntos
Peptídeos Cíclicos , Processamento de Proteína Pós-Traducional , Sequência de Aminoácidos , Peptídeos Cíclicos/químicaRESUMO
Bacteria use small molecules to impose strict regulation over the acquisition, uptake, and sequestration of transition metal ions. Low-abundance nutrient metals, such as Fe(III), need to be scavenged from the environment by high-affinity chelating molecules called siderophores. Conversely, metal ions that become toxic at high concentrations need to be sequestered and detoxified. Often, bacteria produce a suite of compounds that bind various metal ions at different affinities in order to maintain homeostasis. Turnerbactin, a triscatecholate siderophore isolated from the intracellular shipworm symbiont Teredinibacter turnerae T7901, is responsible for iron regulation and uptake. Herein, another series of compounds are described that complex with iron, copper, and molybdenum in solution. Teredinibactins belong to a class of metal-binding molecules that utilize a phenolate-thiazoline moiety in the coordination of metal ions. In contrast to other compounds in this class, such as yersiniabactin, the phenyl ring is decorated with a 2,4-dihydroxy-3-halo substitution pattern. UV-vis absorption spectroscopy based titration experiments with CuCl2 show the formation of an intermediate complex at substoichiometric concentrations and conversion to a copper-bound complex at 1:1 molar equiv.
Assuntos
Compostos Férricos , Sideróforos , Bactérias/metabolismo , Transporte Biológico , Ferro/metabolismo , Sideróforos/químicaRESUMO
Marine tunicates produce defensive amino-acid-derived metabolites, including 2-(3,5-diiodo-4-methoxyphenyl)ethan-1-amine (DIMTA), but their mechanisms of action are rarely known. Using an assay-guided approach, we found that out of the many different sensory cells in the mouse dorsal root ganglion (DRG), DIMTA selectively affected low-threshold cold thermosensors. Whole-cell electrophysiology experiments using DRG cells, channels expressed in Xenopus oocytes, and human cell lines revealed that DIMTA blocks several potassium channels, reducing the magnitude of the afterhyperpolarization and increasing the baseline intracellular calcium concentration [Ca2+]i of low-threshold cold thermosensors. When injected into mice, DIMTA increased the threshold of cold sensation by >3 °C. DIMTA may thus serve as a lead in the further design of compounds that inhibit problems in the cold-sensory system, such as cold allodynia and other neuropathic pain conditions.
Assuntos
Aminas/metabolismo , Canais de Cálcio/metabolismo , Células Receptoras Sensoriais/metabolismo , Aminas/administração & dosagem , Animais , Cálcio/metabolismo , Gânglios Espinais/metabolismo , Masculino , Camundongos , Técnicas de Patch-Clamp , Transdução de Sinais , Sensação Térmica/fisiologia , Urocordados , VertebradosRESUMO
Marine sponges are prolific sources of bioactive natural products, several of which are produced by bacteria symbiotically associated with the sponge host. Bacteria-derived natural products, and the specialized bacterial symbionts that synthesize them, are not shared among phylogenetically distant sponge hosts. This is in contrast to nonsymbiotic culturable bacteria in which the conservation of natural products and natural product biosynthetic gene clusters (BGCs) is well established. Here, we demonstrate the widespread conservation of a BGC encoding a cryptic ribosomally synthesized and post-translationally modified peptide (RiPP) in microbiomes of phylogenetically and geographically dispersed sponges from the Pacific and Atlantic oceans. Detection of this BGC was enabled by mining for halogenating enzymes in sponge metagenomes, which, in turn, allowed for the description of a broad-spectrum regiospecific peptidyl tryptophan-6-brominase which possessed no chlorination activity. In addition, we demonstrate the cyclodehydrative installation of azoline heterocycles in proteusin RiPPs. This is the first demonstration of halogenation and cyclodehydration for proteusin RiPPs and the enzymes catalyzing these transformations were found to competently interact with other previously described proteusin substrate peptides. Within a sponge microbiome, many different generalized bacterial taxa harbored this BGC with often more than 50 copies of the BGC detected in individual sponge metagenomes. Moreover, the BGC was found in all sponges queried that possess high diversity microbiomes but it was not detected in other marine invertebrate microbiomes. These data shed light on conservation of cryptic natural product biosynthetic potential in marine sponges that was not detected by traditional natural product-to-BGC (meta)genome mining.