Secondary or specialized metabolites show extraordinary structural diversity and potent biological activities relevant for clinical and industrial applications. The biosynthesis of these metabolites usually starts with the assembly of a core 'scaffold', which is subsequently modified by tailoring enzymes to define the molecule's final structure and, in turn, its biological activity profile. Knowledge about reaction and substrate specificity of tailoring enzymes is essential for understanding and computationally predicting metabolite biosynthesis, but this information is usually scattered in the literature. Here, we present MITE, the Minimum Information about a Tailoring Enzyme database. MITE employs a comprehensive set of parameters to annotate tailoring enzymes, defining substrate and reaction specificity by the expressive reaction SMARTS (Simplified Molecular Input Line Entry System Arbitrary Target Specification) chemical pattern language. Both human and machine readable, MITE can be used as a knowledge base, for in silico biosynthesis, or to train machine-learning applications, and tightly integrates with existing resources. Designed as a community-driven and open resource, MITE employs a rolling release model of data curation and expert review. MITE is freely accessible at https://mite.bioinformatics.nl/.

Natural products containing vinylogous amino acids are rarely found in nature and often possess significant biological activity. Barnesin A was the first NP reported from an anaerobic bacterium (Sulfurospirillum barnesii) postulated to be biosynthesized by a nonribosomal peptide synthetase (NRPS) polyketide synthases (PKS) hybrid. Containing a vinylogous arginine moiety, the lipodipeptide exhibited nanomolar inhibitory activity against cysteine proteases. While a putative NRPS-PKS hybrid biosynthetic gene cluster (brn) was identified and a trans-acting acyltransferase (trans-AT) domain was postulated, experimental validation remained an open question. Here, we report the production of barnesin A by heterologous expression of the trans-AT domain-dependent NRPS-PKS gene cluster in Escherichia coli. Our findings indicate that the native primary metabolism-derived malonyl CoA-acyl carrier protein transacylase homolog (FabD) functions as a trans-AT in the biosynthesis pathway, while the NRPS-PKS megaenzyme exhibited strict selectivity toward its native phosphopantetheinyl transferase. Metabolome mining further allowed for the description of previously unreported barnesin congeners. The results of this study enabled the establishment of a biosynthetic platform for the generation of novel lipopeptidic vinylogous protease inhibitors.

A targeted sequencing and genome mining approach for delftibactin-like biosynthetic pathways revealed three distinct biosynthetic gene cluster architectures (BGC del, dlc and dlp) encoded in genomes of members of the genus Delftia. Comparative metabolomic analysis guided the isolation and characterization of a yet unreported metallophore, delftichelin A from Delftia deserti DSM1621 (previously named Delftia acidovorans DSM1621). Prediction of BGC architecture and A domain specificity was in line with the structure analysis uncovering previously unreported differences in amino acid composition and modifications. Analysis of bioactivity and metal-binding characteristics demonstrated that delftichelin A shows a preferential affinity for ferric iron, while also exhibiting heavy metal detoxification mechanisms via oxidative degradation, analogous to those reported for the delftibactin family of compounds.

Covering: 2018 to 2025Over recent years, metagenomic-driven studies have revealed an enormous encoded repertoire for the biosynthesis of secondary metabolite scaffolds within host-associated microbiota, yet only a small fraction of these chemical scaffolds has been characterized. This review focuses on recent discoveries of natural products with anti-infective and immunomodulatory properties derived from diverse host-associated microbiomes, covering the period from 2018 to 2025. The selected examples span a wide range of anti-infective and immunomodulatory activities, underscoring the deep integration of microbial secondary metabolism with host physiology, while also highlighting the need for more targeted and efficient combined approaches to fully exploit the predicted biosynthetic capacity of microbiomes for anti-infective research and beyond.

Abstract Bacterial signals control the development of marine algae, yet the molecular basis of these cross-kingdom interactions remains largely unknown. Thallusin is the paradigmatic case: isolated in 2005, it induces rhizoid and cell wall formation in the green seaweed Ulva at picomolar concentrations, but its biosynthesis has remained elusive for two decades. Comparative genomics across five bacterial phyla identifies a conserved set of genes - the eustigmatophyte bacterial operon ( ebo ) - as determinants of thallusin biosynthesis. Isotope labeling, heterologous expression, and gene deletion in Stieleria maiorica show that the aromatic scaffold derives from a cyclitol precursor and L-aspartate, with subsequent prenylation and cyclization. Searching 124,295 prokaryotic genomes identifies producers in eleven bacterial lineages, including soil cyanobacteria, establishing thallusin as a widespread cross-kingdom signal reaching beyond the ocean.

Obesity remains a global health challenge, and novel small-molecule modulators of adipogenesis are urgently needed. Here, we investigated the anti-adipogenic potential of metabolites isolated from termite-associated Streptomyces sp. M45. Chemical investigation of the extract from cultured Streptomyces sp. M45 led to the isolation of six metabolites (1-6). Structures were determined as 2-acetamido-3-hydroxybenzoic acid (1), N-acetyltyramine (2), deoxyuridine (3), cyclo(L-Pro-L-Leu) (4), cyclo(L-Pro-D-Leu) (5), and cyclo(L-Pro-L-Phe) (6) via comprehensive analysis of nuclear magnetic resonance (NMR) spectroscopy and liquid chromatography-mass spectrometry (LC-MS) data. Notably, the structure of the anthranilic acid derivative, 2-acetamido-3-hydroxybenzoic acid (1), was fully elucidated by 1D and 2D NMR, high-resolution electrospray ionization mass spectrometry (HR-ESIMS), and DP4+ probability calculations, allowing us to correct previously reported 1H NMR resonances and to assign 13C NMR signals for the first time. Compounds 1-6 were evaluated for anti-adipogenic activity in differentiated 3T3-L1 cells by assessing lipid accumulation and the expression of adipogenic transcription factors (C/EBPα, C/EBPβ, and SREBP2). Among these, N-acetyltyramine (2) significantly inhibited adipocyte differentiation without impairing cell viability. Mechanistically, compound 2 downregulated the expression of key adipogenic genes including C/EBPα, SREBP2, FASN, UCP2, and leptin. These findings substantiate its potential as a modulator of obesity-related pathways in 3T3-L1 adipocytes.

Covering: up to 2025Metallophores are metal-chelating natural products produced by microorganisms to scavenge essential metal ions in nutrient-limited environments. Among them, yersiniabactin-type metallophores (YTMs) represent a structurally and functionally distinct subgroup with a growing role in host-microbe and microbe-microbe interactions. In contrast to flexible hydroxamate- and carboxylate-type siderophores, YTMs feature a linear, pre-organized arrangement of aryl and five-membered heterocycles, often derived from modular nonribosomal peptide synthetase (NRPS) pathways in combination with polyketide synthase (PKS) domains. Their biosynthesis is encoded by gene clusters that integrate precursor formation, assembly line machinery, and metal transport components. Salicylic acid-derived aryl units and cysteine/serine-derived heterocycles are tailored through oxidation, methylation, and glycosylation, giving rise to complex chelators with a broad metal-binding profile-including Cu(II), Co(II), Ni(II), and Zn(II)-but weaker Fe(III) affinity. Due to structural ambiguity in current terminology, we propose a refined definition for YTMs based on specific connectivity of aryl and heterocyclic units and demonstrated metal chelation. We distinguish YTMs from simpler aryl-hetaryl siderophores such as anguibactin and pre-acinetobactin, and argue against broader umbrella terms like "mixed" or "salicyl-capped" siderophores. This review provides a comprehensive overview of the structural, biosynthetic, and genomic features of YTMs and introduces a classification framework based on a comprehensive biosynthetic pathway survey to facilitate the comparisons across natural product families. Given their prevalence in pathogens prioritized by the World Health Organization, including Pseudomonas aeruginosa and Mycobacterium tuberculosis, YTMs represent promising targets for both ecological and therapeutic exploration.

Specialized or secondary metabolites are small molecules of biological origin, often showing potent biological activities with applications in agriculture, engineering and medicine. Usually, the biosynthesis of these natural products is governed by sets of co-regulated and physically clustered genes known as biosynthetic gene clusters (BGCs). To share information about BGCs in a standardized and machine-readable way, the Minimum Information about a Biosynthetic Gene cluster (MIBiG) data standard and repository was initiated in 2015. Since its conception, MIBiG has been regularly updated to expand data coverage and remain up to date with innovations in natural product research. Here, we describe MIBiG version 4.0, an extensive update to the data repository and the underlying data standard. In a massive community annotation effort, 267 contributors performed 8304 edits, creating 557 new entries and modifying 590 existing entries, resulting in a new total of 3059 curated entries in MIBiG. Particular attention was paid to ensuring high data quality, with automated data validation using a newly developed custom submission portal prototype, paired with a novel peer-reviewing model. MIBiG 4.0 also takes steps towards a rolling release model and a broader involvement of the scientific community. MIBiG 4.0 is accessible online at https://mibig.secondarymetabolites.org/. [GRAPHICS] .

N-acyl tyrosines, a prominent class of N-acyl amino acid biomolecules, are produced by selected species in at least three bacterial phyla: Pseudomonadota, Actinomycetota and Planctomycetota. Long-chain N-acyl tyrosines with a characteristic 2,3-dehydrotyrosine core structure and additional taxon-specific chemical modifications were previously reported under the names thalassotalic acids, kyonggic acids and stieleriacines. However, the underlying pathway for their biosynthesis in the different bacterial taxa remains largely unexplored. Here, we focused on the identification of biosynthetic enzymes in the two known stieleriacine-producing planctomycetal strains of the eponymous genus Stieleria. Comparative genome analyses of stieleriacine-, thalassotalic acid- and kyonggic acid producers suggest a common pathway for N-acyl dehydrotyrosine biosynthesis based on conserved genes encoding a putative adenylyltransferase/cyclase, nitroreductase and the hallmark protein N-acyl amino acid synthase (NasY). The targeted deletion of three predicted nasY genes in Stieleria neptunia indicates that one of the three encoded enzymes predominantly produces stieleriacines. We also confirmed the absolute structure of stieleriacine C by synthesis of its epimer and structural derivatives, which serve as the basis for the future investigation of the biological function of N-acyl tyrosines.

Fish rely on innate immunity, including antimicrobial peptides (AMPs), to combat pathogens. Piscidins are a common AMP family in fish. This study identifies and characterizes a novel piscidin (Gbpis) from gilthead seabream, highlighting its structural uniqueness, antimicrobial function, and immunomodulatory potential. The gbpis cDNA contains a 183-bp open reading frame encoding a 60-amino-acid precursor, classified as a class 1 piscidin. The mature 22-residue peptide (2314.8 Da; pI 10.11) features a disulfide bridge (Cys9-Cys16) critical for activity. Tissue-specific expression analysis revealed Gbpis mRNA in the liver, skin, head kidney, and significant upregulation in the head kidney during Aeromonas hydrophila infection. Recombinant Gbpis, expressed in E. coli with the intact disulfide bond, exhibited potent antimicrobial activity against Streptococcus iniae. Synthetic Gbpis lacking the disulfide bridge was inactive, while the bridged peptide inhibited Gram-positive bacteria, underscoring the bridge's necessity for function. In rainbow trout, Gbpis pre-treatment significantly mitigated the pro-inflammatory response to S. iniae infection. This was demonstrated by a marked reduction in the expression of il-6 and tnf-α in the head kidney. In spleen, Gbpis alone induced a potent il-6 response, which was then significantly dampened following the subsequent S. iniae challenge. These findings establish Gbpis as a novel piscidin with a structurally essential disulfide bond, demonstrating dual roles in pathogen inhibition and mitigating excessive inflammation. This study advances understanding of fish AMPs and highlights their potential in sustainable aquaculture strategies.

Natural products derived from symbiotic microbes remain a rich source of structurally diverse and bioactive molecules. In this study, we report de novo genome sequencing of the termite-associated isolate Micromonospora sp. RB23. Genome mining uncovered a type I polyketide synthase (T1PKS) biosynthetic gene cluster encoding five halogenases, predicted to produce pyrrolomycin-like antimicrobial compounds. Mass-spectrometry-based molecular networking facilitated the identification and isolation of N-methylated pyrrolomycin K and mycothiol-adduct, pyrrolomycin L. Structure elucidation was accomplished based on liquid chromatography high-resolution tandem mass spectrometry (LC-HRMS/MS) alongside 1D and 2D nuclear magnetic resonance (NMR) spectroscopy. Based on the evaluated of antimicrobial activity, we propose that N-methylation and mycothiol-based conjugation in pyrrolomycins are possible detoxification mechanisms that play a role in enhancing self-tolerance.

Integrating organismal interaction studies with advanced genomic and metabolomic approaches offer great promise for discovering novel natural products and their derivatives, yet this strategy remains relatively unexplored. Here, we illustrate its potential by investigating a newly isolated Xylaria strain from a termite colony environment through combined genome and metabolome analyses, complemented by fungal-bacterial coculture experiments. Genome sequencing of the fungal strain allowed us to pinpoint a cytochalasin-related biosynthetic gene cluster responsible for the production of a portfolio of different bioactive epoxy-cytochalasins. Guided by the hypothesis of biosynthetic promiscuity of the underlying nonribosomal peptide synthetase (NRPS), we demonstrated for the first time that the NRPS can accept unnatural ortho- and meta-halogenated phenylalanine derivatives, leading to the isolation of multiple new chlorinated and brominated cytochalasin analogs. Second, based on the hypothesis that structural diversification can arise from interactions with commensal organisms, cocultivation with a termite-associated Streptomyces strain led to the discovery of a previously undescribed aspartic acid-containing cytochalasan derivative, designated xylachalasin A. Isotope labeling experiments revealed that bacterial catabolic activity is responsible for the modification of the fungal-derived cytochalasin. Isolated cytochalasins were also amiable for semisynthesis modifications, which was exemplified by the synthesis of bifunctional probes. Bioassays of a total of 26 isolated and semisynthesized derivatives demonstrated structure-dependent cytotoxicity in some cases with up to 3-fold log differences in potency and generally good plasma stability. Overall, our integrated approach underscores the vast potential of investigating fungal strains from underexplored ecological niches and their organismal interactions, offering new opportunities to discover novel natural products of potential therapeutic relevance and previously unrecognized biochemical processes.

A pink-pigmented, neutrophilic and mesophilic strain, TA3T, was isolated from the hindgut of a fungus-growing termite of the species Macrotermes natalensis. Phylogenetic analysis placed the strain in the family Isosphaeraceae, order Isosphaerales, class Planctomycetia, phylum Planctomycetota. The isolate turned out to be an aerobic chemoorganoheterotroph capable of growth under microaerobic conditions. Cells are non-motile, spherical, and either form shapeless aggregates or grow as single cells. The average cell size (length x width) is 2.5 ± 0.3 μm x 2.3 ± 0.2 μm. Cells divide asymmetrically by budding. Optimum pH and temperature for growth are 7.5 (range 6.0–9.0) and 24 °C (range 18–28 °C), respectively. The strain has a genome size of 7.23 Mbp with 69.3% DNA G + C content and it contains four plasmids. Since the genome of the currently known closest relative Tundrisphaera lichenicola has not been sequenced, the previously characterized type strain P12T was included for genome sequencing. A comparison based on established phylogenetic markers yielded a 16S rRNA gene sequence similarity of 94.8%, an average nucleotide identity of 78.4% and a digital DNA-DNA hybridization (dDDH) value of 20.3%, suggesting a relationship of the two strains on the level of the same genus. Differences in genome-encoded features, e.g. carbohydrate-active enzymes, secondary metabolite-associated biosynthetic gene clusters and plasmid-located genes were analyzed using comparative genomics. Together with whole genome-based phylogenetic analyses and differences in phenotypic characteristics, the data justifies the delineation of the novel isolate from the sole known species in the genus Tundrisphaera. We therefore introduce Tundrisphaera macrotermitis sp. nov. that is represented by TA3T (= CECT 30560T = STH00997T) as the type strain.

Sphingoid bases are important bioactive lipids found in a variety of organisms, serving as the backbone of sphingolipids, which regulate essential physiological processes. Here we describe the total synthesis and structure revision of halisphingosine A, a sphingoid base initially isolated from marine sponges. To address inconsistencies in the NMR interpretation of this natural product, we developed a synthetic route involving a late-stage enantioselective Henry reaction that allows access to multiple stereoisomers of the proposed halisphingosine A core structure. Our library of 32 fully characterized synthetic stereoisomers enabled us to rectify the structure of halisphingosine A as (2R,3R,8R,Z)-2-aminooctadec-9-ene-1,3,8-triol, and to pursue further structure-activity relation (SAR) studies regarding their antimicrobial and cytotoxic potential. In summary, our study offers a yet unreported compound library along with validated analytical datasets of marine sphingoid base derivatives, which significantly affects future ecometabolomic marine research and will facilitate the identification of inhibitors of sphingolipid metabolism or antagonists of sphingolipid base-sensing receptors.

Members of the bacterial phylum Planctomycetota have recently emerged as promising and for the most part untapped sources of novel bioactive compounds. The characterization of more than 100 novel species in the last decade stimulated recent bioprospection studies that start to unveil the chemical repertoire of the phylum. In this study, we performed systematic bioinformatic analyses based on the genomes of all 131 described members of the current phylum focusing on the identification of type III polyketide synthase (PKS) genes. Type III PKSs are versatile enzymes involved in the biosynthesis of a wide array of structurally diverse natural products with potent biological activities. We identified 96 putative type III PKS genes of which 58 are encoded in an operon with genes encoding a putative oxidoreductase and a methyltransferase. Sequence similarities on protein level and the genetic organization of the operon point towards a functional link to the structurally related hierridins recently discovered in picocyanobacteria. The heterologous expression of planctomycetal type III PKS genes from strains belonging to different families in an engineered Corynebacterium glutamicum strain led to the biosynthesis of pentadecyl- and heptadecylresorcinols. Phenotypic assays performed with the heterologous producer strains and a constructed type III PKS gene deletion mutant suggest that the natural function of the identified compounds differs from that confirmed in other bacterial alkylresorcinol producers. Key points • Planctomycetal type III polyketide synthases synthesize long-chain alkylresorcinols. • Phylogenetic analyses suggest an ecological link to picocyanobacterial hierridins. • Engineered C. glutamicum is suitable for an expression of planctomycete-derived genes.

Abstract Background Although Basidiomycota produce pharmaceutically and ecologically relevant natural products, knowledge of how they coordinate their primary and secondary metabolism is virtually non-existent. Upon transition from vegetative mycelium to carpophore formation, mushrooms of the genus Psilocybe use l-tryptophan to supply the biosynthesis of the psychedelic tryptamine alkaloid psilocybin with the scaffold, leading to a strongly increased demand for this particular amino acid as this alkaloid may account for up to 2% of the dry mass. Using Psilocybe mexicana as our model and relying on genetic, transcriptomic, and biochemical methods, this study investigated if l-tryptophan biosynthesis and degradation in P. mexicana correlate with natural product formation. Results A comparative transcriptomic approach of gene expression in P. mexicana psilocybin non-producing vegetative mycelium versus producing carpophores identified the upregulation of l-tryptophan biosynthesis genes. The shikimate pathway genes trpE1, trpD, and trpB (encoding anthranilate synthase, anthranilate phosphoribosyltransferase, and l-tryptophan synthase, respectively) were upregulated in carpophores. In contrast, genes idoA and iasA, encoding indole-2,3-dioxygenase and indole-3-acetaldehyde synthase, i.e., gateway enzymes for l-tryptophan-consuming pathways, were massively downregulated. Subsequently, IasA was heterologously produced in Escherichia coli and biochemically characterized in vitro. This enzyme represents the first characterized microbial l-tryptophan-preferring acetaldehyde synthase. A comparison of transcriptomic data collected in this study with prior data of Psilocybe cubensis showed species-specific differences in how l-tryptophan metabolism genes are regulated, despite the close taxonomic relationship. Conclusions The upregulated l-tryptophan biosynthesis genes and, oppositely, the concomitant downregulated genes encoding l-tryptophan-consuming enzymes reflect a well-adjusted cellular system to route this amino acid toward psilocybin production. Our study has pilot character beyond the genus Psilocybe and provides, for the first time, insight in the coordination of mushroom primary and secondary metabolism.

Uropygial gland secretions of birds consist of host and bacteria derived compounds and play a major sanitary and feather-protective role. Here we report on our microbiome studies of the New Guinean toxic bird Pachycephala schlegelii and the isolation of a member of the Amycolatopsis genus from the uropygial gland secretions. Bioactivity studies in combination with co-cultures, MALDI imaging and HR-MS/MS-based network analyses unveil the basis of its activity against keratinolytic bacteria and fungal skin pathogens. We trace the protective antimicrobial activity of Amycolatopsis sp. PS_44_ISF1 to the production of rifamycin congeners, ciromicin A and of two yet unreported compound families. We perform NMR and HR-MS/MS studies to determine the relative structures of six members belonging to a yet unreported lipopeptide family of pachycephalamides and of one representative of the demiguisins, a new hexapeptide family. We then use a combination of phylogenomic, transcriptomic and knock-out studies to identify the underlying biosynthetic gene clusters responsible for the production of pachycephalamides and demiguisins. Our metabolomics data allow us to map molecular ion features of the identified metabolites in extracts of P. schlegelii feathers, verifying their presence in the ecological setting where they exert their presumed active role for hosts. Our study shows that members of the Actinomycetota may play a role in avian feather protection.

The human microbiome emerges as a promising reservoir for diagnostic markers and therapeutics. Since host-associated microbiomes at various body sites differ and diseases do not occur in isolation, a comprehensive analysis strategy highlighting the full potential of microbiomes should include diverse specimen types and various diseases. To ensure robust data quality and comparability across specimen types and diseases, we employ standardized protocols to generate sequencing data from 1931 prospectively collected specimens, including from saliva, plaque, skin, throat, eye, and stool, with an average sequencing depth of 5.3 gigabases. Collected from 515 patients, these samples yield an average of 3.7 metagenomes per patient. Our results suggest significant microbial variations across diseases and specimen types, including unexpected anatomical sites. We identify 583 unexplored species-level genome bins (SGBs) of which 189 are significantly disease-associated. Of note, the existence of microbial resistance genes in one specimen was indicative of the same resistance genes in other specimens of the same patient. Annotated and previously undescribed SGBs collectively harbor 28,315 potential biosynthetic gene clusters (BGCs), with 1050 significant correlations to diseases. Our combinatorial approach identifies distinct SGBs and BGCs, emphasizing the value of pan-body pan-disease microbiomics as a source for diagnostic and therapeutic strategies. In this large-scale metagenomics study encompassing 3,483 human host-derived samples from seven body sites, researchers identify 583 unreported single-genome bins and report 314 metagenome-disease as well as 814 biosynthetic gene-disease associations.

AbstractBakterien der marinen Roseobacter‐Gruppe spielen eine wichtige Rolle in globalen biogeochemischen Prozessen. Prominente Vetreter dieser Gruppe produzieren schwefelhaltige Aminolipide (SAL), die für die Bildung von Biofilmen und die Besiedlung von Meeresoberflächen von entscheidender Bedeutung sind. Obwohl Genome Mining‐Ansätze und massenspektrometrische Studien homotaurinhaltige Strukturen für eine Gruppe von SALs postulierten, blieben deren relative und absolute Strukturen bisher unbekannt, was biochemische und funktionelle Untersuchungen behinderte. In dieser Studie konnten wir die absoluten Strukturen durch eine Kombination von analytischen Techniken, Isolierungs‐ und Abbauexperimenten sowie Totalsynthese bestimmen. Im Gegensatz zu vorherigen Strukturvorschlägen sind die hier untersuchten Aminolipide durch eine ungewöhnliche N,O‐acylierte Cysteinolsäure Kopfgruppe gekennzeichnet, weshalb wir die Substanzklasse Cysteinolide genannt haben. Durch gezielte Netzwerk‐basierende metabolomische Studien konnten wir zudem die Verteilung und strukturelle Vielfalt von Cysteinoliden in verschiedenee Vertretern der bakteriellen Roseobacter‐Gruppe kartieren. Insgesamt konnten in dieser Studie 14 verschiedene Aminolipide, einschließlich der in dieser Studie isolierten Cysteinolide, synthetisiert werden. Der Vergleich der erhaltenen analytischen Daten ermöglichte tiefergehende strukturelle Einblicke in die Charakteristika diese Substanzgruppe, welche für Studien zum bakteriellen Sulfonolipid‐Stoffwechsel und zu biogeochemischen Nährstoffkreislauf in den Ozeanen von großer Bedeutung sein werden.