A Natural Product Chemist's Guide to Unlocking Silent Biosynthetic Gene Clusters.

Microbial natural products have provided an important source of therapeutic leads and motivated research and innovation in diverse scientific disciplines. In recent years, it has become evident that bacteria harbor a large, hidden reservoir of potential natural products in the form of silent or cryptic biosynthetic gene clusters (BGCs). These can be readily identified in microbial genome sequences but do not give rise to detectable levels of a natural product. Herein, we provide a useful organizational framework for the various methods that have been implemented for interrogating silent BGCs. We divide all available approaches into four categories. The first three are endogenous strategies that utilize the native host in conjunction with classical genetics, chemical genetics, or different culture modalities. The last category comprises expression of the entire BGC in a heterologous host. For each category, we describe the rationale, recent applications, and associated advantages and limitations.

Biosynthesis of selenium-containing small molecules in diverse microorganisms.

Selenium is an essential micronutrient in diverse organisms. Two routes are known for its insertion into proteins and nucleic acids, via selenocysteine and 2-selenouridine, respectively1. However, despite its importance, pathways for specific incorporation of selenium into small molecules have remained elusive. Here we use a genome-mining strategy in various microorganisms to uncover a widespread three-gene cluster that encodes a dedicated pathway for producing selenoneine, the selenium analogue of the multifunctional molecule ergothioneine2,3. We elucidate the reactions of all three proteins and uncover two novel selenium-carbon bond-forming enzymes and the biosynthetic pathway for production of a selenosugar, which is an unexpected intermediate en route to the final product. Our findings expand the scope of biological selenium utilization, suggest that the selenometabolome is more diverse than previously thought, and set the stage for the discovery of other selenium-containing natural products.

The Small-Molecule Language of Dynamic Microbial Interactions.

Although microbes are routinely grown in monocultures in the laboratory, they are almost never encountered as single species in the wild. Our ability to detect and identify new microorganisms has advanced significantly in recent years, but our understanding of the mechanisms that mediate microbial interactions has lagged behind. What makes this task more challenging is that microbial alliances can be dynamic, consisting of multiple phases. The transitions between phases, and the interactions in general, are often mediated by a chemical language consisting of small molecules, also referred to as secondary metabolites or natural products. In this microbial lexicon, the molecules are like words and through their effects on recipient cells they convey meaning. The current review highlights three dynamic microbial interactions in which some of the words and their meanings have been characterized, especially those that mediate transitions in selected multiphasic associations. These systems provide insights into the principles that govern microbial symbioses and a playbook for interrogating similar associations in diverse ecological niches.

Potent and specific antibiotic combination therapy against Clostridioides difficile.

Keratinicyclins and keratinimicins are recently discovered glycopeptide antibiotics. Keratinimicins show broad-spectrum activity against Gram-positive bacteria, while keratinicyclins form a new chemotype by virtue of an unusual oxazolidinone moiety and exhibit specific antibiosis against Clostridioides difficile. Here we report the mechanism of action of keratinicyclin B (KCB). We find that steric constraints preclude KCB from binding peptidoglycan termini. Instead, KCB inhibits C. difficile growth by binding wall teichoic acids (WTAs) and interfering with cell wall remodeling. A computational model, guided by biochemical studies, provides an image of the interaction of KCB with C. difficile WTAs and shows that the same H-bonding framework used by glycopeptide antibiotics to bind peptidoglycan termini is used by KCB for interacting with WTAs. Analysis of KCB in combination with vancomycin (VAN) shows highly synergistic and specific antimicrobial activity, and that nanomolar combinations of the two drugs are sufficient for complete growth inhibition of C. difficile, while leaving common commensal strains unaffected.

Detection of a Kinetically Competent Compound-I Intermediate in the Vancomycin Biosynthetic Enzyme OxyB.

Cytochrome P450 enzymes are abundantly encoded in microbial genomes. Their reactions have two general outcomes, one involving oxygen insertion via a canonical "oxygen rebound" mechanism and a second that diverts from this pathway and leads to a wide array of products, notably intramolecular oxidative cross-links. The antibiotic of-last-resort, vancomycin, contains three such cross-links, which are crucial for biological activity and are installed by the P450 enzymes OxyB, OxyA, and OxyC. The mechanisms of these enzymes have remained elusive in part because of the difficulty in spectroscopically capturing transient intermediates. Using stopped-flow UV/visible absorption and rapid freeze-quench electron paramagnetic resonance spectroscopies, we show that OxyB generates the highly reactive compound-I intermediate, which can react with a model vancomycin peptide substrate in a kinetically competent fashion to generate product. Our results have implications for the mechanism of OxyB and are in line with the notion that oxygen rebound and oxidative cross-links share early steps in their catalytic cycles.

Expanding the Landscape of Noncanonical Amino Acids in RiPP Biosynthesis.

Advancements in DNA sequencing technologies and bioinformatics have enabled the discovery of new metabolic reactions from overlooked microbial species and metagenomic sequences. Using a bioinformatic co-occurrence strategy, we previously generated a network of ∼600 uncharacterized quorum-sensing-regulated biosynthetic gene clusters that code for ribosomally synthesized and post-translationally modified peptide (RiPP) natural products and are tailored by radical S-adenosylmethionine (RaS) enzymes in streptococci. The most complex of these is the GRC subfamily, named after a conserved motif in the precursor peptide and found exclusively in Streptococcus pneumoniae, the causative agent of bacterial pneumonia. In this study, using both in vivo and in vitro approaches, we have elucidated the modifications installed by the grc biosynthetic enzymes, including a ThiF-like adenylyltransferase/cyclase that generates a C-terminal Glu-to-Cys thiolactone macrocycle, and two RaS enzymes, which selectively epimerize the ß-carbon of threonine and desaturate histidine to generate the first instances of l-allo-Thr and didehydrohistidine in RiPP biosynthesis. RaS-RiPPs that have been discovered thus far have stood out for their exotic macrocycles. The product of the grc cluster breaks this trend by generating two noncanonical residues rather than an unusual macrocycle in the peptide substrate. These modifications expand the landscape of nonproteinogenic amino acids in RiPP natural product biosynthesis and motivate downstream biocatalytic applications of the corresponding enzymes.

N-Cα Bond Cleavage Catalyzed by a Multinuclear Iron Oxygenase from a Divergent Methanobactin-like RiPP Gene Cluster.

DUF692 multinuclear iron oxygenases (MNIOs) are an emerging family of tailoring enzymes involved in the biosynthesis of ribosomally synthesized and post-translationally modified peptides (RiPPs). Three members, MbnB, TglH, and ChrH, have been characterized to date and shown to catalyze unusual and complex transformations. Using a co-occurrence-based bioinformatic search strategy, we recently generated a sequence similarity network of MNIO-RiPP operons that encode one or more MNIOs adjacent to a transporter. The network revealed >1000 unique gene clusters, evidence of an unexplored biosynthetic landscape. Herein, we assess an MNIO-RiPP cluster from this network that is encoded in Proteobacteria and Actinobacteria. The cluster, which we have termed mov (for methanobactin-like operon in Vibrio), encodes a 23-residue precursor peptide, two MNIOs, a RiPP recognition element, and a transporter. Using both in vivo and in vitro methods, we show that one MNIO, homologous to MbnB, installs an oxazolone-thioamide at a Thr-Cys dyad in the precursor. Subsequently, the second MNIO catalyzes N-Cα bond cleavage of the penultimate Asn to generate a C-terminally amidated peptide. This transformation expands the reaction scope of the enzyme family, marks the first example of an MNIO-catalyzed modification that does not involve Cys, and sets the stage for future exploration of other MNIO-RiPPs.

Bicyclostreptins are radical SAM enzyme-modified peptides with unique cyclization motifs.

Microbial natural products comprise diverse architectures that are generated by equally diverse biosynthetic strategies. In peptide natural products, amino acid sidechains are frequently used as sites of modification to generate macrocyclic motifs. Backbone amide groups, among the most stable of biological moieties, are rarely used for this purpose. Here we report the discovery and biosynthesis of bicyclostreptins-peptide natural products from Streptococcus spp. with an unprecedented structural motif consisting of a macrocyclic ß-ether and a heterocyclic sp3-sp3 linkage between a backbone amide nitrogen and an adjacent α-carbon. Both reactions are installed, in that order, by two radical S-adenosylmethionine (RaS) metalloenzymes. Bicyclostreptins are produced at nM concentrations and are potent growth regulation agents in Streptococcus thermophilus. Our results add a distinct and unusual chemotype to the growing family of ribosomal peptide natural products, expand the already impressive catalytic scope of RaS enzymes, and provide avenues for further biological studies in human-associated streptococci.

Piperacillin triggers virulence factor biosynthesis via the oxidative stress response in Burkholderia thailandensis.

Natural products have been an important source of therapeutic agents and chemical tools. The recent realization that many natural product biosynthetic genes are silent or sparingly expressed during standard laboratory growth has prompted efforts to investigate their regulation and develop methods to induce their expression. Because it is difficult to intuit signals that induce a given biosynthetic locus, we recently implemented a forward chemical-genetic approach to identify such inducers. In the current work, we applied this approach to nine silent biosynthetic loci in the model bacterium Burkholderia thailandensis to systematically screen for elicitors from a library of Food and Drug Administration-approved drugs. We find that ß-lactams, fluoroquinolones, antifungals, and, surprisingly, calcimimetics, phenothiazine antipsychotics, and polyaromatic antidepressants are the most effective global inducers of biosynthetic genes. Investigations into the mechanism of stimulation of the silent virulence factor malleicyprol by the ß-lactam piperacillin allowed us to elucidate the underlying regulatory circuits. Low-dose piperacillin causes oxidative stress, thereby inducing redox-sensing transcriptional regulators, which activate malR, a pathway-specific positive regulator of the malleicyprol gene cluster. Malleicyprol is thus part of the OxyR and SoxR regulons in B. thailandensis, allowing the bacterium to initiate virulence in response to oxidative stress. Our work catalogs a diverse array of elicitors and a previously unknown regulatory input for secondary metabolism in B. thailandensis.

Trapping a cross-linked lysine-tryptophan radical in the catalytic cycle of the radical SAM enzyme SuiB.

The radical S-adenosylmethionine (rSAM) enzyme SuiB catalyzes the formation of an unusual carbon-carbon bond between the sidechains of lysine (Lys) and tryptophan (Trp) in the biosynthesis of a ribosomal peptide natural product. Prior work on SuiB has suggested that the Lys-Trp cross-link is formed via radical electrophilic aromatic substitution (rEAS), in which an auxiliary [4Fe-4S] cluster (AuxI), bound in the SPASM domain of SuiB, carries out an essential oxidation reaction during turnover. Despite the prevalence of auxiliary clusters in over 165,000 rSAM enzymes, direct evidence for their catalytic role has not been reported. Here, we have used electron paramagnetic resonance (EPR) spectroscopy to dissect the SuiB mechanism. Our studies reveal substrate-dependent redox potential tuning of the AuxI cluster, constraining it to the oxidized [4Fe-4S]2+ state, which is active in catalysis. We further report the trapping and characterization of an unprecedented cross-linked Lys-Trp radical (Lys-Trp•) in addition to the organometallic Ω intermediate, providing compelling support for the proposed rEAS mechanism. Finally, we observe oxidation of the Lys-Trp• intermediate by the redox-tuned [4Fe-4S]2+ AuxI cluster by EPR spectroscopy. Our findings provide direct evidence for a role of a SPASM domain auxiliary cluster and consolidate rEAS as a mechanistic paradigm for rSAM enzyme-catalyzed carbon-carbon bond-forming reactions.

Hirocidins, Cytotoxic Metabolites from Streptomyces hiroshimensis, Induce Mitochondrion-mediated Apoptosis.

Recent advances in whole genome sequencing have revealed an immense microbial potential for the production of therapeutic small molecules, even from well-known producers. To access this potential, we subjected prominent antimicrobial producers to alternative antiproliferative assays using persistent cancer cell lines. Described herein is our discovery of hirocidins, novel secondary metabolites from Streptomyces hiroshimensis with antiproliferative activities against colon and persistent breast cancer cells. Hirocidin A is an unusual nine-membered carbocyclic maleimide and hirocidins B and C are relatives with an unprecedented, bridged azamacrocyclic backbone. Mode of action studies show that hirocidins trigger mitochondrion-dependent apoptosis by inducing expression of the key apoptotic effector caspase-9. The discovery of new cytotoxins contributes to scaffold diversification in anticancer drug discovery and the reported modes of action and concise total synthetic route for variant A set the stage for unraveling specific targets and biochemical interactions of the hirocidins.

Structural Characterization and Ligand-Induced Conformational Changes of SenB, a Se-Glycosyltransferase Involved in Selenoneine Biosynthesis.

Selenium (Se) is an essential micronutrient that is found naturally in proteins, nucleic acids, and natural products. Unlike selenoproteins and selenonucleic acids, little is known about the structures of biosynthetic enzymes that incorporate Se into small molecules. Here, we report the X-ray crystal structure of SenB, the first known Se-glycosyltransferase that was recently found to be involved in the biosynthesis of the Se-containing metabolite selenoneine. SenB catalyzes C-Se bond formation using selenophosphate and an activated uridine diphosphate sugar as a Se and glycosyl donor, respectively, making it the first known selenosugar synthase and one of only four bona fide C-Se bond-forming enzymes discovered to date. Our crystal structure, determined to 2.25 Å resolution, reveals that SenB is a type B glycosyltransferase, displaying the prototypical fold with two globular Rossmann-like domains and a catalytic interdomain cleft. By employing complementary structural biology techniques, we find that SenB undergoes both local and global substrate-induced conformational changes, demonstrating a significant increase in α-helicity and a transition to a more compact conformation. Our results provide the first structure of SenB and set the stage for further biochemical characterization in the future.

A microbial metabolite synergizes with endogenous serotonin to trigger C. elegans reproductive behavior.

Natural products are a major source of small-molecule therapeutics, including those that target the nervous system. We have used a simple serotonin-dependent behavior of the roundworm Caenorhabditis elegans, egg laying, to perform a behavior-based screen for natural products that affect serotonin signaling. Our screen yielded agonists of G protein-coupled serotonin receptors, protein kinase C agonists, and a microbial metabolite not previously known to interact with serotonin signaling pathways: the disulfide-bridged 2,5-diketopiperazine gliotoxin. Effects of gliotoxin on egg-laying behavior required the G protein-coupled serotonin receptors SER-1 and SER-7, and the Gq ortholog EGL-30. Furthermore, mutants lacking serotonergic neurons and mutants that cannot synthesize serotonin were profoundly resistant to gliotoxin. Exogenous serotonin restored their sensitivity to gliotoxin, indicating that this compound synergizes with endogenous serotonin to elicit behavior. These data show that a microbial metabolite with no structural similarity to known serotonergic agonists potentiates an endogenous serotonin signal to affect behavior. Based on this study, we suggest that microbial metabolites are a rich source of functionally novel neuroactive molecules.

Vitamin B3 Triggers Biosynthesis of Secondary Metabolite Dormancy Signals in Streptococcus suis.

Human-associated streptococci have not been viewed as productive sources of natural products. Against expectation, bioinformatic searches recently revealed a large collection of diverse biosynthetic gene clusters coding for ribosomally synthesized and post-translationally modified peptides (RiPPs) in streptococcal genomes. The most abundant of these, the tqq gene cluster, is specific to Streptococcus suis, a burdensome agricultural pathogen and zoonotic agent. Herein, we used high-throughput elicitor screening to identify both small molecule elicitors and products of the tqq cluster. We show that the B3 vitamin niacin effectively elicits the tqq cluster leading to the biosynthesis of a family of RiPP natural products, which we termed threoglucins and characterized structurally. The defining feature of threoglucins is an aliphatic ether bond giving rise to a substituted 1,3-oxazinane heterocycle in the peptide backbone. Isolation of 22 congeners of threoglucins facilitated structure activity relationship studies, demonstrating the requirement for the oxazinane substructure and a Trp-Tyr C-terminal dyad for biological activity, namely antibiotic persistence and allolysis at low and high doses, respectively. Potential therapeutic applications of threoglucins are discussed.

Bioinformatic Atlas of Radical SAM Enzyme-Modified RiPP Natural Products Reveals an Isoleucine-Tryptophan Crosslink.

Ribosomally synthesized and post-translationally modified peptides (RiPPs) are a growing family of natural products with diverse activities and structures. RiPP classes are defined by the tailoring enzyme, which can introduce a narrow range of modifications or a diverse set of alterations. In the latter category, RiPPs synthesized by radical S-adenosylmethionine (SAM) enzymes, known as RaS-RiPPs, have emerged as especially divergent. A map of all RaS-RiPP gene clusters does not yet exist. Moreover, precursor peptides remain difficult to predict using computational methods. Herein, we have addressed these challenges and report a bioinformatic atlas of RaS-RiPP gene clusters in available microbial genome sequences. Using co-occurrence of RaS enzymes and transporters from varied families as a bioinformatic hook in conjunction with an in-house code to identify precursor peptides, we generated a map of ∼15,500 RaS-RiPP gene clusters, which reveal a remarkable diversity of syntenies pointing to a tremendous range of enzymatic and natural product chemistries that remain to be explored. To assess its utility, we examined one family of gene clusters encoding a YcaO enzyme and a RaS enzyme. We find the former is noncanonical, contains an iron-sulfur cluster, and installs a novel modification, a backbone amidine into the precursor peptide. The RaS enzyme was also found to install a new modification, a C-C crosslink between the unactivated terminal δ-methyl group of Ile and a Trp side chain. The co-occurrence search can be applied to other families of RiPPs, as we demonstrate with the emerging DUF692 di-iron enzyme superfamily.

Cytotoxic alkyl-quinolones mediate surface-induced virulence in Pseudomonas aeruginosa.

Surface attachment, an early step in the colonization of multiple host environments, activates the virulence of the human pathogen P. aeruginosa. However, the downstream toxins that mediate surface-dependent P. aeruginosa virulence remain unclear, as do the signaling pathways that lead to their activation. Here, we demonstrate that alkyl-quinolone (AQ) secondary metabolites are rapidly induced upon surface association and act directly on host cells to cause cytotoxicity. Surface-induced AQ cytotoxicity is independent of other AQ functions like quorum sensing or PQS-specific activities like iron sequestration. We further show that packaging of AQs in outer-membrane vesicles (OMVs) increases their cytotoxicity to host cells but not their ability to stimulate downstream quorum sensing pathways in bacteria. OMVs lacking AQs are significantly less cytotoxic, suggesting these molecules play a role in OMV cytotoxicity, in addition to their previously characterized role in OMV biogenesis. AQ reporters also enabled us to dissect the signal transduction pathways downstream of the two known regulators of surface-dependent virulence, the quorum sensing receptor, LasR, and the putative mechanosensor, PilY1. Specifically, we show that PilY1 regulates surface-induced AQ production by repressing the AlgR-AlgZ two-component system. AlgR then induces RhlR, which can induce the AQ biosynthesis operon under specific conditions. These findings collectively suggest that the induction of AQs upon surface association is both necessary and sufficient to explain surface-induced P. aeruginosa virulence.

Induction of Diverse Cryptic Fungal Metabolites by Steroids and Channel Blockers.

Fungi offer a deep source of natural products but remain underutilized. Most biosynthetic gene clusters (BGCs) that can be detected are silent or "cryptic" in standard lab cultures and their products are thus not interrogated in routine screens. As genetic alterations are difficult and some strains can only be grown on agar, we have herein applied an agar-based high-throughput chemical genetic screen to identify inducers of fungal BGCs. Using R. solani and S. sclerotiorum as test cases, we report 13 cryptic metabolites in four compound groups, including sclerocyclane, a natural product with a novel scaffold. Steroids were the best elicitors and follow-up studies showed that plant-steroids trigger sclerocyclane synthesis, which shows antibiotic activity against B. plantarii, an ecological competitor of S. sclerotiorum. Our results open new paths to exploring the chemical ecology of fungal-plant interactions and provide a genetics-free approach for uncovering cryptic fungal metabolites.

Momomycin, an Antiproliferative Cryptic Metabolite from the Oxytetracycline Producer Streptomyces rimosus.

Natural products provide an important source of pharmaceuticals and chemical tools. Traditionally, assessment of unexplored microbial phyla has led to new natural products. However, with every new microbe, the number of orphan biosynthetic gene clusters (BGC) grows. As such, the more difficult proposition is finding new molecules from well-studied strains. Herein, we targeted Streptomyces rimosus, the widely-used oxytetracycline producer, for the discovery of new natural products. Using MALDI-MS-guided high-throughput elicitor screening (HiTES), we mapped the global secondary metabolome of S. rimosus and structurally characterized products of three cryptic BGCs, including momomycin, an unusual cyclic peptide natural product with backbone modifications and several non-canonical amino acids. We elucidated important aspects of its biosynthesis and evaluated its bioactivity. Our studies showcase HiTES as an effective approach for unearthing new chemical matter from "drained" strains.

Structural Elucidation of Cryptic Algaecides in Marine Algal-Bacterial Symbioses by NMR Spectroscopy and MicroED.

Microbial secondary metabolite discovery is often conducted in pure monocultures. In a natural setting, however, where metabolites are constantly exchanged, biosynthetic precursors are likely provided by symbionts or hosts. In the current work, we report eight novel and architecturally unusual secondary metabolites synthesized by the bacterial symbiont Phaeobacter inhibens from precursors that, in a native context, would be provided by their algal hosts. Three of these were produced at low titres and their structures were determined de novo using the emerging microcrystal electron diffraction method. Some of the new metabolites exhibited potent algaecidal activity suggesting that the bacterial symbiont can convert algal precursors, tryptophan and sinapic acid, into complex cytotoxins. Our results have important implications for the parasitic phase of algal-bacterial symbiotic interactions.

A genetics-free method for high-throughput discovery of cryptic microbial metabolites.

Bacteria contain an immense untapped trove of novel secondary metabolites in the form of 'silent' biosynthetic gene clusters (BGCs). These can be identified bioinformatically but are not expressed under normal laboratory growth conditions. Methods to access their products would dramatically expand the pool of bioactive compounds. We report a universal high-throughput method for activating silent BGCs in diverse microorganisms. Our approach relies on elicitor screening to induce the secondary metabolome of a given strain and imaging mass spectrometry to visualize the resulting metabolomes in response to ~500 conditions. Because it does not require challenging genetic, cloning, or culturing procedures, this method can be used with both sequenced and unsequenced bacteria. We demonstrate the power of the approach by applying it to diverse bacteria and report the discovery of nine cryptic metabolites with potentially therapeutic bioactivities, including a new glycopeptide chemotype with potent inhibitory activity against a pathogenic virus.