Christian Paetz

Faecal bile acids (BAs) are increasingly used in archaeological studies to detect domesticated species and reconstruct herding practices. However, it is unclear how environmental factors influence BA profiles and how much these profiles vary across regions. This uncertainty raises questions about their reliability for species-specific identifications across diverse settings. To address this, we analysed faecal BA profiles from free-grazing sheep ( Ovis aries ) across a wide range of climatic and elevational zones in Iceland, Germany, Tanzania, Iran, Mongolia, and Tibet. To test whether closely related species living under similar environmental conditions can be distinguished based on their BA profiles, we also analysed goats ( Capra aegagrus ssp. hircus ) from Tanzania and blue sheep ( Pseudois nayaur ) from Tibet, each compared to their respective co-occurring domestic sheep. We find significant regional differences in sheep primary, secondary, and oxo-BA profiles, with BA abundances influenced by altitude and precipitation. For instance, cholic acid varies with precipitation, while deoxycholic acid and lithocholic acid are significantly influenced by elevation. Oxo-BAs consistently account for 50–80% of total BAs in these animal groups, comparable to cattle (~ 60%) and much higher than in monogastric species (9–25%), suggesting their potential for separating ruminants from non-ruminants. While no individual BA or ratio serves as a universal marker for sheep, intra-regional comparisons suggest that goats and sheep in Tanzania, and blue sheep and domesticated sheep in Tibet, can be distinguished based on their faecal BA profiles. These findings show that caution is needed when applying modern BA reference data across regions. Species identification based on one region’s samples may lead to misinterpretation elsewhere. Because BA composition is shaped by both species and environment, robust interpretation requires a regionally grounded, multi-proxy approach.

Diterpene resin acids are abundant oleoresin compounds in conifer trees and persistent pollutants in pulp mill effluents. The bacterium Pseudomonas abietaniphila BKME-9 has previously been reported to metabolize abietane-type diterpene resin acids, but the complete catabolic pathway and the identities of downstream metabolites are not yet fully elucidated. In this study, time-resolved metabolomics was used to characterize the metabolism of dehydroabietic acid (1) by P. abietaniphila. In addition to the known intermediates 7β-hydroxy-dehydroabietic acid (2), 7-oxo-dehydroabietic acid (3), and 11,12-dihydroxy-7-oxoabieta-8,13-dien-18-oic acid (4), four previously undescribed diterpenoid acids were detected at later incubation times. These new metabolites were identified as 5-hydroxy-dehydroabietic acid (5), 5,7β-dihydroxyabietan-18-oic acid (6), 5-hydroxy-pimara-8-en-18-oic acid (7), and 5-hydroxy-7-oxo-pimara-8-en-18-oic acid (8) by spectroscopic analysis. Time-course data revealed sequential formation and accumulation of these compounds through new oxidations and rearrangements of dehydroabietic acid. These findings expand the known diversity of microbial diterpene metabolites and provide insights into the metabolic network of P. abietaniphila involved in transforming plant-derived diterpenoids.

After consumption by herbivores, plant antimicrobial defense compounds may enhance herbivore immunity to pathogenic microbes. In conifer-bark beetle interactions, beetles ingest large quantities of phloem tissue containing high concentrations of antimicrobial phenolic glucosides, such as stilbenes and flavonoids. It is not known, however, if these compounds increase bark beetle resistance to pathogens. We showed that Eurasian spruce bark beetles ( Ips typographus ) attacking Norway spruce ( Picea abies ) hydrolyze phenolic glucosides to their corresponding aglucones increasing their antifungal activity. However, the entomopathogen Beauveria bassiana , a natural fungal parasite of these beetles, detoxifies stilbene and flavonoid aglucones by forming methylglucoside derivatives. A two-step pathway involving a UDP-glycosyltransferase and an O -methyltransferase produces phenolic O -methylglucosides that are no longer toxic to B. bassiana and are stable to β-glucosidase action. Compared to wild-type strains of B. bassiana , mutant strains knocked out in the genes of this pathway exhibited decreased methylglucoside formation, slower growth on medium containing phenolic compounds, and reduced virulence toward bark beetles. Hence, methylglucosylation of plant-derived phenolics is a detoxification process that significantly increases the ability of B. bassiana to parasitize host insects consuming plant tissue high in phenolics, such as conifer phloem. This is one of the few examples of an entomopathogen that is able to resist the plant-derived defenses of an insect host.

Major secondary metabolites of Physalis plants, epoxidized withanolides, have a potent feeding deterrent and growth inhibitory effect on most herbivorous insects. Caterpillars of the specialist moth species Heliothis (Chloridea) subflexa consume only Physalis fruits, whereas the closely related generalist Heliothis (Chloridea) virescens feeds on 14 different plant families, but not Physalis . The two species have different physiological responses to dietary withanolides, so it is wondered whether they metabolize withanolides differently. The Physalis peruviana plants are grown in a [ ¹³ C]CO ₂ ‐supplied atmosphere, 4 β ‐hydroxywithanolide E is isolated and purified from the leaves, and the compound is fed to the caterpillars. Subsequent high‐performance liquid chromatography with diode array UV‐vis detection coupled to high‐resolution electrospray ionization mass spectrometry (HPLC‐DAD‐HRESIMS) and nuclear magnetic resonance (NMR) analyses of the main metabolite isolated from the frass show that both species convert 4 β ‐hydroxywithanolide E mainly to withanolide S, probably by the action of an epoxide hydrolase. Withanolide S is completely characterized regarding its NMR and electronic circular dichroism data. To date, this is the first study to analyze the fate of withanolides after ingestion by insects.

Fungal biodiversity significantly influences ecosystem dynamics through various interactions with plants, ranging from pathogenic to mutually beneficial associations. This study explores the fungal diversity associated with an ornamental orchid genus Epidendrum that is widely propagated globally but native to northern South America. Root samples were collected from Epidendrum in diverse geographic locations: Brazil, Colombia, Germany, Spain and six South African provinces. Fungal biodiversity was catalogued from the genomic DNA extracted from these roots using fungal-specific primers and Illumina MiSeq sequencing. Bioinformatic and statistical analyses revealed significant fungal diversity in the roots, with distinct dominant orders in each geographic region. Among the South African samples, significant differences were found in alpha diversity indices and species richness. Even though samples originating from different provinces overlapped in the PCoA plot, PERMANOVA indicated a significant difference in the fungal biodiversity, which was further supported by PERMDISP. In the global dataset, alpha diversity indices were insignificant, but species richness was. In the PCoA plot, data points clustered by sampling sites, indicating substantial differences in fungal biodiversity between the samples. This was validated by PERMANOVA and PERMDISP analyses. Outcomes from the core fungal analyses showed Epidendrum retained a conserved set of fungal orders from its native habitat when it transitioned to exotic regions, while it also formed new associations with local fungal communities in these introduced regions. These findings highlight the role of both core and region-specific fungal communities in the ecological adaptability and success of this widely planted orchid genus.

Here, we use transcriptomic data from seeds of Musella lasiocarpa to identify five enzymes involved in the formation of dihydrocurcuminoids. Characterization of the substrate specificities of the enzymes reveals two distinct dihydrocurcuminoid pathways leading to phenylphenalenones and linear diarylheptanoid derivatives, the major seed metabolites. Furthermore, we demonstrate the stepwise conversion of dihydrobisdemethoxycurcumin to the phenylphenalenone 4 '-hydroxylachnanthocarpone by feeding intermediates to M. lasiocarpa root protein extract.

Aposematic animals rely on diverse secondary metabolites for defence. Various hypotheses, such as competition, life history and multifunctionality, have been posited to explain defence variability and diversity. We investigate the compound selectivity hypothesis using large milkweed bugs, Oncopeltus fasciatus , to determine if distinct cardenolides vary in toxicity to different predators. We quantify cardenolides in the bug’s defensive secretions and body tissues and test the individual compounds against predator target sites, the Na ⁺ /K ⁺ -ATPases, that are predicted to differ in sensitivity. Frugoside, gofruside, glucopyranosyl frugoside and glucopyranosyl gofruside were the dominant cardenolides in the body tissues of the insects, whereas the two monoglycosidic cardenolides—frugoside and gofruside—were the most abundant in the defensive fluid. These monoglycosidic cardenolides were highly toxic (IC ₅₀ < 1 μM) to an invertebrate and a sensitive vertebrate enzyme, in comparison to the glucosylated compounds. Gofruside was the weakest inhibitor for a putatively resistant vertebrate predator. Glucopyranosyl calotropin, found in only 60% of bugs, was also an effective inhibitor of sensitive vertebrate enzymes. Our results suggest that the compounds sequestered by O. fasciatus probably provide consistency in protection against a range of predators and underscore the need to consider predator communities in prey defence evolution.

Medicinal compounds from plants include bicyclo[3.3.1]nonane derivatives, the majority of which are polycyclic polyprenylated acylphloroglucinols (PPAPs). Prototype molecules are hyperforin, the antidepressant constituent of St. John’s wort, and garcinol, a potential anticancer compound. Their complex structures have inspired innovative chemical syntheses, however, their biosynthesis in plants is still enigmatic. PPAPs are divided into two subclasses, named type A and B. Here we identify both types in Hypericum sampsonii plants and isolate two enzymes that regiodivergently convert a common precursor to pivotal type A and B products. Molecular modelling and substrate docking studies reveal inverted substrate binding modes in the two active site cavities. We identify amino acids that stabilize these alternative binding scenarios and use reciprocal mutagenesis to interconvert the enzymatic activities. Our studies elucidate the unique biochemistry that yields type A and B bicyclo[3.3.1]nonane cores in plants, thereby providing key building blocks for biotechnological efforts to sustainably produce these complex compounds for preclinical development.

Phenylphenalenones (PPs) are complex polycyclic natural products that play an important role in the chemical defense system of banana and plantain (Musaceae). Although suggestions for how plants synthesize the PP scaffold were first proposed more than 50 years ago, no biosynthetic information is yet available at the enzyme level. Here, we use transcriptomic data from seeds of Musella lasiocarpa , the Chinese dwarf banana, to identify five biosynthetic genes involved in the formation of dihydrocurcuminoids. Characterization of the substrate specificities of the enzymes reveals two distinct dihydrocurcuminoid pathways leading to the two types of major aromatic seed metabolites, the PPs and the linear diarylheptanoid derivatives. Furthermore, through multiple rounds of feeding potential intermediates to M. lasiocarpa root protein extract, followed by high-resolution mass spectrometry profiling, product isolation, and NMR-based elucidation, we demonstrate the stepwise conversion of a dihydrocurcumin-type precursor to the PP 4’-hydroxylachnanthocarpone. In contrast to the commonly hypothesized Diels–Alder cyclization mechanism, we propose an unexpected two-step cyclization route to the PP scaffold.

Phenylphenalenones (PhPNs), phytoalexins in wild bananas (Musaceae), are known to act against various pathogens. However, the abundance of PhPNs in many Musaceae plants of economic importance is low. Knowledge of the biosynthesis of PhPNs and the application of biosynthetic approaches to improve their yield is vital for fighting banana diseases. However, the processes of PhPN biosynthesis, especially those involved in methylation modification, remain unclear. Musella lasiocarpa is a herbaceous plant belonging to Musaceae, and due to the abundant PhPNs, their biosynthesis in M. lasiocarpa has been the subject of much attention. In this study, we assembled a telomere-to-telomere gapless genome of M. lasiocarpa as the reference, and further integrated transcriptomic and metabolomic data to mine the candidate genes involved in PhPN biosynthesis. To elucidate the diversity of PhPNs in M. lasiocarpa, three screened O-methyltransferases (Ml01G0494, Ml04G2958, and Ml08G0855) by phylogenetic and expressional clues were subjected to in vitro enzymatic assays. The results show that the three were all novel O-methyltransferases involved in the biosynthesis of PhPN phytoalexins, among which Ml08G0855 was proved to function as a multifunctional enzyme targeting multiple hydroxyl groups in PhPN structure. Moreover, we tested the antifungal activity of PhPNs against Fusarium oxysporum and found that the methylated modification of PhPNs enhanced their antifungal activity. These findings provide valuable genetic resources in banana breeding and lay a foundation for improving disease resistance through molecular breeding.