ABSTRACT
All organisms produce specialized organic molecules, ranging from small volatile chemicals to large gene-encoded peptides, that have evolved to provide them with diverse cellular and ecological functions. As natural products, they are broadly applied in medicine, agriculture and nutrition. The rapid accumulation of genomic information has revealed that the metabolic capacity of virtually all organisms is vastly underappreciated. Pioneered mainly in bacteria and fungi, genome mining technologies are accelerating metabolite discovery. Recent efforts are now being expanded to all life forms, including protists, plants and animals, and new integrative omics technologies are enabling the increasingly effective mining of this molecular diversity.
Subject(s)
Bacteria/genetics , Biological Products/chemistry , Drug Discovery/methods , Fungi/genetics , Genome , Genomics/methods , Plants/genetics , Animals , Biological Products/metabolism , Biological Products/therapeutic use , Computational Biology/methods , HumansABSTRACT
Sea sponges are the largest marine source of small-molecule natural products described to date. Sponge-derived molecules, such as the chemotherapeutic eribulin, the calcium-channel blocker manoalide, and antimalarial compound kalihinol A, are renowned for their impressive medicinal, chemical, and biological properties. Sponges contain microbiomes that control the production of many natural products isolated from these marine invertebrates. In fact, all genomic studies to date investigating the metabolic origins of sponge-derived small molecules concluded that microbes-not the sponge animal host-are the biosynthetic producers. However, early cell-sorting studies suggested the sponge animal host may play a role particularly in the production of terpenoid molecules. To investigate the genetic underpinnings of sponge terpenoid biosynthesis, we sequenced the metagenome and transcriptome of an isonitrile sesquiterpenoid-containing sponge of the order Bubarida. Using bioinformatic searches and biochemical validation, we identified a group of type I terpene synthases (TSs) from this sponge and multiple other species, the first of this enzyme class characterized from the sponge holobiome. The Bubarida TS-associated contigs consist of intron-containing genes homologous to sponge genes and feature GC percentage and coverage consistent with other eukaryotic sequences. We identified and characterized TS homologs from five different sponge species isolated from geographically distant locations, thereby suggesting a broad distribution amongst sponges. This work sheds light on the role of sponges in secondary metabolite production and speaks to the possibility that other sponge-specific molecules originate from the animal host.
Subject(s)
Biological Products , Microbiota , Porifera , Animals , Porifera/genetics , Aquatic Organisms/genetics , Microbiota/genetics , Metagenome , PhylogenyABSTRACT
Domoic acid (DA), the causative agent of amnesic shellfish poisoning, is produced by select organisms within two distantly related algal clades: planktonic diatoms and red macroalgae. The biosynthetic pathway to isodomoic acid A was recently solved in the harmful algal bloom-forming diatom Pseudonitzschia multiseries, establishing the genetic basis for the global production of this potent neurotoxin. Herein, we sequenced the 507-Mb genome of Chondria armata, the red macroalgal seaweed from which DA was first isolated in the 1950s, identifying several copies of the red algal DA (rad) biosynthetic gene cluster. The rad genes are organized similarly to the diatom DA biosynthesis cluster in terms of gene synteny, including a cytochrome P450 (CYP450) enzyme critical to DA production that is notably absent in red algae that produce the simpler kainoid neurochemical, kainic acid. The biochemical characterization of the N-prenyltransferase (RadA) and kainoid synthase (RadC) enzymes support a slightly altered DA biosynthetic model in C. armata via the congener isodomoic acid B, with RadC behaving more like the homologous diatom enzyme despite higher amino acid similarity to red algal kainic acid synthesis enzymes. A phylogenetic analysis of the rad genes suggests unique origins for the red macroalgal and diatom genes in their respective hosts, with native eukaryotic CYP450 neofunctionalization combining with the horizontal gene transfer of N-prenyltransferases and kainoid synthases to establish DA production within the algal lineages.
Subject(s)
Dimethylallyltranstransferase/genetics , Dimethylallyltranstransferase/metabolism , Kainic Acid/analogs & derivatives , Neurotoxins/metabolism , Rhodophyta/metabolism , Biological Evolution , Biosynthetic Pathways/genetics , Diatoms/genetics , Diatoms/metabolism , Harmful Algal Bloom/physiology , Kainic Acid/metabolism , Multigene Family/genetics , Neurotoxins/genetics , Phylogeny , Shellfish Poisoning/metabolismABSTRACT
Covering: 1997 to 2023The shikimate pathway is the metabolic process responsible for the biosynthesis of the aromatic amino acids phenylalanine, tyrosine, and tryptophan. Seven metabolic steps convert phosphoenolpyruvate (PEP) and erythrose 4-phosphate (E4P) into shikimate and ultimately chorismate, which serves as the branch point for dedicated aromatic amino acid biosynthesis. Bacteria, fungi, algae, and plants (yet not animals) biosynthesize chorismate and exploit its intermediates in their specialized metabolism. This review highlights the metabolic diversity derived from intermediates of the shikimate pathway along the seven steps from PEP and E4P to chorismate, as well as additional sections on compounds derived from prephenate, anthranilate and the synonymous aminoshikimate pathway. We discuss the genomic basis and biochemical support leading to shikimate-derived antibiotics, lipids, pigments, cofactors, and other metabolites across the tree of life.
Subject(s)
Cyclohexanecarboxylic Acids , Cyclohexenes , Shikimic Acid , Shikimic Acid/analogs & derivatives , Shikimic Acid/metabolism , Molecular Structure , Chorismic Acid/metabolism , Phosphoenolpyruvate/metabolism , Sugar Phosphates/metabolism , Bacteria/metabolism , Fungi/metabolism , Plants/metabolismABSTRACT
Octocorals are major contributors of terpenoid chemical diversity in the ocean. Natural products from other sessile marine animals are primarily biosynthesized by symbiotic microbes rather than by the host. Here, we challenge this long-standing paradigm by describing a monophyletic lineage of animal-encoded terpene cyclases (TCs) ubiquitous in octocorals. We characterized 15 TC enzymes from nine genera, several of which produce precursors of iconic coral-specific terpenoids, such as pseudopterosin, lophotoxin and eleutherobin. X-ray crystallography revealed that coral TCs share conserved active site residues and structural features with bacterial TCs. The identification of coral TCs enabled the targeted identification of the enzyme that constructs the coral-exclusive capnellane scaffold. Several TC genes are colocalized with genes that encode enzymes known to modify terpenes. This work presents an example of biosynthetic capacity in the kingdom Animalia that rivals the chemical complexity generated by plants, unlocking the biotechnological potential of octocorals for biomedical applications.
Subject(s)
Anthozoa , Animals , Bacteria/genetics , Terpenes/chemistryABSTRACT
The marine microbial natural product salinosporamide A (marizomib) is a potent proteasome inhibitor currently in clinical trials for the treatment of brain cancer. Salinosporamide A is characterized by a complex and densely functionalized γ-lactam-ß-lactone bicyclic warhead, the assembly of which has long remained a biosynthetic mystery. Here, we report an enzymatic route to the salinosporamide core catalyzed by a standalone ketosynthase (KS), SalC. Chemoenzymatic synthesis of carrier protein-tethered substrates, as well as intact proteomics, allowed us to probe the reactivity of SalC and understand its role as an intramolecular aldolase/ß-lactone synthase with roles in both transacylation and bond-forming reactions. Additionally, we present the 2.85-Å SalC crystal structure that, combined with site-directed mutagenesis, allowed us to propose a bicyclization reaction mechanism. This work challenges our current understanding of the role of KS enzymes and establishes a basis for future efforts toward streamlined production of a clinically relevant chemotherapeutic.
Subject(s)
Biological Products , Lactams , Biological Products/pharmacology , Lactones/chemistry , Proteasome Inhibitors , Pyrroles/pharmacologyABSTRACT
The Natural Product Domain Seeker (NaPDoS) webtool detects and classifies ketosynthase (KS) and condensation domains from genomic, metagenomic, and amplicon sequence data. Unlike other tools, a phylogeny-based classification scheme is used to make broader predictions about the polyketide synthase (PKS) and nonribosomal peptide synthetase (NRPS) genes in which these domains are found. NaPDoS is particularly useful for the analysis of incomplete biosynthetic genes or gene clusters, as are often observed in poorly assembled genomes and metagenomes, or when loci are not clustered, as in eukaryotic genomes. To help support the growing interest in sequence-based analyses of natural product biosynthetic diversity, here we introduce version 2 of the webtool, NaPDoS2, available at http://napdos.ucsd.edu/napdos2. This update includes the addition of 1417 KS sequences, representing a major expansion of the taxonomic and functional diversity represented in the webtool database. The phylogeny-based KS classification scheme now recognizes 41 class and subclass assignments, including new type II PKS subclasses. Workflow modifications accelerate run times, allowing larger datasets to be analyzed. In addition, default parameters were established using statistical validation tests to maximize KS detection and classification accuracy while minimizing false positives. We further demonstrate the applications of NaPDoS2 to assess PKS biosynthetic potential using genomic, metagenomic, and PCR amplicon datasets. These examples illustrate how NaPDoS2 can be used to predict biosynthetic potential and detect genes involved in the biosynthesis of specific structure classes or new biosynthetic mechanisms.
Subject(s)
Biological Products , Polyketide Synthases , Software , Genome , Metagenomics/methods , Peptide Synthases/genetics , Peptide Synthases/chemistry , Phylogeny , Polyketide Synthases/genetics , Polyketide Synthases/chemistry , Web BrowserABSTRACT
The biosynthetic installation of halogen atoms is largely performed by oxidative halogenases that target a wide array of electron-rich substrates, including aromatic compounds and conjugated systems. Halogenated alkyne-containing molecules are known to occur in Nature; however, halogen atom installation on the terminus of an alkyne has not been demonstrated in enzyme catalysis. Herein, we report the discovery and characterization of an alkynyl halogenase in natural product biosynthesis. We show that the flavin-dependent halogenase from the jamaicamide biosynthetic pathway, JamD, is not only capable of terminal alkyne halogenation on a late-stage intermediate en route to the final natural product but also has broad substrate tolerance for simple to complex alkynes. Furthermore, JamD is specific for terminal alkynes over other electron-rich aromatic substrates and belongs to a newly identified family of halogenases from marine cyanobacteria, indicating its potential as a chemoselective biocatalyst for the formation of haloalkynes.
Subject(s)
Biological Products , Halogenation , Halogens , Alkynes , CatalysisABSTRACT
The logical and effective discovery of macrolactams, structurally unique natural molecules with diverse biological activities, has been limited by a lack of targeted search methods. Herein, a targeted discovery method for natural macrolactams was devised by coupling genomic signature-based PCR screening of a bacterial DNA library with spectroscopic signature-based early identification of macrolactams. DNA library screening facilitated the efficient selection of 43 potential macrolactam-producing strains (3.6% of 1,188 strains screened). The PCR amplicons of the amine-deprotecting enzyme-coding genes were analyzed to predict the macrolactam type (α-methyl, α-alkyl, or ß-methyl) produced by the hit strains. 1H-15N HSQC-TOCSY NMR analysis of 15N-labeled culture extracts enabled macrolactam detection and structural type assignment without any purification steps. This method identified a high-titer Micromonospora strain producing salinilactam (1), a previously reported α-methyl macrolactam, and two Streptomyces strains producing new α-alkyl and ß-methyl macrolactams. Subsequent purification and spectroscopic analysis led to the structural revision of 1 and the discovery of muanlactam (2), an α-alkyl macrolactam with diene amide and tetraene chromophores, and concolactam (3), a ß-methyl macrolactam with a [16,6,6]-tricyclic skeleton. Detailed genomic analysis of the strains producing 1-3 identified putative biosynthetic gene clusters and pathways. Compound 2 displayed significant cytotoxicity against various cancer cell lines (IC50 = 1.58 µM against HCT116), whereas 3 showed inhibitory activity against Staphylococcus aureus sortase A. This genomic and spectroscopic signature-based method provides an efficient search strategy for new natural macrolactams and will be generally applicable for the discovery of nitrogen-bearing natural products.
Subject(s)
Streptomyces , Molecular Structure , Lactams, Macrocyclic/pharmacology , Lactams, Macrocyclic/chemistry , Streptomyces/metabolism , Genomics , Polymerase Chain Reaction , Multigene FamilyABSTRACT
Isonitrile-containing natural products have garnered attention for their manifold bioactivities but are difficult to detect and isolate due to the chemical lability of the isonitrile functional group. Here, we used the isonitrile-chlorooxime ligation (INC) in a reactivity-based screening (RBS) protocol for the detection and isolation of alkaloid and terpene isonitriles in the cyanobacterium Fischerella ambigua and a marine sponge of the order Bubarida, respectively. A trifunctional probe bearing a chlorooxime moiety, a UV active aromatic moiety, and a bromine label facilitated the chemoselective reaction with isonitriles, UV-Vis spectroscopic detection, and mass spectrometric analysis. The INC-based RBS allowed for the detection, isolation, and structural elucidation of isonitriles in microgram quantities.
Subject(s)
Alkaloids , Biological Products , Porifera , Animals , Biological Products/chemistryABSTRACT
Multidomain enzymes orchestrate two or more catalytic activities to carry out metabolic transformations with increased control and speed. Here, we report the design and development of a genome-mining approach for targeted discovery of biochemical transformations through the analysis of co-occurring enzyme domains (CO-ED) in a single protein. CO-ED was designed to identify unannotated multifunctional enzymes for functional characterization and discovery based on the premise that linked enzyme domains have evolved to function collaboratively. Guided by CO-ED, we targeted an unannotated predicted ThiF-nitroreductase di-domain enzyme found in more than 50 proteobacteria. Through heterologous expression and biochemical reconstitution, we discovered a series of natural products containing the rare oxazolone heterocycle and characterized their biosynthesis. Notably, we identified the di-domain enzyme as an oxazolone synthetase, validating CO-ED-guided genome mining as a methodology with potential broad utility for both the discovery of unusual enzymatic transformations and the functional annotation of multidomain enzymes.
Subject(s)
Oxazolone/metabolism , Peptide Synthases/metabolism , Molecular Structure , Oxazolone/chemistry , Peptide Synthases/chemistryABSTRACT
Prenylation is a common biological reaction in all domains of life wherein prenyl diphosphate donors transfer prenyl groups onto small molecules as well as large proteins. The enzymes that catalyze these reactions are structurally distinct from ubiquitous terpene cyclases that, instead, assemble terpenes via intramolecular rearrangements of a single substrate. Herein, we report the structure and molecular details of a new family of prenyltransferases from marine algae that repurposes the terpene cyclase structural fold for the N-prenylation of glutamic acid during the biosynthesis of the potent neurochemicals domoic acid and kainic acid. We solved the X-ray crystal structure of the prenyltransferase found in domoic acid biosynthesis, DabA, and show distinct active site binding modifications that remodel the canonical magnesium (Mg2+)-binding motif found in terpene cyclases. We then applied our structural knowledge of DabA and a homologous enzyme from the kainic acid biosynthetic pathway, KabA, to reengineer their isoprene donor specificities (geranyl diphosphate [GPP] versus dimethylallyl diphosphate [DMAPP]) with a single amino acid change. While diatom DabA and seaweed KabA enzymes share a common evolutionary lineage, they are distinct from all other terpene cyclases, suggesting a very distant ancestor to the larger terpene synthase family.
Subject(s)
Alkyl and Aryl Transferases/chemistry , Diatoms/enzymology , Dimethylallyltranstransferase/chemistry , Kainic Acid/analogs & derivatives , Neurotoxins/biosynthesis , Alkyl and Aryl Transferases/genetics , Alkyl and Aryl Transferases/metabolism , Amino Acid Substitution , Binding Sites , Diatoms/metabolism , Dimethylallyltranstransferase/genetics , Dimethylallyltranstransferase/metabolism , Glutamic Acid/metabolism , Kainic Acid/metabolism , Magnesium/metabolism , Prenylation , Protein BindingABSTRACT
Vanadium-dependent haloperoxidases (VHPOs) from Streptomyces bacteria differ from their counterparts in fungi, macroalgae, and other bacteria by catalyzing organohalogenating reactions with strict regiochemical and stereochemical control. While this group of enzymes collectively uses hydrogen peroxide to oxidize halides for incorporation into electron-rich organic molecules, the mechanism for the controlled transfer of highly reactive chloronium ions in the biosynthesis of napyradiomycin and merochlorin antibiotics sets the Streptomyces vanadium-dependent chloroperoxidases apart. Here we report high-resolution crystal structures of two homologous VHPO family members associated with napyradiomycin biosynthesis, NapH1 and NapH3, that catalyze distinctive chemical reactions in the construction of meroterpenoid natural products. The structures, combined with site-directed mutagenesis and intact protein mass spectrometry studies, afforded a mechanistic model for the asymmetric alkene and arene chlorination reactions catalyzed by NapH1 and the isomerase activity catalyzed by NapH3. A key lysine residue in NapH1 situated between the coordinated vanadate and the putative substrate binding pocket was shown to be essential for catalysis. This observation suggested the involvement of the ε-NH2, possibly through formation of a transient chloramine, as the chlorinating species much as proposed in structurally distinct flavin-dependent halogenases. Unexpectedly, NapH3 is modified post-translationally by phosphorylation of an active site His (τ-pHis) consistent with its repurposed halogenation-independent, α-hydroxyketone isomerase activity. These structural studies deepen our understanding of the mechanistic underpinnings of VHPO enzymes and their evolution as enantioselective biocatalysts.
Subject(s)
Streptomyces , Vanadium , Anti-Bacterial Agents/chemistry , Catalysis , Isomerases , Vanadium/chemistryABSTRACT
Aetokthonotoxin (AETX) is a cyanobacterial neurotoxin that causes vacuolar myelinopathy, a neurological disease that is particularly deadly to bald eagles in the United States. The recently characterized AETX is structurally unique among cyanotoxins and is composed of a pentabrominated biindole nitrile. Herein we report the discovery of an efficient, five-enzyme biosynthetic pathway that the freshwater cyanobacterium Aetokthonos hydrillicola uses to convert two molecules of tryptophan to AETX. We demonstrate that the biosynthetic pathway follows a convergent route in which two functionalized indole monomers are assembled and then reunited by biaryl coupling catalyzed by the cytochrome P450 AetB. Our results revealed enzymes with novel biochemical functions, including the single-component flavin-dependent tryptophan halogenase AetF and the iron-dependent nitrile synthase AetD.
Subject(s)
Indoles , Neurotoxins , Nitriles , Cyanobacteria/genetics , Cyanobacteria/metabolism , Cytochrome P-450 Enzyme System/metabolism , Indoles/metabolism , Multigene Family , Neurotoxins/biosynthesis , Nitriles/metabolism , Oxidoreductases/metabolism , Tryptophan/metabolism , Tryptophanase/metabolismABSTRACT
Harmful cyanobacterial blooms (cyanoHABs) cause recurrent toxic events in global watersheds. Although public health agencies monitor the causal toxins of most cyanoHABs and scientists in the field continue developing precise detection and prediction tools, the potent anticholinesterase neurotoxin, guanitoxin, is not presently environmentally monitored. This is largely due to its incompatibility with widely employed analytical methods and instability in the environment, despite guanitoxin being among the most lethal cyanotoxins. Here, we describe the guanitoxin biosynthesis gene cluster and its rigorously characterized nine-step metabolic pathway from l-arginine in the cyanobacterium Sphaerospermopsis torques-reginae ITEP-024. Through environmental sequencing data sets, guanitoxin (gnt) biosynthetic genes are repeatedly detected and expressed in municipal freshwater bodies that have undergone past toxic events. Knowledge of the genetic basis of guanitoxin biosynthesis now allows for environmental, biosynthetic gene monitoring to establish the global scope of this neurotoxic organophosphate.
Subject(s)
Cyanobacteria , Cyanobacteria/genetics , Cyanobacteria/metabolism , Cyanobacteria Toxins , Environmental Monitoring , Fresh Water , Multigene FamilyABSTRACT
Modular nonribosomal peptide synthetase (NRPS) and polyketide synthase (PKS) enzymatic assembly lines are large and dynamic protein machines that generally effect a linear sequence of catalytic cycles. Here, we report the heterologous reconstitution and comprehensive characterization of two hybrid NRPS-PKS assembly lines that defy many standard rules of assembly line biosynthesis to generate a large combinatorial library of cyclic lipodepsipeptide protease inhibitors called thalassospiramides. We generate a series of precise domain-inactivating mutations in thalassospiramide assembly lines, and present evidence for an unprecedented biosynthetic model that invokes intermodule substrate activation and tailoring, module skipping and pass-back chain extension, whereby the ability to pass the growing chain back to a preceding module is flexible and substrate driven. Expanding bidirectional intermodule domain interactions could represent a viable mechanism for generating chemical diversity without increasing the size of biosynthetic assembly lines and challenges our understanding of the potential elasticity of multimodular megaenzymes.
Subject(s)
Multigene Family , Peptide Synthases/metabolism , Peptides, Cyclic/biosynthesis , Catalysis , Chromatography, Liquid , Cloning, Molecular , Elasticity , Gene Deletion , Genetic Complementation Test , Mass Spectrometry , Mutation , Polyketide Synthases/metabolism , Protein Domains , Proteobacteria/enzymology , Substrate SpecificityABSTRACT
The implementation of ortho-quinone methide (o-QM) intermediates in complex molecule assembly represents a remarkably efficient strategy designed by Nature and utilized by synthetic chemists. o-QMs have been taken advantage of in biomimetic syntheses for decades, yet relatively few examples of o-QM-generating enzymes in natural product biosynthetic pathways have been reported. The biosynthetic enzymes that have been discovered thus far exhibit tremendous potential for biocatalytic applications, enabling the selective production of desirable compounds that are otherwise intractable or inherently difficult to achieve by traditional synthetic methods. Characterization of this biosynthetic machinery has the potential to shine a light on new enzymes capable of similar chemistry on diverse substrates, thus expanding our knowledge of Nature's catalytic repertoire. The presently known o-QM-generating enzymes include flavin-dependent oxidases, hetero-Diels-Alderases, S-adenosyl-l-methionine-dependent pericyclases, and α-ketoglutarate-dependent nonheme iron enzymes. In this review, we discuss their diverse enzymatic mechanisms and potential as biocatalysts in constructing natural product molecules such as cannabinoids.
Subject(s)
Biological Products , Indolequinones , Biocatalysis , Catalysis , Indolequinones/chemistryABSTRACT
Ortho-quinone methides (o-QMs) are reactive intermediates in biosynthesis that give rise to a variety of intra- and intermolecular cyclization/addition products in bacteria, fungi, and plants. Herein, we report a new metabolic deviation of an o-QM intermediate in a benzylic dehydrogenation reaction that links the newly described marine bacterial natural products dihydrotetrachlorizine and tetrachlorizine. We discovered these novel dichloropyrrole-containing compounds from actinomycete strain AJS-327 that unexpectedly harbors in its genome a biosynthetic gene cluster (BGC) of striking similarity to that of chlorizidine, another marine alkaloid bearing a different carbon skeleton. Heterologous expression of the homologous flavin-dependent oxidoreductase enzymes Tcz9 and Clz9 revealed their native functions in tetrachlorizine and chlorizidine biosynthesis, respectively, supporting divergent oxidative dehydrogenation and pyrrolizine-forming reactions. Swapping these berberine bridge enzyme-like oxidoreductases, we produced cyclized and dehydrogenated analogs of tetrachlorizine and chlorizidine, including a dearomatized chlorizidine analog that stabilizes an o-QM via conjugation with a 3H-pyrrolizine ring.
Subject(s)
Indolequinones/chemistry , Actinobacteria/genetics , Actinobacteria/metabolism , Cyclization , Genes, Fungal , Hydrogen/chemistry , Oxidation-ReductionABSTRACT
Covering: Up to December 2020Enzymatic halogenation reactions are essential for the production of thousands of halogenated natural products. However, in recent years, scientists discovered several halogenases that transiently incorporate halogen atoms in intermediate biosynthetic molecules to activate them for further chemical reactions such as cyclopropanation, terminal alkyne formation, C-/O-alkylation, biaryl coupling, and C-C rearrangements. In each case, the halogen atom is lost in the course of biosynthesis to the final product and is hence termed "cryptic". In this review, we provide an overview of our current knowledge of cryptic halogenation reactions in natural product biosynthesis.
Subject(s)
Biological Products/metabolism , Biological Products/chemistry , HalogenationABSTRACT
Covering: 2016 to 2021With genetic information available for hundreds of thousands of organisms in publicly accessible databases, scientists have an unprecedented opportunity to meticulously survey the diversity and inner workings of life. The natural product research community has harnessed this breadth of sequence information to mine microbes, plants, and animals for biosynthetic enzymes capable of producing bioactive compounds. Several orthogonal genome mining strategies have been developed in recent years to target specific chemical features or biological properties of bioactive molecules using biosynthetic, resistance, or transporter proteins. These "biosynthetic hooks" allow researchers to query for biosynthetic gene clusters with a high probability of encoding previously undiscovered, bioactive compounds. This review highlights recent case studies that feature orthogonal approaches that exploit genomic information to specifically discover bioactive natural products and their gene clusters.