Structure and Biosynthesis of Hectoramide B, a Linear Depsipeptide from Marine Cyanobacterium Moorena producens JHB Discovered via Coculture with Candida albicans.

The tropical marine cyanobacterium Moorena producens JHB is a prolific source of secondary metabolites with potential biomedical utility. Previous studies on this strain led to the discovery of several novel compounds such as hectochlorins and jamaicamides. However, bioinformatic analyses of its genome indicate the presence of numerous cryptic biosynthetic gene clusters that have yet to be characterized. To potentially stimulate the production of novel compounds from this strain, it was cocultured with Candida albicans. From this experiment, we observed the increased production of a new compound that we characterize here as hectoramide B. Bioinformatic analysis of the M. producens JHB genome enabled the identification of a putative biosynthetic gene cluster responsible for hectoramide B biosynthesis. This work demonstrates that coculture competition experiments can be a valuable method to facilitate the discovery of novel natural products from cyanobacteria.

microbeMASST: a taxonomically informed mass spectrometry search tool for microbial metabolomics data.

microbeMASST, a taxonomically informed mass spectrometry (MS) search tool, tackles limited microbial metabolite annotation in untargeted metabolomics experiments. Leveraging a curated database of >60,000 microbial monocultures, users can search known and unknown MS/MS spectra and link them to their respective microbial producers via MS/MS fragmentation patterns. Identification of microbe-derived metabolites and relative producers without a priori knowledge will vastly enhance the understanding of microorganisms' role in ecology and human health.

Open access repository-scale propagated nearest neighbor suspect spectral library for untargeted metabolomics.

Despite the increasing availability of tandem mass spectrometry (MS/MS) community spectral libraries for untargeted metabolomics over the past decade, the majority of acquired MS/MS spectra remain uninterpreted. To further aid in interpreting unannotated spectra, we created a nearest neighbor suspect spectral library, consisting of 87,916 annotated MS/MS spectra derived from hundreds of millions of MS/MS spectra originating from published untargeted metabolomics experiments. Entries in this library, or "suspects," were derived from unannotated spectra that could be linked in a molecular network to an annotated spectrum. Annotations were propagated to unknowns based on structural relationships to reference molecules using MS/MS-based spectrum alignment. We demonstrate the broad relevance of the nearest neighbor suspect spectral library through representative examples of propagation-based annotation of acylcarnitines, bacterial and plant natural products, and drug metabolism. Our results also highlight how the library can help to better understand an Alzheimer's brain phenotype. The nearest neighbor suspect spectral library is openly available for download or for data analysis through the GNPS platform to help investigators hypothesize candidate structures for unknown MS/MS spectra in untargeted metabolomics data.

A Taxonomically-informed Mass Spectrometry Search Tool for Microbial Metabolomics Data.

MicrobeMASST, a taxonomically-informed mass spectrometry (MS) search tool, tackles limited microbial metabolite annotation in untargeted metabolomics experiments. Leveraging a curated database of >60,000 microbial monocultures, users can search known and unknown MS/MS spectra and link them to their respective microbial producers via MS/MS fragmentation patterns. Identification of microbial-derived metabolites and relative producers, without a priori knowledge, will vastly enhance the understanding of microorganisms' role in ecology and human health.

Enzymatic Halogenation of Terminal Alkynes.

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.

Structure and Biosynthesis of Hectoramide B, a Linear Depsipeptide from the Marine Cyanobacterium Moorena producens JHB Discovered via Co-culture with Candida albicans.

The tropical marine cyanobacterium Moorena producens JHB is a prolific source of secondary metabolites with potential biomedical utility. Previous studies of this strain led to the discovery of several novel compounds such as the hectochlorins and jamaicamides; however, bioinformatic analyses of its genome suggested that there were many more cryptic biosynthetic gene clusters yet to be characterized. To potentially stimulate the production of novel compounds from this strain, it was co-cultured with Candida albicans. From this experiment, we observed the increased production of a new compound that we characterize here as hectoramide B. Bioinformatic analysis of the M. producens JHB genome enabled the identification of a putative biosynthetic gene cluster responsible for hectoramide B biosynthesis. This work demonstrates that co-culture competition experiments can be a valuable method to facilitate the discovery of novel natural products from cyanobacteria.

MIBiG 3.0: a community-driven effort to annotate experimentally validated biosynthetic gene clusters.

With an ever-increasing amount of (meta)genomic data being deposited in sequence databases, (meta)genome mining for natural product biosynthetic pathways occupies a critical role in the discovery of novel pharmaceutical drugs, crop protection agents and biomaterials. The genes that encode these pathways are often organised into biosynthetic gene clusters (BGCs). In 2015, we defined the Minimum Information about a Biosynthetic Gene cluster (MIBiG): a standardised data format that describes the minimally required information to uniquely characterise a BGC. We simultaneously constructed an accompanying online database of BGCs, which has since been widely used by the community as a reference dataset for BGCs and was expanded to 2021 entries in 2019 (MIBiG 2.0). Here, we describe MIBiG 3.0, a database update comprising large-scale validation and re-annotation of existing entries and 661 new entries. Particular attention was paid to the annotation of compound structures and biological activities, as well as protein domain selectivities. Together, these new features keep the database up-to-date, and will provide new opportunities for the scientific community to use its freely available data, e.g. for the training of new machine learning models to predict sequence-structure-function relationships for diverse natural products. MIBiG 3.0 is accessible online at https://mibig.secondarymetabolites.org/.

Tuaimenals B-H, Merosesquiterpenes from the Irish Deep-Sea Soft Coral Duva florida with Bioactivity against Cervical Cancer Cell Lines.

Previous chemical investigation of the Irish deep-sea soft coral Duva florida led to the identification of tuaimenal A (10), a new merosesquiterpene containing a highly substituted chromene core and modest cytotoxicity against cervical cancer. Further MS/MS and NMR-guided investigation of this octocoral has resulted in the isolation and characterization of seven additional tuaimenal analogs, B-H (1-7), as well as two known A-ring aromatized steroids (8, 9), and additional tuaimenal A (10). Tuaimenals B, F, and G (1, 5, 6), bearing an oxygen at the C5 position, as well as monocyclic tuaimenal H (7), show increased cervical cancer inhibition profiles in comparison to that of 10. Tuaimenal G further displayed potent, selective cytotoxicity with an EC50 value of 0.04 µM against the C33A cell line compared to the CaSki cell line (EC50 20 µM). These data reveal the anticancer properties of tuaimenal analogs and suggest unique antiproliferation mechanisms across these secondary metabolites.

Integrated Metabolomic-Genomic Workflows Accelerate Microbial Natural Product Discovery.

The pairing of analytical chemistry with genomic techniques represents a new wave in natural product chemistry. With an increase in the availability of sequencing and assembly of microbial genomes, interrogation into the biosynthetic capability of producers with valuable secondary metabolites is possible. However, without the development of robust, accessible, and medium to high throughput tools, the bottleneck in pairing metabolic potential and compound isolation will continue. Several innovative approaches have proven useful in the nascent stages of microbial genome-informed drug discovery. Here, we consider a number of these approaches which have led to prioritization of strain targets and have mitigated rediscovery rates. Likewise, we discuss integration of principles of comparative evolutionary studies and retrobiosynthetic predictions to better understand biosynthetic mechanistic details and link genome sequence to structure. Lastly, we discuss advances in engineering, chemistry, and molecular networking and other computational approaches that are accelerating progress in the field of omic-informed natural product drug discovery. Together, these strategies enhance the synergy between cutting edge omics, chemical characterization, and computational technologies that pitch the discovery of natural products with pharmaceutical and other potential applications to the crest of the wave where progress is ripe for rapid advances.

Tuaimenal A, a Meroterpene from the Irish Deep-Sea Soft Coral Duva florida, Displays Inhibition of the SARS-CoV-2 3CLpro Enzyme.

Cold water benthic environments are a prolific source of structurally diverse molecules with a range of bioactivities against human disease. Specimens of a previously chemically unexplored soft coral, Duva florida, were collected during a deep-sea cruise that sampled marine invertebrates along the Irish continental margin in 2018. Tuaimenal A (1), a cyclized merosesquiterpenoid representing a new carbon scaffold with a highly substituted chromene core, was discovered through exploration of the soft coral secondary metabolome via NMR-guided fractionation. The absolute configuration was determined through vibrational circular dichroism. Functional biochemical assays and in silico docking experiments found tuaimenal A selectively inhibits the viral main protease (3CLpro) of SARS-CoV-2.

Discovery of an Antarctic Ascidian-Associated Uncultivated Verrucomicrobia with Antimelanoma Palmerolide Biosynthetic Potential.

The Antarctic marine ecosystem harbors a wealth of biological and chemical innovation that has risen in concert over millennia since the isolation of the continent and formation of the Antarctic circumpolar current. Scientific inquiry into the novelty of marine natural products produced by Antarctic benthic invertebrates led to the discovery of a bioactive macrolide, palmerolide A, that has specific activity against melanoma and holds considerable promise as an anticancer therapeutic. While this compound was isolated from the Antarctic ascidian Synoicum adareanum, its biosynthesis has since been hypothesized to be microbially mediated, given structural similarities to microbially produced hybrid nonribosomal peptide-polyketide macrolides. Here, we describe a metagenome-enabled investigation aimed at identifying the biosynthetic gene cluster (BGC) and palmerolide A-producing organism. A 74-kbp candidate BGC encoding the multimodular enzymatic machinery (hybrid type I-trans-AT polyketide synthase-nonribosomal peptide synthetase and tailoring functional domains) was identified and found to harbor key features predicted as necessary for palmerolide A biosynthesis. Surveys of ascidian microbiome samples targeting the candidate BGC revealed a high correlation between palmerolide gene targets and a single 16S rRNA gene variant (R = 0.83 to 0.99). Through repeated rounds of metagenome sequencing followed by binning contigs into metagenome-assembled genomes, we were able to retrieve a nearly complete genome (10 contigs) of the BGC-producing organism, a novel verrucomicrobium within the Opitutaceae family that we propose here as "Candidatus Synoicihabitans palmerolidicus." The refined genome assembly harbors five highly similar BGC copies, along with structural and functional features that shed light on the host-associated nature of this unique bacterium. IMPORTANCE Palmerolide A has potential as a chemotherapeutic agent to target melanoma. We interrogated the microbiome of the Antarctic ascidian, Synoicum adareanum, using a cultivation-independent high-throughput sequencing and bioinformatic strategy. The metagenome-encoded biosynthetic machinery predicted to produce palmerolide A was found to be associated with the genome of a member of the S. adareanum core microbiome. Phylogenomic analysis suggests the organism represents a new deeply branching genus, "Candidatus Synoicihabitans palmerolidicus," in the Opitutaceae family of the Verrucomicrobia phylum. The Ca. Synoicihabitans palmerolidicus 4.29-Mb genome encodes a repertoire of carbohydrate-utilizing and transport pathways, a chemotaxis system, flagellar biosynthetic capacity, and other regulatory elements enabling its ascidian-associated lifestyle. The palmerolide producer's genome also contains five distinct copies of the large palmerolide biosynthetic gene cluster that may provide structural complexity of palmerolide variants.

Bioinformatic and Mechanistic Analysis of the Palmerolide PKS-NRPS Biosynthetic Pathway From the Microbiome of an Antarctic Ascidian.

Complex interactions exist between microbiomes and their hosts. Increasingly, defensive metabolites that have been attributed to host biosynthetic capability are now being recognized as products of host-associated microbes. These unique metabolites often have bioactivity targets in human disease and can be purposed as pharmaceuticals. Polyketides are a complex family of natural products that often serve as defensive metabolites for competitive or pro-survival purposes for the producing organism, while demonstrating bioactivity in human diseases as cholesterol lowering agents, anti-infectives, and anti-tumor agents. Marine invertebrates and microbes are a rich source of polyketides. Palmerolide A, a polyketide isolated from the Antarctic ascidian Synoicum adareanum, is a vacuolar-ATPase inhibitor with potent bioactivity against melanoma cell lines. The biosynthetic gene clusters (BGCs) responsible for production of secondary metabolites are encoded in the genomes of the producers as discrete genomic elements. A candidate palmerolide BGC was identified from a S. adareanum microbiome-metagenome based on a high degree of congruence with a chemical structure-based retrobiosynthetic prediction. Protein family homology analysis, conserved domain searches, active site and motif identification were used to identify and propose the function of the ∼75 kbp trans-acyltransferase (AT) polyketide synthase-non-ribosomal synthase (PKS-NRPS) domains responsible for the stepwise synthesis of palmerolide A. Though PKS systems often act in a predictable co-linear sequence, this BGC includes multiple trans-acting enzymatic domains, a non-canonical condensation termination domain, a bacterial luciferase-like monooxygenase (LLM), and is found in multiple copies within the metagenome-assembled genome (MAG). Detailed inspection of the five highly similar pal BGC copies suggests the potential for biosynthesis of other members of the palmerolide chemical family. This is the first delineation of a biosynthetic gene cluster from an Antarctic microbial species, recently proposed as Candidatus Synoicihabitans palmerolidicus. These findings have relevance for fundamental knowledge of PKS combinatorial biosynthesis and could enhance drug development efforts of palmerolide A through heterologous gene expression.

Uncovering the Core Microbiome and Distribution of Palmerolide in Synoicum adareanum Across the Anvers Island Archipelago, Antarctica.

Polar marine ecosystems hold the potential for bioactive compound biodiscovery, based on their untapped macro- and microorganism diversity. Characterization of polar benthic marine invertebrate-associated microbiomes is limited to few studies. This study was motivated by our interest in better understanding the microbiome structure and composition of the ascidian, Synoicum adareanum, in which palmerolide A (PalA), a bioactive macrolide with specificity against melanoma, was isolated. PalA bears structural resemblance to a hybrid nonribosomal peptide-polyketide that has similarities to microbially-produced macrolides. We conducted a spatial survey to assess both PalA levels and microbiome composition in S. adareanum in a region of the Antarctic Peninsula near Anvers Island (64° 46'S, 64° 03'W). PalA was ubiquitous and abundant across a collection of 21 ascidians (3 subsamples each) sampled from seven sites across the Anvers Island Archipelago. The microbiome composition (V3-V4 16S rRNA gene sequence variants) of these 63 samples revealed a core suite of 21 bacterial amplicon sequence variants (ASVs)-20 of which were distinct from regional bacterioplankton. ASV co-occurrence analysis across all 63 samples yielded subgroups of taxa that may be interacting biologically (interacting subsystems) and, although the levels of PalA detected were not found to correlate with specific sequence variants, the core members appeared to occur in a preferred optimum and tolerance range of PalA levels. These results, together with an analysis of the biosynthetic potential of related microbiome taxa, describe a conserved, high-latitude core microbiome with unique composition and substantial promise for natural product biosynthesis that likely influences the ecology of the holobiont.

Precision medicine of aneurysmal subarachnoid hemorrhage, vasospasm and delayed cerebral ischemia.

INTRODUCTION: Precision medicine provides individualized treatment of diseases through leveraging patient-to-patient variation. Aneurysmal subarachnoid hemorrhage carries tremendous morbidity and mortality with cerebral vasospasm and delayed cerebral ischemia proving devastating and unpredictable. Lack of treatment measures for these conditions could be improved through precision medicine. Areas covered: Discussed are the pathophysiology of CV and DCI, treatment guidelines, and evidence for precision medicine used for prediction and prevention of poor outcomes following aSAH. A PubMed search was performed using keywords cerebral vasospasm or delayed cerebral ischemia and either biomarkers, precision medicine, metabolomics, proteomics, or genomics. Over 200 peer-reviewed articles were evaluated. The studies presented cover biomarkers identified as predictive markers or therapeutic targets following aSAH. Expert commentary: The biomarkers reviewed here correlate with CV, DCI, and neurologic outcomes after aSAH. Though practical use in clinical management of aSAH is not well established, using these biomarkers as predictive tools or therapeutic targets demonstrates the potential of precision medicine.