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Lupins are high-protein crops that are rapidly gaining interest as hardy alternatives to soybean; however, they accumulate antinutritional alkaloids of the quinolizidine type (QAs). Lupin domestication was enabled by the discovery of genetic loci conferring low QA levels (sweetness), but the precise identity of the underlying genes remains uncertain. We show that pauper, the most common sweet locus in white lupin, encodes an acetyltransferase (AT) unexpectedly involved in the early QA pathway. In pauper plants, a single-nucleotide polymorphism (SNP) strongly impairs AT activity, causing pathway blockage. We corroborate our hypothesis by replicating the pauper chemotype in narrow-leafed lupin via mutagenesis. Our work adds a new dimension to QA biosynthesis and establishes the identity of a lupin sweet gene for the first time, thus facilitating lupin breeding and enabling domestication of other QA-containing legumes.

Naturstoffer har gennem tiden været en vigtig kilde til lægemidler, og mange lægemidler, der bruges i dag, har sin oprindelse i naturen. Specielt endofytiske svampe har i nyere tid haft øget fokus grundet deres evne til at producere mange forskellige metabolitter. Planter af slægten Eremophila har været brugt som medicinske planter af Australiens Aboriginals, og metabolitter fra disse planter har udvist blandt andet antibakteriel, antidiabetisk og anticancer aktivitet. I dette projekt er ni endofytiske svampe, som er isoleret fra rødder af Eremophila arter, undersøgt for ændringer i produktionen af metabolitter, når de blev dyrket under forskellige forhold med ”OneStrain-MAny-Compounds” (OSMAC) metoden. Kemisk profilering for de ni svampe dyrket på syv forskellige medier blev udført med analytisk skala HPLC. Fra de kemiske profiler kunne det ses, at metabolitproduktionen ændredes signifikant for nogle svampe, mens den for andre svampe ikke blev påvirket betydeligt. Ekstrakter fra otte svampe blev undersøgt med et disk-diffusions assay for antibakteriel aktivitet mod S. aureus og P. aeruginosa, hvor ekstrakter fra svamp 7 udviste inhiberende aktivitet over for S. aureus. På ekstrakt fra denne svamp blev der udført High-resolution inhibition profiling, for at identificere aktive stoffer. I biochromatogrammet kunne det ses, at to stoffer var ansvarlige for den inhibitoriske aktivitet. Disse stoffer blev isoleret med semipræparativ HPLC og derefter blev de videre undersøgt med MS- og NMR-eksperimenter. Kun eksperimenter for det ene af de to aktive stoffer resulterede i tilstrækkelige data for strukturopklaring. Fra disse eksperimenter blev citrinin, en svampe-metabolit kendt for antibakteriel aktivitet, identificeret. Der sås en betydelig forskel i den kemiske profil fra svamp 1 dyrket på medie, der indeholdt en epigenetisk modifier, sammenlignet med når den blev dyrket på medie uden epigenetisk, Natural products have been a valuable source of drugs throughout history, and many drugs used today originate from nature. Especially endophytic fungi have gained attention in recent times due to their ability to produce a wide variety of secondary metabolites. Plants of the Eremophila genus have been used as traditional medicine by the Aboriginal Peoples of Australia, and metabolites from these plants have shown many different activities such as antibacterial, antidiabetic, and anticancer activity. In this project, nine endophytes from roots of Eremophila species were investigated regarding changes in their metabolite production when cultivated under different conditions with the “OneStrain-Many-Compounds” (OSMAC) method. Chemical profiles from extracts of the nine fungi cultivated on seven different media were constructed with analytical scale HPLC. Here, it was seen that some fungi changed significantly when the growth medium changed, while the metabolite production of other fungi was not affected. Extracts from eight endophytes were tested with disk-diffusion assay for inhibitory effect on S. aureus and P. aeruginosa, where fungus 7 showed inhibition of S. aureus. High-resolution inhibition profiling was performed on extracts from this fungus to identify the active compounds in the extract. In the biochromatogram it was seen that two compounds exhibited inhibitory activity. These compounds were isolated with semi-preparative HPLC for further investigation by NMR and MS experiments. Only one of the active compounds displayed sufficient data for structure elucidation, and this compound was found to be the fungal metabolite, citrinin, which is also known to have antibacterial activity. Furthermore, extract from fungus 1 grown on medium containing the epigenetic modifier, 5- azacytidine, were further investigated, due to the significant changes in the chemical profile compared to the che

Gennem tiden er naturprodukter blevet anvendt som medicin til at behandle forskellige sygdomme og lidelser. Selv i dag bliver naturprodukter brugt som inspirationskilde, idet 25% af alle lægemidler godkendt mellem 1981 og 2019 enten er fundet direkte i naturen eller er derivater af naturprodukter. For antibakterielle lægemidler var dette tal 55%, og der er derfor et stort potentiale i at søge efter nye antibakterielle lægemidler i naturen. Ydermere, er der med den stigende antibiotikaresistens et øget behov for at udvikle disse antibakterielle stoffer og opdage nye. 9 endofytiske svampe blev groet på 7 forskellige vækstmedier via OSMAC-metoden og ekstrakter fra disse endofytter blev analyseret med analytisk HPLC. Alle kemiske profiler blev grundigt gennemgået og sammenlignet, og det blev observeret, at flere af svampene havde forskellige kemiske profiler som afhang af vækstmediet de var groet på. Specielt én svamp producerede flere forskellige metabolitter og særligt på et af medierne, og det blev derfor valgt at undersøge denne svamp nærmere. Ekstrakt fra denne svamp blev derfor fraktioneret på semipræparativ HPLC og adskillige metabolitter blev opsamlet og isoleret. En af disse metabolitter blev analyseret ved 1D og 2D NMR-spektroskopi og strukturopklaret. Dette stof blev fundet i litteraturen og viste sig at være det kendte stof fusarin A. Der blev udført en screening af alle ekstrakter for antibakteriel aktivitet mod både grampositive og gramnegative bakterier. Ingen af svampene udviste antibakterielle egenskaber mod gramnegative bakterier og kun én svamp udviste antibakterielle egenskaber mod grampositive bakterier. Der blev udført high-resolution inhibition profilering på to ekstrakter fra denne svamp for at undersøge de aktive metabolitter og biokromatogrammer blev dannet ud fra resultaterne. Biokromatogrammerne viste at to metabolitter stod for størstedelen af de antibakterielle egen, Throughout history, natural products have been used for medicinal purposes to treat various ailments and diseases. Even to this day, natural products are used as inspiration in drug discovery, as 25% of all drugs approved in the last 40 years were either unaltered natural products or natural product derivates. For antibacterial agents, this number was 55% and therefore there is a massive potential in searching for new antibacterial drug leads in nature. Furthermore, with the rising in antibacterial resistance, there is a need for increased drug development and drug discovery in this field. 9 endophytic fungi were cultivated on 7 different media in accordance with the OSMAC approach and extracts from the endophytes were analyzed by analytical-scale HPLC. All chemical profiles were thoroughly examined and compared, and it was seen that several fungi had different chemical profiles depending on the medium they were cultivated on. Especially one fungus appeared to produce various metabolites on one of the media and was therefore chosen for further investigation. Extract of this fungus was subjected to fractionation by semipreparative HPLC and several metabolites were isolated. One of these metabolites was subjected to 1D and 2D NMR spectroscopy and the structure was elucidated. The compound was found in literature as the known polyketide of fusarin A. Furthermore, the extracts were screened for antibacterial effect on both gram-positive and gramnegative bacteria. None of the fungi showed any antibacterial effect towards gram-negative bacteria and only one fungus showed antibacterial activity towards gram-positive bacteria. Two extracts of this fungus were subjected to high-resolution inhibition profiling to investigate which metabolites were responsible for the antibacterial effects and biochromatograms were constructed of the results. These showed that two metabolites were the major contributors to

Discovery of sustainable and benign-by-design drugs to combat emerging health pandemics calls for new analytical technologies to explore the chemical and pharmacological properties of Nature's unique chemical space. Here, we present a new analytical technology workflow, polypharmacology-labeled molecular networking (PLMN), where merged positive and negative ionization tandem mass spectrometry-based molecular networking is linked with data from polypharmacological high-resolution inhibition profiling for easy and fast identification of individual bioactive constituents in complex extracts. The crude extract of Eremophila rugosa was subjected to PLMN analysis for the identification of antihyperglycemic and antibacterial constituents. Visually easy-interpretable polypharmacology scores and poly pharmacology pie charts as well as microfractionation variation scores of each node in the molecular network provided direct information about each constituent's activity in the seven assays included in this proof-of-concept study. A total of 27 new non canonical nerylneryl diphosphate-derived diterpenoids were identified. Serrulatane ferulate esters were shown to be associated with antihyperglycemic and antibacterial activities, including some showing synergistic activity with oxacillin in clinically relevant (epidemic) methicillin-resistant Staphylococcus aureus strains and some showing saddle-shaped binding to the active site of protein tyrosine phosphatase 1B. PLMN is scalable in the number and types of assays included and thus holds potential for a paradigm shift toward polypharmacological natural-products-based drug discovery.