Multiple mutations in the Nav1.4 sodium channel of New Guinean toxic birds provide autoresistance to deadly batrachotoxin.

Toxicity has evolved multiple times across the tree of life and serves important functions related to hunting, defence and parasite deterrence. Toxins are produced either in situ by the toxic organism itself or associated symbionts, or acquired through diet. The ability to exploit toxins from external sources requires adaptations that prevent toxic effects on the consumer (autoresistance). Here, we examine genomic adaptations that could facilitate autoresistance to the diet-acquired potent neurotoxic alkaloid batrachotoxin (BTX) in New Guinean toxic birds. Our work documents two new toxic bird species and shows that toxic birds carry multiple mutations in the SCN4A gene that are under positive selection. This gene encodes the most common vertebrate muscle Nav channel (Nav1.4). Molecular docking results indicate that some of the mutations that are present in the pore-forming segment of the Nav channel, where BTX binds, could reduce its binding affinity. These mutations should therefore prevent the continuous opening of the sodium channels that BTX binding elicits, thereby preventing muscle paralysis and ultimately death. Although these mutations are different from those present in Neotropical Phyllobates poison dart frogs, they occur in the same segments of the Nav1.4 channel. Consequently, in addition to uncovering a greater diversity of toxic bird species than previously known, our work provides an intriguing example of molecular-level convergent adaptations allowing frogs and birds to ingest and use the same neurotoxin. This suggests that genetically modified Nav1.4 channels represent a key adaptation to BTX tolerance and exploitation across vertebrates.

GNPS-Guided Discovery of Madurastatin Siderophores from the Termite-Associated Actinomadura sp. RB99.

In this study, we analyzed if Actinomadura sp. RB99 produces siderophores that that could be responsible for the antimicrobial activity observed in co-cultivation studies. Dereplication of high-resolution tandem mass spectrometry (HRMS/MS) and global natural product social molecular networking platform (GNPS) analysis of fungus-bacterium co-cultures resulted in the identification of five madurastatin derivatives (A1, A2, E1, F, and G1), of which were four new derivatives. Chemical structures were unambiguously confirmed by HR-ESI-MS, 1D and 2D NMR experiments, as well as MS/MS data and their absolute structures were elucidated based on Marfey's analysis, DP4+ probability calculation and total synthesis. Structure analysis revealed that madurastatin E1 (2) contained a rare 4-imidazolidinone cyclic moiety and madurastatin A1 (5) was characterized as a Ga3+ -complex. The function of madurastatins as siderophores was evaluated using the fungal pathogen Cryptococcus neoformans as model organism. Based on homology models, we identified the putative NRPS-based gene cluster region of the siderophores in Actinomadura sp. RB99.

Targeted Isolation of Saalfelduracin B-D from Amycolatopsis saalfeldensis Using LC-MS/MS-Based Molecular Networking.

High-resolution tandem mass spectrometry (HR-MS2)-based metabolomic studies of Amycolatopsis saalfeldensis, isolated from the "Saalfelder Feengrotten" caves in Germany, led to the isolation of three ribosomally synthesized and post-translationally modified type II thiopeptides, saalfelduracin B-D (1-3) and the known saalfelduracin A (4). The structures of all four compounds were determined by comparative two-dimensional NMR analysis and high-resolution tandem mass spectrometry.

Targeted Discovery of Tetrapeptides and Cyclic Polyketide-Peptide Hybrids from a Fungal Antagonist of Farming Termites.

Herein, we report the targeted isolation and characterization of four linear nonribosomally synthesized tetrapeptides (pseudoxylaramide A-D) and two cyclic nonribosomal peptide synthetase-polyketide synthase-derived natural products (xylacremolide A and B) from the termite-associated stowaway fungus Pseudoxylaria sp. X187. The fungal strain was prioritized for further metabolic analysis based on its taxonomical position and morphological and bioassay data. Metabolic data were dereplicated based on high-resolution tandem mass spectrometry data and global molecular networking analysis. The structure of all six new natural products was elucidated based on a combination of 1D and 2D NMR analysis, Marfey's analysis and X-ray crystallography.

Polyhalogenation of Isoflavonoids by the Termite-Associated Actinomadura sp. RB99.

Based on high-resolution tandem mass spectrometry (HR-MS2) and global natural products social molecular networking (GNPS), we found that plant-derived daidzein and genistein derivatives are polyhalogenated by termite-associated Actinomadura species RB99. MS-guided purification from extracts of bacteria grown under optimized conditions led to the isolation of eight polychlorinated isoflavones, including six unreported derivatives, and seven novel polybrominated derivatives, two of which showed antimicrobial activity.

Molecular networking and computational NMR analyses uncover six polyketide-terpene hybrids from termite-associated Xylaria isolates.

Fungi constitute the Earth's second most diverse kingdom, however only a small percentage of these have been thoroughly examined and categorized for their secondary metabolites, which still limits our understanding of the ecological chemical and pharmacological potential of fungi. In this study, we explored members of the co-evolved termite-associated fungal genus Xylaria and identified a family of highly oxygenated polyketide-terpene hybrid natural products using an MS/MS molecular networking-based dereplication approach. Overall, we isolated six no yet reported xylasporin derivatives, of which xylasporin A (1) features a rare cyclic-carbonate moiety. Extensive comparative spectrometric (HRMS2) and spectroscopic (1D and 2D NMR) studies allowed to determine the relative configuration across the xylasporin family, which was supported by chemical shift calculations of more than 50 stereoisomers and DP4+ probability analyses. The absolute configuration of xylasporin A (1) was also proposed based on TDDFT-ECD calculations. Additionally, we were able to revise the relative and absolute configurations of co-secreted xylacremolide B produced by single x-ray crystallography. Comparative genomic and transcriptomic analysis allowed us to deduce the putative biosynthetic assembly line of xylasporins in the producer strain X802, and could guide future engineering efforts of the biosynthetic pathway.

Heterologous expression of the cryptic mdk gene cluster and structural revision of maduralactomycin A.

After conducting an in silico analysis of the cryptic mdk cluster region and performing transcriptomic studies, an integrative Streptomyces BAC Vector containing the mdk gene sequence was constructed. The heterologous expression of the mdk cluster in Streptomyces albus J1074 resulted in the production of the angucyclic product, seongomycin, which allowed for the assesment of its antibacterial, antiproliferative, and antiviral activities. Heterologous production was further confirmed by targeted knock-out experiments involving key regulators of the biosynthetic pathways. We were further able to revise the core structure of maduralactomycin A, using a computational approach.

Adaptations of Pseudoxylaria towards a comb-associated lifestyle in fungus-farming termite colonies.

Characterizing ancient clades of fungal symbionts is necessary for understanding the evolutionary process underlying symbiosis development. In this study, we investigated a distinct subgeneric taxon of Xylaria (Xylariaceae), named Pseudoxylaria, whose members have solely been isolated from the fungus garden of farming termites. Pseudoxylaria are inconspicuously present in active fungus gardens of termite colonies and only emerge in the form of vegetative stromata, when the fungus comb is no longer attended ("sit and wait" strategy). Insights into the genomic and metabolic consequences of their association, however, have remained sparse. Capitalizing on viable Pseudoxylaria cultures from different termite colonies, we obtained genomes of seven and transcriptomes of two Pseudoxylaria isolates. Using a whole-genome-based comparison with free-living members of the genus Xylaria, we document that the association has been accompanied by significant reductions in genome size, protein-coding gene content, and reduced functional capacities related to oxidative lignin degradation, oxidative stress responses and secondary metabolite production. Functional studies based on growth assays and fungus-fungus co-cultivations, coupled with isotope fractionation analysis, showed that Pseudoxylaria only moderately antagonizes growth of the termite food fungus Termitomyces, and instead extracts nutrients from the food fungus biomass for its own growth. We also uncovered that Pseudoxylaria is still capable of producing structurally unique metabolites, which was exemplified by the isolation of two novel metabolites, and that the natural product repertoire correlated with antimicrobial and insect antifeedant activity.

GNPS-guided discovery of xylacremolide C and D, evaluation of their putative biosynthetic origin and bioactivity studies of xylacremolide A and B.

Targeted HRMS2-GNPS-based metabolomic analysis of Pseudoxylaria sp. X187, a fungal antagonist of the fungus-growing termite symbiosis, resulted in the identification of two lipopeptidic congeners of xylacremolides, named xylacremolide C and D, which are built from d-phenylalanine, l-proline and an acetyl-CoA starter unit elongated by four malonyl-CoA derived ketide units. The putative xya gene cluster was identified from a draft genome generated by Illumina and PacBio sequencing and RNAseq studies. Biological activities of xylacremolide A and B were evaluated and revealed weak histone deacetylase inhibitory (HDACi) and antifungal activities, as well as moderate protease inhibition activity across a panel of nine human, viral and bacterial proteases.

The Termite Fungal Cultivar Termitomyces Combines Diverse Enzymes and Oxidative Reactions for Plant Biomass Conversion.

Macrotermitine termites have domesticated fungi in the genus Termitomyces as their primary food source using predigested plant biomass. To access the full nutritional value of lignin-enriched plant biomass, the termite-fungus symbiosis requires the depolymerization of this complex phenolic polymer. While most previous work suggests that lignocellulose degradation is accomplished predominantly by the fungal cultivar, our current understanding of the underlying biomolecular mechanisms remains rudimentary. Here, we provide conclusive omics and activity-based evidence that Termitomyces employs not only a broad array of carbohydrate-active enzymes (CAZymes) but also a restricted set of oxidizing enzymes (manganese peroxidase, dye decolorization peroxidase, an unspecific peroxygenase, laccases, and aryl-alcohol oxidases) and Fenton chemistry for biomass degradation. We propose for the first time that Termitomyces induces hydroquinone-mediated Fenton chemistry (Fe2+ + H2O2 + H+ → Fe3+ + •OH + H2O) using a herein newly described 2-methoxy-1,4-dihydroxybenzene (2-MH2Q, compound 19)-based electron shuttle system to complement the enzymatic degradation pathways. This study provides a comprehensive depiction of how efficient biomass degradation by means of this ancient insect's agricultural symbiosis is accomplished. IMPORTANCE Fungus-growing termites have optimized the decomposition of recalcitrant plant biomass to access valuable nutrients by engaging in a tripartite symbiosis with complementary contributions from a fungal mutualist and a codiversified gut microbiome. This complex symbiotic interplay makes them one of the most successful and important decomposers for carbon cycling in Old World ecosystems. To date, most research has focused on the enzymatic contributions of microbial partners to carbohydrate decomposition. Here, we provide genomic, transcriptomic, and enzymatic evidence that Termitomyces also employs redox mechanisms, including diverse ligninolytic enzymes and a Fenton chemistry-based hydroquinone-catalyzed lignin degradation mechanism, to break down lignin-rich plant material. Insights into these efficient decomposition mechanisms reveal new sources of efficient ligninolytic agents applicable for energy generation from renewable sources.

Metabolic Pathway Rerouting in Paraburkholderia rhizoxinica Evolved Long-Overlooked Derivatives of Coenzyme F420.

Coenzyme F420 is a specialized redox cofactor with a negative redox potential. It supports biochemical processes like methanogenesis, degradation of xenobiotics, and the biosynthesis of antibiotics. Although well-studied in methanogenic archaea and actinobacteria, not much is known about F420 in Gram-negative bacteria. Genome sequencing revealed F420 biosynthetic genes in the Gram-negative, endofungal bacterium Paraburkholderia rhizoxinica, a symbiont of phytopathogenic fungi. Fluorescence microscopy, high-resolution LC-MS, and structure elucidation by NMR demonstrated that the encoded pathway is active and yields unexpected derivatives of F420 (3PG-F420). Further analyses of a biogas-producing microbial community showed that these derivatives are more widespread in nature. Genetic and biochemical studies of their biosynthesis established that a specificity switch in the guanylyltransferase CofC reprogrammed the pathway to start from 3-phospho-d-glycerate, suggesting a rerouting event during the evolution of F420 biosynthesis. Furthermore, the cofactor activity of 3PG-F420 was validated, thus opening up perspectives for its use in biocatalysis. The 3PG-F420 biosynthetic gene cluster is fully functional in Escherichia coli, enabling convenient production of the cofactor by fermentation.