Search

Vanadium-dependent haloperoxidases (VHPOs) from

An important factor dictating coral fitness is the quality of bacteria associated with corals and coral reefs. One way that bacteria benefit corals is by stimulating the larval to juvenile life cycle transition of settlement and metamorphosis. Tetrabromopyrrole (TBP) is a small molecule produced by bacteria that stimulates metamorphosis in a range of coral species. A standing debate remains, however, about whether TBP biosynthesis from livePseudoalteromonasbacteria is the primary stimulant of coral metamorphosis. In this study, we create aPseudoalteromonassp. PS5 mutant lacking the TBP brominase gene,bmp2. Using this mutant, we confirm that thebmp2gene is critical for TBP biosynthesis inPseudoalteromonassp. PS5. Mutation of this gene ablates the bacterium’s ability in live cultures to stimulate the metamorphosis of the stony coralPorites astreoides. We further demonstrate that expression of TBP biosynthesis genes is strongest in stationary and biofilm modes of growth, wherePseudoalteromonassp. PS5 might exist within surface-attached biofilms on the sea floor. Finally, we create a modular transposon plasmid for genomic integration and fluorescent labeling ofPseudoalteromonassp. PS5 cells. Our results functionally link a TBP biosynthesis gene from live bacteria to a morphogenic effect in corals. The genetic techniques established here provide new tools to explore coral-bacteria interactions and could help to inform future decisions about utilizing marine bacteria or their products for restoring degraded coral reefs.

Cyclic arginine noncanonical amino acids (ncAAs) are found in several actinobacterial peptide natural products with therapeutically useful antibacterial properties. The preparation of ncAAs like enduracididine and capreomycidine currently takes multiple biosynthetic or chemosynthetic steps, thus limiting the commercial availability and applicability of these cyclic guanidine-containing amino acids. We recently discovered and characterized the biosynthetic pathway of guanitoxin, a potent freshwater cya-nobacterial neurotoxin, that contains an arginine-derived cyclic guanidine phosphate within its highly polar structure. The ncAA L-enduracididine is an early intermediate in guanitoxin biosynthesis and is produced by GntC, a unique pyridoxal-5’-phosphate (PLP)-dependent enzyme. GntC catalyzes a cyclodehydration from a stereoselectively γ-hydroxylated L-arginine precursor via a reaction that functionally and mechanistically diverges from previously established actinobacterial cyclic arginine ncAA pathways. Herein, we interrogate L-enduracididine biosynthesis from the cyanobacteriumSphaerospermopsis torques-reginaeITEP-024 using spectroscopic, stable isotope labeling techniques, and X-ray crystal structure-guided site-directed mutagenesis. GntC initially facilitates the reversible deprotonations of the α- and β-positions of its substrate prior to catalyzing an irreversible diastereoselective dehydration and subsequent intramolecular cyclization. The comparison ofholo-and substrate bound GntC structures and activity assays on sitespecific mutants further identified amino acid residues that contribute to the overall catalytic mechanism. These interdisciplinary efforts at structurally and functionally characterizing GntC enables an improved understanding of how Nature divergently produces cyclic arginine ncAAs and generates additional tools for their biocatalytic production and downstream biological applications.

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.

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-β-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/β-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.

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.

A conspicuous roadblock to studying marine bacteria for fundamental research and biotechnology is a lack of modular synthetic biology tools for their genetic manipulation. Here, we applied, and generated new parts for, a modular plasmid toolkit to study marine bacteria in the context of symbioses and host-microbe interactions. To demonstrate the utility of this plasmid system, we genetically manipulated the marine bacteriumPseudoalteromonas luteoviolacea, which stimulates the metamorphosis of the model tubeworm,Hydroides elegans. Using these tools, we quantified constitutive and native promoter expression, developed reporter strains that enable the imaging of host-bacteria interactions, and used CRISPR interference (CRISPRi) to knock down a secondary metabolite and a host-associated gene. We demonstrate the broader utility of this modular system for rapidly creating and iteratively testing genetic tractability by modifying marine bacteria that are known to be associated with diverse host-microbe symbioses. These efforts enabled the successful transformation of twelve marine strains across two Proteobacteria classes, four orders and ten genera. Altogether, the present study demonstrates how synthetic biology strategies enable the investigation of marine microbes and marine host-microbe symbioses with broader implications for environmental restoration and biotechnology.

Bromopyrroles (BrPyr) are synthesized naturally by marine sponge symbionts and produced anthropogenically as byproducts of wastewater treatment. BrPyr interact with ryanodine receptors (RYRs) and sarco/endoplasmic reticulum (SR/ER) Ca2+-ATPase (SERCA). Influences of BrPyr on the neuronal network activity remain uncharted. BrPyr analogues with differing spectra of RYR/SERCA activities were tested using RYR-null or RYR1-expressing HEK293 and murine cortical neuronal/glial cocultures (NGCs) loaded with Fluo-4 to elucidate their mechanisms altering Ca2+ dynamics. The NGC electrical spike activity (ESA) was measured from NGCs plated on multielectrode arrays. Nanomolar tetrabromopyrrole (TBP, 1) potentiated caffeine-triggered Ca2+ release independent of extracellular [Ca2+] in RYR1-HEK293, whereas higher concentrations produce slow and sustained rise in cytoplasmic [Ca2+] independent of RYR1 expression. TBP, 2,3,5-tribromopyrrole (2), pyrrole (3), 2,3,4-tribromopyrrole (4), and ethyl 4-bromopyrrole-2-carboxylate (5) added acutely to NGC showed differential potency; rank order TBP (IC50 ≈ 220 nM) > 2 ≫ 5, whereas 3 and 4 were inactive at 10 μM. TBP >2 μM elicited sustained elevation of cytoplasmic [Ca2+] and loss of neuronal viability. TBP did not alter network ESA. BrPyr from marine and anthropogenic sources are ecological signaling molecules and emerging anthropogenic pollutants of concern to environmental and human health that potently alter ER Ca2+ dynamics and warrant further investigation in vivo.

Aromatic amines in nature are typically installed with Glu or Gln as the nitrogen donor. Here we report a pathway that features glycyl-tRNA instead. During the biosynthesis of pyrroloiminoquinone-type natural products such as ammosamides, peptide-aminoacyl tRNA ligases append amino acids to the C-terminus of a ribosomally synthesized peptide. First, $${\mathrm{Amm}}{{{\mathrm{B}}}}_{{{\mathrm{C}}}}^{{{{\mathrm{Trp}}}}}$$ adds Trp in a Trp-tRNA-dependent reaction and the flavoprotein AmmC1 then carries out three hydroxylations of the indole ring of Trp. After oxidation to the corresponding ortho-hydroxy para-quinone, $${\mathrm{Amm}}{{{\mathrm{B}}}}_{{{\mathrm{D}}}}^{{{{\mathrm{Gly}}}}}$$ attaches Gly to the indole ring in a Gly-tRNA dependent fashion. Subsequent decarboxylation and hydrolysis results in an amino-substituted indole. Similar transformations are catalysed by orthologous enzymes from Bacillus halodurans. This pathway features three previously unknown biochemical processes using a ribosomally synthesized peptide as scaffold for non-ribosomal peptide extension and chemical modification to generate an amino acid-derived natural product. Biosynthesis of aromatic amines typically uses glutamic acid or glutamine as the nitrogen donor. Now, a biosynthetic pathway to aromatic amines has been reported that uses glycyl-tRNA as the nitrogen donor. The myosin-targeting ammosamide is made via a pathway that involves the post-translational modification of a tryptophan, which is added to the end of a peptide in a tryptophanyl-tRNA-dependent step.

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., Chemistry - A European Journal, 29 (6), ISSN:0947-6539, ISSN:1521-3765

Harmful algal blooms (HABs) of the toxic haptophytePrymnesium parvumare a recurrent problem in many inland and estuarine waters around the world. Strains ofP. parvumvary in the toxins they produce and in other physiological traits associated with HABs, but the genetic basis for this variation is unknown. To investigate genome diversity in this morphospecies, we generated genome assemblies for fifteen phylogenetically and geographically diverse strains ofP. parvumincluding Hi-C guided, near-chromosome level assemblies for two strains. Comparative analysis revealed considerable DNA content variation between strains, ranging from 115 Mbp to 845 Mbp. Strains included haploids, diploids, and polyploids, but not all differences in DNA content were due to variation in genome copy number. Haploid genome size between strains of different chemotypes differed by as much as 243 Mbp. Syntenic and phylogenetic analyses indicate that UTEX 2797, a common laboratory strain from Texas, is a hybrid that retains two phylogenetically distinct haplotypes. Investigation of gene families variably present across strains identified several functional categories associated with metabolism, including candidates for the biosynthesis of toxic metabolites, as well as genome size variation, including recent proliferations of transposable elements. Together, our results indicate thatP. parvumis comprised of multiple cryptic species. These genomes provide a robust phylogenetic and genomic framework for investigations into the eco-physiological consequences of the intra- and inter-specific genetic variation present inP. parvumand demonstrate the need for similar resources for other HAB-forming morphospecies.SIGNIFICANCE STATEMENTHarmful algal blooms (HABs) are a global concern. Efforts to understand the genetic basis of traits associated with the success of HAB-forming species are limited by a dearth of genomic resources. In this paper we present genomes for fifteen strains ofPrymnesium parvum, a toxic alga that causes ecosystem and societally disruptive HABs around the world. We uncover an unprecedented amount of sequence-level, gene family, and genome architecture evolution inP. parvumand provide evidence for both cryptic speciation and hybridization. These results illustrate how both inter- and intraspecific genetic variation can be dramatically underestimated in a protist morphospecies. More work is needed to understand the eco-physiological consequences of hidden genetic diversity inP. parvumand HAB-forming species more generally.

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.

Covering: 2016 to 2021 With 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., The continual growth of publicly available genomic databases offers researchers unprecedented bioinformatic opportunities. This review examines different approaches to mining genomic data for the targeted discovery of bioactive natural products.

Covering: Up to December 2020 Enzymatic 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.

Bacterial hormones, such as the iconic gamma-butyrolactone A-factor, are essential signaling molecules that regulate diverse physiological processes, including specialized metabolism. These low molecular weight compounds are common in Streptomyces species and display species–specific structural differences. Recently, unusual gamma-butyrolactone natural products called salinipostins were isolated from the marine actinomycete genus Salinispora based on their antimalarial properties. As the salinipostins possess a rare phosphotriester motif of unknown biosynthetic origin, we set out to explore its construction by the widely conserved 9-gene spt operon in Salinispora species. We show through a series of in vivo and in vitro studies that the spt gene cluster dually encodes the salinipostins and newly identified natural A-factor-like gamma-butyrolactones (Sal-GBLs). Remarkably, homologous biosynthetic gene clusters are widely distributed among many actinomycete genera, including Streptomyces, suggesting the significance of this operon in bacteria.

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.

Genetic engineering of natural product biosynthetic gene clusters represents an attractive approach to access new and complex bioactive small molecules. However, due to the large number and size of some genes involved in specialized metabolism, notably those encoding modular polyketide synthase and nonribosomal peptide synthetase megaproteins, it remains difficult to introduce precise genetic mutations to probe domain activity or alter chemical product formation. Here, we report the development and validation of a robust method combining oligonucleotide recombineering and CRISPR/Cas9 targeting for rapid site-directed mutagenesis of cloned pathways, which can be directly transferred to a heterologous host for expression. We rapidly generated 12 point mutations and identified several important determinants of successful mutagenesis, including the protospacer/PAM sequence and presence of regions of local homology. Our approach may be broadly applicable for researchers interested in probing natural product biosynthesis or performing pathway engineering.

Streptococcus mutans is a common constituent of dental plaque and a major etiologic agent of dental caries (tooth decay). In this study, we elucidated the biosynthetic pathway encoded by muc, a hybrid polyketide synthase and nonribosomal peptide synthetase (PKS/NRPS) biosynthetic gene cluster (BGC), present in a number of globally distributed S. mutans strains. The natural products synthesized by muc included three N-acyl tetramic acid compounds (reutericyclin and two novel analogues) and an unacylated tetramic acid (mutanocyclin). Furthermore, the enzyme encoded by mucF was identified as a novel class of membrane-associated aminoacylases and was responsible for the deacylation of reutericyclin to mutanocyclin. A large number of hypothetical proteins across a broad diversity of bacteria were homologous to MucF, suggesting that this may represent a large family of unexplored acylases. Finally, S. mutans utilized the reutericyclin produced by muc to impair the growth of neighboring oral commensal bacteria. Since S. mutans must be able to out-compete these health-associated organisms to persist in the oral microbiota and cause disease, the competitive advantage conferred by muc suggests that this BGC is likely to be involved in S. mutans ecology and therefore dental plaque dysbiosis and the resulting caries pathogenesis.

Covering: Up to January 2020Meroterpenoids derived from the polyketide 1,3,6,8-tetrahydroxynaphthalene (THN) are complex natural products produced exclusively by Streptomyces bacteria. These antibacterial compounds include the napyradiomycins, merochlorins, marinones, and furaquinocins and have inspired many attempts at their chemical synthesis. In this review, we highlight the role played by biosynthetic studies in the stimulation of biomimetic and, ultimately, chemoenzymatic total syntheses of these natural products. In particular, the application of genome mining techniques to marine Streptomyces bacteria led to the discovery of unique prenyltransferase and vanadium-dependent haloperoxidase enzymes that can be used as highly selective biocatalysts in fully enzymatic total syntheses, thus overcoming the limitations of purely chemical reagents.

Motivated by the biosynthesis of azamerone, we report the first example of a diazo-Hooker reaction, which involves the formation of a phthalazine ring system by the oxidative rearrangement of a diazoketone. Computational studies indicate that the diazo-Hooker reaction proceeds via an 8π-electro-cyclization followed by ring contraction and aromatization. The biosynthetic origin of the diazoketone functional group was also chemically mimicked using a related natural product, naphterpin, as a model system.

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

Bromopyrroles (BrPyr) are synthesized naturally by marine sponge symbionts and produced anthropogenically as byproducts of wastewater treatment. BrPyr interact with ryanodine receptors (RYRs) and sarco/endoplasmic reticulum (SR/ER) Ca(2+)-ATPase (SERCA). Influences of BrPyr on the neuronal network activity remain uncharted. BrPyr analogues with differing spectra of RYR/SERCA activities were tested using RYR-null or RYR1-expressing HEK293 and murine cortical neuronal/glial cocultures (NGCs) loaded with Fluo-4 to elucidate their mechanisms altering Ca(2+) dynamics. The NGC electrical spike activity (ESA) was measured from NGCs plated on multielectrode arrays. Nanomolar tetrabromopyrrole (TBP, 1) potentiated caffeine-triggered Ca(2+) release independent of extracellular [Ca(2+)] in RYR1-HEK293, whereas higher concentrations produce slow and sustained rise in cytoplasmic [Ca(2+)] independent of RYR1 expression. TBP, 2,3,5-tribromopyrrole (2), pyrrole (3), 2,3,4-tribromopyrrole (4), and ethyl 4-bromopyrrole-2-carboxylate (5) added acutely to NGC showed differential potency; rank order TBP (IC(50) ≈ 220 nM) > 2 ≫ 5, whereas 3 and 4 were inactive at 10 μM. TBP >2 μM elicited sustained elevation of cytoplasmic [Ca(2+)] and loss of neuronal viability. TBP did not alter network ESA. BrPyr from marine and anthropogenic sources are ecological signaling molecules and emerging anthropogenic pollutants of concern to environmental and human health that potently alter ER Ca(2+) dynamics and warrant further investigation in vivo.

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.

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-β-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/β-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.

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 inpoorly assembled genomes and metagenomes, or when loci are not clustered, as in eukaryotic genomes. To help support the growing interest in sequence-based analyses ofnatural product biosynthetic diversity, here we introduceversion 2 of the webtool, NaPDoS2, available at http://napdos.ucsd.edu/napdos2. This update includes the additionof 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.

Multidomain enzymes are cellular machines that orchestrate two or more catalytic activities to carry out metabolic transformations with increased control and speed. Our understanding of these enzymes’ capabilities drives progress in fundamental metabolic research, biocatalysis, and human health. Here, we report the development of a new genome mining approach for the targeted discovery of novel 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 new natural products containing the rare oxazolone (azlactone) heterocycle and characterized the di-domain enzyme as the first reported oxazolone synthetase in biology. This enzyme has the potential to become a valuable biocatalyst for the production of versatile oxazolone synthetic intermediates. This proof-of-principle experiment validates CO-ED-guided genome mining as a new method with potential broad utility for both the discovery of novel enzymatic transformations and the functional gene annotation of multidomain enzymes.TOC graphic

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 inter-module 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 inter-module 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 multi-modular megaenzymes.

Kainic acid, the flagship member of the kainoid family of natural neurochemicals, is a widely used neuro-pharmacological agent that helped unravel the key role of ionotropic glutamate receptors, including the kainate receptor, in the central nervous system. Worldwide shortages of this seaweed natural product in the year 2000 prompted numerous chemical syntheses, including scalable preparations with as few as six-steps. Herein we report the discovery and characterization of the concise two-enzyme biosynthetic pathway to kainic acid from l-glutamic acid and dimethylallyl pyrophosphate in red macroalgae and show that the biosynthetic genes are co-clustered in genomes of Digenea simplex and Palmaria palmata. Moreover, we applied a key biosynthetic α-ketoglutarate-dependent dioxygenase enzyme in a biotransformation methodology to efficiently construct kainic acid on the gram scale. This study establishes both the feasibility of mining seaweed genomes for their biotechnological prowess.

l-4-Chlorokynurenine (l-4-Cl-Kyn) is a neuropharmaceutical drug candidate that is in development for the treatment of major depressive disorder. Recently, this amino acid was naturally found as a residue in the lipopeptide antibiotic taromycin. Herein, we report the unprecedented conversion of l-tryptophan into l-4-Cl-Kyn catalyzed by four enzymes in the taromycin biosynthetic pathway from the marine bacterium Saccharomonospora sp. CNQ-490. We used genetic, biochemical, structural, and analytical techniques to establish l-4-Cl-Kyn biosynthesis, which is initiated by the flavin-dependent tryptophan chlorinase Tar14 and its flavin reductase partner Tar15. This work revealed the first tryptophan 2,3-dioxygenase (Tar13) and kynurenine formamidase (Tar16) enzymes that are selective for chlorinated substrates. The substrate scope of Tar13, Tar14, and Tar16 was examined and revealed intriguing promiscuity, thereby opening doors for the targeted engineering of these enzymes as useful biocatalysts.

Enzymatic dehalogenation is an important and well-studied biological process in both the detoxification and catabolism of small molecules, many of which are anthropogenic in origin. However, dedicated dehalogenation reactions that replace a halogen atom with a hydrogen are rare in the biosynthesis of natural products. In fact, the debrominase Bmp8 is the only known example. It catalyzes the reductive debromination of the coral settlement cue and the potential human toxin 2,3,4,5-tetrabromopyrrole as part of the biosynthesis of the antibiotic pentabromopseudilin. Using a combination of protein crystallography, mutagenesis, and computational modeling, we propose a catalytic mechanism for Bmp8 that is reminiscent of that catalyzed by human deiodinases in the maintenance of thyroid hormones. The identification of the key catalytic residues enabled us to recognize divergent functional homologues of Bmp8. Characterization of one of these homologues demonstrated its debromination activity even though it is found in a completely distinct genomic context. This observation suggests that additional enzymes outside those associated with the tetrabromopyrrole biosynthetic pathway may be able to alter the lifetime of this compound in the environment.

Enzyme-mediated cascade reactions are widespread in biosynthesis. To facilitate comparison with the mechanistic categorizations of cascade reactions by synthetic chemists and delineate the common underlying chemistry, we discuss four types of enzymatic cascade reactions: those involving nucleophilic, electrophilic, pericyclic, and radical reactions. Two subtypes of enzymes that generate radical cascades exist at opposite ends of the oxygen abundance spectrum. Iron-based enzymes use O(2) to generate high valent iron-oxo species to homolyze unactivated C–H bonds in substrates to initiate skeletal rearrangements. At anaerobic end, enzymes reversibly cleave S-adenosylmethionine (SAM) to generate the 5’-deoxyadenosyl radical as a powerful oxidant to initiate C–H bond homolysis in bound substrates. The latter enzymes are termed radical SAM enzymes. We categorize the former as “thwarted oxygenases”.

The production of pentabromopseudilin and related brominated compounds by Pseudoalteromonas spp. has recently been linked to the bmp biosynthetic gene cluster. This study explored the distribution and evolutionary history of this gene cluster in the genus Pseudoalteromonas. A phylogeny of the genus revealed numerous clades that do not contain type strains, suggesting considerable species level diversity has yet to be described. Comparative genomics revealed four distinct versions of the gene cluster distributed among 19 of the 101 Pseudoalteromonas genomes examined. These were largely localized to the least inclusive clades containing the Pseudoalteromonas luteoviolacea and Pseudoalteromonas phenolica type strains and show clear evidence of gene and gene cluster loss in certain lineages. Bmp gene phylogeny is largely congruent with the Pseudoalteromonas species phylogeny, suggesting vertical inheritance within the genus. However, the gene cluster is found in three different genomic environments suggesting either chromosomal rearrangement or multiple acquisition events. Bmp conservation within certain lineages suggests the encoded products are highly relevant to the ecology of these bacteria.

Sequential enzymatic reactions on substrates tethered to carrier proteins (CPs) generate thiotemplated building blocks that are then delivered to nonribosomal peptide synthetases (NRPSs) to generate peptidic natural products. The underlying diversity of these thiotemplated building blocks is the principal driver of the chemical diversity of NRPS-derived natural products. Structural insights into recognition of CPs by tailoring enzymes that generate these building blocks are sparse. Here we present the crystal structure of a flavin-dependent prolyl oxidase that furnishes thiotemplated pyrrole as the product, in complex with its cognate CP in the holo and product-bound states. The thiotemplated pyrrole is an intermediate that is well-represented in natural product biosynthetic pathways. Our results delineate the interactions between the CP and the oxidase while also providing insights into the stereospecificity of the enzymatic oxidation of the prolyl heterocycle to the aromatic pyrrole. Biochemical validation of the interaction between the CP and the oxidase demonstrates that NRPSs recognize and bind to their CPs using interactions quite different from those of fatty acid and polyketide biosynthetic enzymes. Our results posit that structural diversity in natural product biosynthesis can be, and is, derived from subtle modifications of primary metabolic enzymes.

International audience; With contributions from the global natural product (NP) research community, and continuing the Raw Data Initiative, this review collects a comprehensive demonstration of the immense scientific value of disseminating raw nuclear magnetic resonance (NMR) data, independently of, and in parallel with, classical publishing outlets. A comprehensive compilation of historic to present-day cases as well as contemporary and future applications show that addressing the urgent need for a repository of publicly accessible raw NMR data has the potential to transform natural products (NPs) and associated fields of chemical and biomedical research. The call for advancing open sharing mechanisms for raw data is intended to enhance the transparency of experimental protocols, augment the reproducibility of reported outcomes, including biological studies, become a regular component of responsible research, and thereby enrich the integrity of NP research and related fields.

Thiotetronate-containing natural products, including thiolactomycin, thiotetromycin, and thiotetroamide, are potent, broad-spectrum antibacterial compounds that target fatty acid synthesis in bacteria. Natural modifications at the C-5 dialkyl position in this molecular series result in pronounced bioactivity differences. The C-5 acetamide-containing thiotetroamide, which is the more potent antibacterial agent in this family, is biosynthesized from the C-5 ethyl analogue thiotetromycin via a unique two-enzyme process involving the cytochrome P450-amidotransferase enzyme pair TtmP-TtmN. Herein we synthesized a focused library of 17 novel thiotetromycin derivatives differing at the 5-position alkyl substituent to investigate their biological activities and their reactivity towards the hydroxylase TtmP. Although we observed marginal anti-tuberculosis activity, select thiotetromycin analogues showed antibacterial activity against an Escherichia coli ΔtolC strain with IC(50) values in a range of 1.9 – 36 µg/mL. Additional screening efforts highlighted select thiotetronate analogues as inhibitors of the cancer-associated enzyme nicotinamide N-methyltransferase (NNMT), with a unique scaffold compared to previously identified NNMT inhibitors. In vitro assays further showed that the TtmP P450 was capable of resolving racemic substrate mixtures and had modest promiscuity to hydroxylate derivatives with variable alkyl chains; however triple oxidation to a carboxylic acid remained specific for the natural thiotetromycin substrate. The tendency of TtmP to accept a range of unnatural substrates for hydroxylation makes it an interesting target for P450 engineering towards broader applications.

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.

International audience; Genomics and metabolomics are widely used to explore specialized metabolite diversity. The Paired Omics Data Platform is a community initiative to systematically document links between metabolome and (meta)genome data, aiding identification of natural product biosynthetic origins and metabolite structures.

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 inbacteria 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.