Integrating genomics and metabolomics to accelerate the discovery of anti-MRSA natural products from the endophytic fungus Neocucurbitaria sp. VM-36

Integrating genomics and metabolomics to accelerate the discovery of anti-MRSA natural products from the endophytic fungus Neocucurbitaria sp. 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VM-36 Xiao Li , Rosario del Carmen Flores-Vallejo , Ting He , View ORCID Profile Jan Maarten van Dijl , View ORCID Profile Kristina Haslinger doi: https://doi.org/10.1101/2025.10.09.681339 Xiao Li a Department of Chemical and Pharmaceutical Biology, Groningen Research Institute of Pharmacy, University of Groningen , Antonius Deusinglaan 1, 9713 AV Groningen, The Netherlands ; Find this author on Google Scholar Find this author on PubMed Search for this author on this site Rosario del Carmen Flores-Vallejo b Department of Medical Microbiology and Infection Prevention, University of Groningen, University Medical Center Groningen , Hanzeplein 1, Groningen 9700RB, The Netherlands ; Find this author on Google Scholar Find this author on PubMed Search for this author on this site Ting He a Department of Chemical and Pharmaceutical Biology, Groningen Research Institute of Pharmacy, University of Groningen , Antonius Deusinglaan 1, 9713 AV Groningen, The Netherlands ; Find this author on Google Scholar Find this author on PubMed Search for this author on this site Jan Maarten van Dijl b Department of Medical Microbiology and Infection Prevention, University of Groningen, University Medical Center Groningen , Hanzeplein 1, Groningen 9700RB, The Netherlands ; Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Jan Maarten van Dijl Kristina Haslinger a Department of Chemical and Pharmaceutical Biology, Groningen Research Institute of Pharmacy, University of Groningen , Antonius Deusinglaan 1, 9713 AV Groningen, The Netherlands ; Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Kristina Haslinger For correspondence: k.haslinger{at}rug.nl Abstract Full Text Info/History Metrics Supplementary material Preview PDF Abstract Endophytic fungi in medicinal plants are a rich source of bioactive natural products. Herein, we performed a comprehensive genomic and metabolic analysis of an uncharacterized endophytic fungus Neocucurbitaria sp. VM-36. Whole-genome sequencing and comparative analysis of the encoded biosynthetic gene clusters with six Cucurbitariaceae strains predicted its potential to produce compounds related to griseofulvin, usnic acid, hypothemycin, and phomasetin. Untargeted metabolomics confirmed several of these predictions with the presence of phomasetin analogs and isousnic acid, and uncovered a diverse range of other secondary metabolites, including specialized lipids, amino acids, and peptides, such as cyclic hexapeptides. We successfully isolated the main compound ( 1 ), a phomasetin analog, and show that it has bactericidal activity against different methicillin-resistant and -sensitive Staphylococcus aureus strains comparable in strength to vancomycin and daptomycin. Checkerboard assays with these compounds revealed mostly indifferent interactions. These findings demonstrate the antibacterial potential of compound 1 and Neocucurbitaria sp. VM-36. Download figure Open in new tab 1. Introduction Antibiotic resistance has become a global problem threatening human health 1 . Pathogenic bacteria can cause illnesses by producing toxins that neutralize the human immune defenses and damage healthy tissue, thereby causing harmful infections and posing a serious threat to public health 2 . While antibiotics were initially effective in treating bacterial infections, their widespread use has triggered the rapid transfer of antibiotic resistance genes between bacteria and led to prevalent bacterial resistance against clinically applied drugs 3 . For instance, certain Staphylococcus aureus strains, such as methicillin-resistant S. aureus (MRSA), have acquired the mecA gene, which encodes a low-affinity penicillin-binding protein (PBP2a) that allows cell wall synthesis to continue despite the presence of β-lactams 4 . Moreover, many bacterial pathogens can nowadays withstand multiple antibacterial agents, making it extremely difficult to treat the infections they cause 3 . This applies in particular to the so-called ESKAPE pathogens, which include Enterococcus faecium , S. aureus , Klebsiella pneumoniae , Acinetobacter baumannii , Pseudomonas aeruginosa and Enterobacter spp. Therefore, there is a pressing need to discover new antimicrobial agents. Endophytic fungi from medicinal plants are rich resources of novel drug leads 5 . So far, antimicrobial agents with diverse chemical scaffolds have been discovered in such endophytes, including polyketides, alkaloids, terpenoids, lactones, anthraquinones, quinones, glycosides, steroids and lignans 6 . Previously, we isolated endophytic fungi from Vinca minor , a medicinal plant from the Apocynaceae family, to obtain an in-house fungal library and conducted in-depth genomic and metabolomic analyses of two interesting fungi, namely Fusarium sp. VM-40 7 and Cosmosporella sp. VM-42 8 . An extensive screening of our fungal library for antibacterial activity also uncovered another fascinating fungus, denoted isolate VM-36. In particular, initial disk diffusion assays showed that its crude ethyl acetate (EtOAc) extract produced a significant inhibition zone against the indicator bacterium Bacillus subtilis 168. Furthermore, internal transcribed spacer (ITS) sequencing analysis indicated that the fungus belongs to Neocucurbitaria , a little studied genus from the Cucurbitariaceae family. So far, only 20 reports on this genus can be retrieved from the PubMed database, with the earliest publication dating to 2018. Most of these publications focus on fungal ecology, with only two studies addressing fungal metabolites. In particular, a total of 15 diterpenes and their derivatives were isolated from the marine fungus Neocucurbitaria unguis-hominis FS685, including the neocucurbols A-H and neocucurbins A-G 9 , 10 . However, these compounds showed neither antibacterial activity against Escherichia coli and S. aureus , nor anticancer activity against human cancer cell lines, including the SF-268 (glioblastoma carcinoma), MCF-7 (breast cancer), HepG-2 and A549 (both liver cancer cancer) cell lines 9 , 10 . Altogether, the limited information that is currently available on Neocucurbitaria species indicates that fungi belonging to this genus still offer great opportunity for investigation. In the present study, we conducted an in-depth analysis of the genome and metabolome of Neocucurbitaria sp. VM-36 to explore its capabilities for producing antibacterial secondary metabolites (SMs). We sequenced the whole genome of this fungus using Oxford Nanopore Technology, followed by contiguous assembly and annotation. We further predicted its biosynthetic gene clusters (BGCs) by AntiSMASH. We applied high-resolution, high-performance liquid chromatography-coupled tandem mass spectrometry (LC-MS/MS) and molecular networking for the analysis of its SMs. Besides, we isolated the dominant compound 1 from large-scale fermentation cultures and characterized it by 1D and 2D nuclear magnetic resonance (NMR) spectroscopy, Marfey’s method, and experimental and computational electronic circular dichroism (ECD). We also evaluated the antibacterial activity of compound 1 against several S. aureus strains, including interactions with vancomycin and daptomycin and potentially hemolytic effects on human red blood cells. Lastly, we propose the biosynthetic pathway of compound 1 by combining the obtained genome and metabolome data. Altogether, our study provides new insights into the biosynthetic potential of Neocucurbitaria sp. VM-36 and the possible development of new potent antibacterial agents for healthcare or agriculture. 2. Materials and Methods 2.1. Fungus Isolation, Morphology, and Phylogenetic Tree Analysis The fungus Neocucurbitaria sp. VM-36 was isolated from healthy leaves of Vinca minor in November, 2021 (Groningen, The Netherlands) as previously described 7 . The strain was subsequently stored at 8 L in the department of Chemical and Pharmaceutical Biology at the University of Groningen. To investigate the morphology of this fungus, we inoculated it onto potato dextrose agar (PDA), malt extract agar (MEA), synthetically nutrient-poor agar (SNA), and Sabouraud dextrose agar (SDA) plates. After incubation at 25 °C for 28 days, as well as 77 days for SDA, we determined the colony characteristics, including color, diameter, and mycelium morphology. Fungal hyphae were stained with lactophenol blue dye, and their microscopic features were examined with an optical microscope (Olympus BX41). Digital images were captured using a Leica camera (Heerbrugg, Switzerland) connected to the microscope. For the molecular identification of this fungus, we compared the sequence of its Internal Transcribed Spacer (ITS) region to 18 Neocucurbitaria strains, with Fusarium oxysporum Fo47 used as the outgroup, following the method described in our recent publication 7 . We furthermore performed a multi-locus phylogenetic analysis using three barcode sequences: ITS, the RNA polymerase II subunits 2 ( rpb2 ), and the large ribosomal subunit (LSU). Genome sequences of nine Cucurbitariaceae family strains, obtained from the Joint Genome Institute Genome Portal (JGI, https://genome.jgi.doe.gov/portal/ ) and NCBI ( https://www.ncbi.nlm.nih.gov/ ), were included alongside F. oxysporum Fo47 used as an outgroup. Sequences from the three loci were concatenated and aligned using ClustalW, and the Maximum Likelihood (ML) tree was generated in MEGA (v11) with 1000 bootstrap replicates 11 . 2.2. Whole-Genome Sequencing, Assembly, Gene Prediction, and Annotation DNA extraction, library preparation, sequencing, and assembly were conducted as described recently 7 . The raw reads were base-called using Guppy version 6.1.5 (Oxford Nanopore Technologies, Oxford, UK) in GPU mode using the dna_r10. 4_ e8.1_ sup. cfg model 12 . The base-called reads were subsequently filtered to a minimum length of 2 kb and a minimum quality of Q10 using NanoFilt (version 2.8.0) 13 . NanoPlot (version 1.40.0) 13 was used to evaluate the filtered reads. Assembly was performed using Flye (version 2.9-b1778). The quality of the genome assembly was evaluated using QUAST v5.1.0rc1 14 . Bandage (version 0.8.1) 15 was used to visualize the newly assembled genome of Neocucurbitaria sp. VM-36 (Figure S1). The draft assembly was subsequently polished in two rounds: first using Racon version 1.4.10 with default settings 16 and then Medaka version 0.11.5 with default settings. The completeness of the assembly was evaluated using BUSCO 5.4.3 (ascomycota_odb10 dataset). Genome annotation was carried out using the online platform Genome Sequence Annotation Server (GenSAS, https://www.gensas.org ), which provides a pipeline for whole-genome structural and functional annotation 17 . The sequencing data and genome assembly for this study were deposited in the European Nucleotide Archive at EMBL-EBI under accession number PRJEB97715. Gene Ontology (GO) annotation 18 , carbohydrate-active enzymes (CAZymes) annotation 19 , tRNA 20 and rRNA prediction ( https://github.com/tseemann/barrnap ) were further performed. The biosynthetic potential of Neocucurbitaria sp. VM-36, along with that of six other strains with available whole genome sequences, including Cucurbitaria berberidis CBS 394.84, Neocucurbitaria cava IMI 356814, Parafenestella ontariensis EI-6, Pyrenochaeta sp. DS3sAY3a, Pyrenochaeta inflorescentiae CORFU0001, and Fenestella fenestrata ATCC 66461, was analyzed by antiSMASH fungal version 7.0.1 21 under relaxed detection strictness and default settings. 2.3. Extraction of Secondary Metabolites and Molecular Networking-based LC-MS analysis Fungal mycelium of Neocucurbitaria sp. VM-36 was separately transferred to two different culture media, including PDA solid medium ( ϕ 35 mmX10 mm plate) and DPY liquid medium (20 mL medium in Erlenmeyer flask). Solid plates were incubated at 25 □ for 28 days, and their SMs were extracted with organic solvent as recently described 7 . Fungal mycelium in DPY liquid medium was cultured at 25 □ for 28 days, with shaking at 130 rpm. The fungal mycelium and broth were mixed with 20 mL EtOAc, sonicated and rotated in a rotating mixer for 1 h, respectively. The organic phase was subsequently collected and dried under a gentle stream of N 2 . The dried extract was resuspended in 1 mL of 1:1 MeOH-MilliQ water ( v / v ) and filtered with 0.45 µm polytetrafluoroethylene filters (Screening Devices BV company, The Netherlands). LC-MS/MS analysis of crude EtOAc extracts was performed as described before 7 . Fresh PDA and DPY media were used as blank control groups. The acquired data was processed by Thermo Scientific FreeStyle software version 1.8. Untargeted metabolomic analysis was conducted using the online workflow on the Global Natural Products Social Molecular Networking (GNPS) website ( https://gnps.ucsd.edu/ ) according to our previously described method 7 , 22 . The raw mass spectrometry data were deposited on GNPS under the accession number MassIVE ID: MSV000098902. A molecular networking analysis ( https://gnps.ucsd.edu/ProteoSAFe/status.jsp?task=9099def02462433487a26b71afb487e4 ) was conducted using the default settings in GNPS, except that the precursor ion mass tolerance and the MS/MS fragment ion tolerance were both set to 0.02 Da. The results were visualized in Cytoscape version 3.9.1 23 . Nodes from the blank PDA and DPY media were used as background and omitted to obtain the final molecular network. 2.4. Fermentation, Extraction, and Isolation The fungus Neocucurbitaria sp. VM-36 was grown on PDA plates at 25 □ for 28 days. 5 mycelial discs were used to inoculate a 3 □ Erlenmeyer flask containing DPY medium. 7 flasks with a total of 13.0 □ medium were cultured in a shaker at 25 □ at a speed of 130 rpm. After 30 days, the broth and mycelium were separated by filtration. The mycelium was ultrasonicated with EtOAc (3 times, 0.52 □ organic solvent each time, 1h, 30 L). The broth was extracted by EtOAc using liquid-liquid extraction (3 times, 6.5 □ solvent each time). The EtOAc extracts were combined and evaporated to dryness using a rotary evaporator (Hei-VAP Core Rotary Evaporators, Heidolph Instruments GmbH & CO. KG, Germany) to obtain a total of 1404 mg crude extract. The crude extract was further dissolved in 80% acetonitrile-water solvent and centrifuged to obtain the supernatant, which was used for MPLC separation (BUCHI Reveleris™ X2-UV System) on a C 18 column (FlashPure EcoFlex C 18 12 g column from Buchi®). The analytes were eluted with solution A (water) and solution B (acetonitrile) using a step-gradient program (10 min, 0 % B; 5 min, 0%-20% B; 10 min, 20% B; 5 min, 20%-40% B; 10 min, 40% B; 5 min, 40%-60% B; 10 min, 60% B; 5 min, 60%-80% B; 10 min, 80% B; 5 min, 80%-90% B; 10 min, 90% B) to give 7 fractions. The flow rate was 10.0 mL/min. UV detection was set at 210, 230, and 294 nm. Fractions F and G were separately concentrated by a rotary evaporator under reduced pressure at 35 L, and further purified by a preparative LC (Shimadzu LC-8A preparative liquid chromatograph system, SCL-10A vp system controller, SPD-10A UV-vis detector, SIL-10AP auto injector, and FRC-10A fraction collector) on a C 18 column (VarioPrep high-performance liquid chromatography (HPLC) Separation Column VP Nucleodur 100-5 C 18 ec, 10 × 250 mm, 5 μm particle size) with 75% ACN isocratic elution. Column temperature was set at 40 □. The flow rate was 7 mL/min, and UV detection was set at 290 nm. 15 fractions were obtained after separation. In order to obtain pure compounds, fractions 9, 10, and 11 were purified on a Shimadzu LC-10AT high-performance liquid chromatography system equipped with a Shimadzu SPD-M20A diode array detector and eluted on a C 18 column (EC 100/4.6 NUCLEOSHELL RP 18, 4.6 × 100 mm, 2.7 µm particle size) with isocratic 75% ACN at 0.8 mL/min flow rate, to yield compound 1 (114 mg, retention time (RT) = 8.5 min). 2.5. Structure Characterization 6 mg compound 1 was dissolved in DMSO-d 6 and used for NMR analysis. NMR spectra were obtained using a Bruker Ascend Evo TM 600 NMR spectrometer (Bruker-Biospin, Billerica, MA, USA). Chemical shifts (0) are reported in parts per million (ppm) and coupling constants ( J ) are reported in hertz (Hz). MestReNova 14 was used for data analysis. Marfey’s method was used to determine the amino acid configuration in compound 1 according to the reported method 24 with minor modification as outlined in the Supplementary Methods. UV and circular dichroism (CD) spectra were obtained on a JASCO V-660 spectrometer and JASCO J-815 CD spectrometer (JASCO corp., Tokyo, Japan), respectively. The wavelength scan was set to 210-400 nm. ECD calculations were performed following a reported reference 25 with minor modifications as described below. Conformational sampling was carried out using RDKit ( https://www.rdkit.org/ ) (ETKDGv3), and resulting conformers were energy-minimized with the MMFF94 force field. Further geometry optimization and frequency analysis were performed at the B3LYP/6-31G(d) level with the PCM solvent model for EtOH. TD-DFT calculations were performed for excited-state properties. Conformers without imaginary frequencies were subjected to ECD calculations at the B3LYP/6-31+G(d), performed without applying a solvent model due to the large computational cost and frequent convergence difficulties encountered when including solvent effects. ECD spectra were simulated using SpecDis with a half-bandwidth of 0.16–0.3 eV and averaged according to Boltzmann populations. All quantum chemical calculations were performed with Gaussian 16. Experimental and calculated spectra were compared for configuration assignment. 2.6. Antibacterial susceptibility assay Several bacterial strains were used in this assay (Table S1), including the following strains from the American Type Culture Collection (ATCC) and the National Collection of Type Cultures (NCTC): S. aureus ATCC 29213 (methicillin sensitive strain, MSSA), S. aureus NCTC 8325 (MSSA), Enterococcus faecalis ATCC 29212, Enterobacter cloacae ATCC 13047, Klebsiella pneumoniae ATCC 13883, Acinetobacter baumannii ATCC 17978, Pseudomonas aeruginosa ATCC 27853, and Escherichia coli ATCC 25922. In addition, we used S. aureus strain Newman 26 , S. aureus NE1688 (Sle1) (MRSA) from the Nebraska transposon library 27 , which carries a transposon insertion in the sle1 gene 28 , and the two clinical MRSA isolates D15 and D17, which were respectively associated with community- and hospital-acquired infections 29 . The minimum inhibitory concentrations (MICs) and minimum bactericidal concentrations (MBCs) of compound 1 were determined using the broth microdilution method in 96-well microtiter plates as described in the Supplementary Methods. The lowest concentration of compound 1 that completely inhibited bacterial growth was determined as the MIC, and the MBC was determined as the lowest concentration of compound 1 that reduced the viable bacterial count on agar plates by 99.9%. Vancomycin and daptomycin were used as positive controls. Bacteria treated with the respective vehicles were used as negative control. Bacterial growth kinetics of compound 1 , vancomycin, and daptomycin against 1 MSSA and 4 MRSA strains at final concentrations of 2, 4, and 8 µg/mL were monitored by measuring the optical density at 600 nm (OD 600 ) values. Bacterial viability before compound addition and at the endpoint was determined by colony-forming unit (CFU) enumeration, as detailed in the Supplementary Methods. All experiments were performed in triplicate. The drug interaction of compound 1 with daptomycin or vancomycin against the three MRSA strains, USA300, D15 and D17 was determined using the checkerboard assay in 96 well microplates as described in the Supplementary Methods. 2.7. In vitro cytotoxicity on human erythrocytes To evaluate the effect of compound 1 on human erythrocytes, a hemolysis assay was performed as detailed in the Supplementary Methods. Briefly, compound 1 was dissolved in DMSO at concentrations of 200, 100, 10, and 1 μg/mL. DMSO at 1%, 0.5%, 0.05% and 0.005% v/v concentrations were used as blanks. Sodium dodecyl sulfate at a final concentration of 0.1% w/v was used as the positive control and Dulbecco’s phosphate-buffered saline (DPBS) pH 7.2 served as a negative control. Vancomycin and daptomycin were included at the same final concentrations as compound 1 . Double-distilled water and NaCl 0.9% w/v were evaluated as their blanks, respectively. All groups were tested in triplicates. 2.8. Medical ethics committee approval Blood donations from healthy volunteers were collected based on written informed consent with approval of the medical ethics committee of the University Medical Center Groningen (UMCG; approval no. Metc2012-375), and in accordance with the Declaration of Helsinki guidelines. 3. Results 3.1. Morphological Characterization of Neocucurbitaria sp. VM-36 The Neocucurbitaria sp. VM-36 was cultured on different nutrient media and the macroscopic features of its colonies were recorded ( Figure 1A ). PDA and MEA media appeared to be more favorable for mycelial growth and conidia production compared with SNA and SDA media. On PDA plates, the colonies presented a diameter of 27-37 mm after 28 days with black mycelium and a textured center. On MEA plates, the colonies reached a diameter of 35-40 mm with a brown and white floccose mycelium and a non-hardened center. The fungus formed smaller colonies on SNA plates, with a diameter of 15-25 mm and white velvety aerial hyphae. Colonies on SDA plates were only 12-20 mm in diameter and had a markedly different yellow color. They appeared like solid particles at first and only at the final stage of the 28-day cultivation, a gray-white mycelium emerged on the top. Accordingly, we continued to culture the fungus on SDA plates until day 77 at which time point the colonies reached a diameter of 28-37 mm with brown mycelium and a textured center. On the reverse side the colonies were brown-yellow in the center and black in the periphery. Download figure Open in new tab Figure 1. Macroscopic (A) and microscopic (B) characteristics of Neocucurbitaria sp. VM-36 on different culture media (PDA, MEA, SDA, and SNA), incubated at 25 °C for 28 days or 77 days (SDA-2). (1) Hyphae; (2) Pycnidium-Conidiomata; (3) Pycnidia; (4) Conidiophores; (5) Conidia; and (6) Chlamydospore chains formed on SDA medium. Scale bars = 50 µm. Microscopy revealed that the fungus formed spherical pycnidia, an asexual structure lined with conidiophores for producing asexual conidiospores ( Figure 1B ). In addition, Neocucurbitaria sp. VM-36 also produces chlamydospores when grown on SDA plates, which support the survival under adverse conditions due to the formation of particularly thick walls. 3.2. Genomic analysis To characterize the genetic make-up of Neocucurbitaria sp. VM-36, we sequenced, assembled, annotated, and analyzed its whole genome (Table S2). With the highly complete (97.2 % according to BUSCO) and contiguous assembly with only 19 contigs at hand, we performed additional phylogenetic analyses to more accurately locate our isolate in the Neocucurbitaria genus. Both single-locus and multi-locus analyses consistently showed that Neocucurbitaria sp. VM-36 is closely related to N. salicis-albae CBS 144611 ( Figure 2 ), a species previously isolated from a Salix alba twig in Germany 30 . Download figure Open in new tab Figure 2. Phylogenetic analysis of Neocucurbitaria sp. VM-36. (A) ML phylogram of Neocucurbitaria sp. VM-36 based on the ITS sequence. (B) Maximum Likelihood (ML) phylogenetic tree based on concatenated nucleotide sequences of rpb2 , ITS, and LSU. F. oxysporum Fo47 was used as an outgroup in both analyses. Functional annotation of the assembly revealed that the genome encodes 11,817 proteins, 29 rRNAs and 120 tRNAs. Based on Gene Ontology (GO) analysis the top 50 terms are categorized into biological processes (24%), molecular functions (52%), and cellular components (24 %) (Figure S2A). Among these, carbohydrate metabolism was prominently represented, with 224 genes annotated under “carbohydrate metabolic process,” 694 under “hydrolase activity,” and 129 under “pectate lyase activity”. More in-depth analysis revealed the presence of 571 CAZyme-related genes. This highlights the ecological role and biotechnological potential of Neocucurbitaria sp. VM-36 in plant biomass decomposition and carbohydrate metabolism. This potential appears to be shared with other Cucurbitariaceae species (Figure S2B), suggesting conserved metabolic strategies likely shaped by shared ecological niches and evolutionary pressures. To also explore the biosynthetic potential of Neocucurbitaria sp. VM-36, we predicted the presence of BGCs in its genome and compared them to the predicted BGCs of six strains in the Cucurbitariaceae family. A total of 34 putative BGCs were identified in Neocucurbitaria sp. VM-36, a number comparable to the average BGC count (36) predicted across other Cucurbitariaceae fungi ( Figure 3A , Table S3). These BGCs were categorized into five major types: 10 polyketide synthase (PKS)-type BGCs, 9 ribosomally synthesized and post-translationally modified peptide (RiPP)-like BGCs, 9 non-ribosomal peptide synthetase (NRPS) and NRPS-like BGCs, 3 terpene-type BGCs, and 3 hybrid BGCs (2 NRPS+T1PKS and 1 T1PKS+ fungal-RiPP-like). Annotation against the MIBiG database revealed that 11 of the 34 BGCs were similar to known biosynthetic pathways ( Figure 3B , Figure S3). Notably, BGCs associated with choline and metachelin C production were conserved across all seven analyzed fungi, suggesting a common biosynthetic capability within the family. Download figure Open in new tab Figure 3. Comparative analysis of predicted BGCs in 7 strains of the Cucurbitariaceae family. (A) Number of BGCs in each genome by BGC type; (B) Presence (blue) or absence (white) of known BGCs in the 7 strains and chemical structures associated with 4 of the known BGCs which were only identified in Neocucurbitaria sp. VM-36. Additionally, BGCs responsible for the biosynthesis of squalestatin S1, secalonic acids, scytalone/T3HN, (-)-mellein, and phyllostictin A were identified not only in Neocucurbitaria sp. VM-36 but also in select species within the Cucurbitariaceae family. Importantly, four BGCs, putatively involved in the production of compounds related to hypothemycin, usnic acid, griseofulvin, and phomasetin, were unique to Neocucurbitaria sp. VM-36. These unique clusters suggest a distinct biosynthetic repertoire, potentially contributing to novel bioactivities (Supplementary Note S1). 3.3. Molecular Networking-Based Secondary Metabolite Identification So far, only 15 compounds have been isolated from Neocucurbitaria strains, encompassing macrocyclic and phomactin diterpenes and their derivates 9 , 10 , and a total of 28 compounds were isolated from the Cucurbitariaceae family (Table S4, Figure S4). Such few reports on SMs from this family aroused our interests for exploring the chemical compounds formed by Neocucurbitaria sp. VM-36 and their biosynthetic origin in this fungus. Thus, we performed an untargeted metabolomic analysis of fungal crude extracts from PDA and DPY medium, compared to medium blank controls. The final molecular network consists of 1521 nodes and 1896 edges ( Figure 4 , Table S5 and Figure S5). 19 compounds were putatively identified via library matching with existing MS2 databases and 7 compounds were inferred from matched adjacent nodes ( Figure 4 and 5 ). Download figure Open in new tab Figure 4. Molecular network of SMs in crude EtOAc extracts of Neocucurbitaria sp. VM-36 grown in PDA solid medium and DPY liquid medium. (A) Overview of the molecular network, with annotated SMs; (B) Molecular network of tetramic acid components containing decalin rings (cluster 5). Node colors represent signals in samples from PDA solid medium (green) and DPY liquid medium (red). Darker shades of node borders indicate higher metabolite abundance in samples based on peak area integration of the base peak, and purple represents the highest abundance. Edges represent the structural similarity between nodes. Download figure Open in new tab Download figure Open in new tab Figure 5. Chemical structures of annotated compounds putatively identified in the crude extracts of Neocucurbitaria sp. VM-36 grown in PDA solid medium and DPY liquid medium (* indicates putative structures according to the molecular network). The major constituents with high abundance in the crude extracts are annotated to be CJ-21058 analogues in cluster 5 ( Figure 4B ), which are tetramic acid-type components containing decalin rings or DTAs (decalin-containing tetramic acids). Based on the MS2 fragments (Table S5), we inferred 7 additional tetramic acid-type compounds (Supplementary Note S2). Among the less abundant compounds, a total of 9 lipids, including compounds 3 (DG(16:1/18:2/0:0)), 4 (DG(18:2/0:0/18:2)), 6 (AEG(o-16:3/15:0)), 8 (AEG(o-18:4/18:2)), 12 (linoleic acid), 14 (9(10)-EpOME), 16 (9S-Hydroxy-10E,12Z,15Z-octadecatrienoic acid), 18 (phytosphingosine), and 19 (1-linoleoylglycerol), are annotated as singleton nodes in the molecular network. Six amino acid-containing or peptide compounds, including compounds 2 (FruLeuIle), 5 (Asp-Leu), 7 (4-[5-[[4-[5-[acetyl(hydroxy)amino]pentylamino]-4-oxobutanoyl]-hydroxyamino] pentylamino]-4-oxobutanoic acid), 11 (N-[3-[5,8-bis[3-[acetyl(hydroxy)amino]propyl]-14-(hydroxymethyl)-3,6,9,12,15,18-hexaoxo-1,4, 7,10,13,16-hexazacyclooctadec-2-yl]propyl]-N-hydroxyacetamide/ desferriferricrocin), 13 (Arg-C18:2), and 15 (Gly-C18:2), are also found scattered across the network. Compound 11 is a cyclic hexa-peptide hydroxamate siderophore, with the general formula RC(O)N(OH)RL ( N -hydroxy amides) 31 . Compounds of this type do not only help fungi to chelate iron for survival under iron-restricted conditions, but they have also attracted attention due to their medicinal value as potent antibacterial or antitumor agents 31 . In Neocucurbitaria sp. VM-36, we hypothesize that the BGC encoded in region 8.2 (hybrid BGC of NRP-metallophore and NRPS) is likely to be responsible for producing 11 . Although there are no results with Known-Cluster-Blast, the core NRPS gene in the BGC shows amino acid similarity with an epichloenin A synthetase (MIBIG protein-AET13875.1, BGC0001250), with 26% identity and 82.7% coverage. Its enzymatic product, epichloenin A, is an extracellular siderophore related to ferrirubin, which is required to maintain the mutualistic interaction of the endophyte Epichloe festucae with the perennial ryegrass Lolium perenne 32 . Specifically, secretion of epichloenin A enables the symbiotic fungus to compete for plant iron while avoiding fungal over-growth inside the host plant 32 . Moreover, another core gene in the NRP-metallophore BGC of Neocucurbitaria sp. VM-36 shares similarity with an L-ornithine_N-monooxygenase (MIBiG protein CAJ96465.1) from BGC0000330, with 42% amino acid identity and 87% coverage, suggesting that it is probably responsible for forming an N-hydroxylated and N-acylated ornithine moiety in 11 33 . Node 10 is predicted to be the [M + H + CH 3 OH] + adduct of isousnic acid, an isomer of usnic acid. The cluster in region 20.2 in Neocucurbitaria sp. VM-36 has a Known-Cluster-Blast hit with BGC0002483, producing usnic acid, and 33% of its encoded enzymes show amino acid similarity with the query sequence. In particular, it encodes enzymes similar to the non-reducing PKS (methylphloracetophenone synthase) and a cytochrome p450 (methylphloracetophenone oxidase), which are responsible for the production of usnic acid 34 . Usnic acid, a toxic dibenzofuran, has been discovered in lichens and was extensively reported to have a variety of pharmacological effects, including remarkable antimicrobial, antitumor, antiviral, and antiparasitic activities 35 , 36 . It is active against Gram-positive bacteria, such as B. subtilis and S. aureus , by inhibiting RNA and DNA synthesis and blocking DNA replication and elongation, but it is not active against Gram-negative bacteria 36 . Although usnic and isousnic acid are regioisomers, the latter is comparatively poorly investigated, with only a few studies reporting on its antimicrobial and anti-inflammatory properties 37 , 38 . Overall, many clusters in the molecular network, especially with three or more nodes, could not be assigned to compound classes or specific compounds. They are potentially new compounds and need to be further explored by compound isolation and structural identification. 3.4. Structural Characterization of Compound 1 Compound 1 ( Figure 6 ) is the most abundant constituent of Neocucurbitaria sp. VM-36. It is predicted to have a tetramic acid-containing decalin scaffold based on its MS2 fragmentation. We purified it from large-scale fungal culture as a white amorphous powder. Its molecular formula was established to be C 23 H 33 NO 4 based on its m/z 388.2489 value ([M + H] + ion, calculated 388.2488), with 8 degrees of unsaturation. The absorbance spectrum of compound 1 in EtOH with maxima at A max (£) 252 (4.05) and 291.8 (4.08) nm ( Figure 6C ) is similar to the structurally related compound phomasetin 39 . Furthermore, the absolute configuration of the serine-derived moiety in compound 1 is also □ (Figure S6A), as known for phomasetin 39 . Download figure Open in new tab Figure 6. Physico-chemical characterization of compound 1 . Chemical structure with key two-dimensional NMR correlations (COSY and HMBC (A) and NOESY (B)); UV spectrum (C) and experimental and calculated ECD spectra (D). Next, we determined the 3D structure of compound 1 by NMR 24 and ECD. According to the 1D NMR ( Table 1 and Figures S7-S8) and MS2 data (Table S5), compound 1 has one -C 2 H 2 unit less and two sp 2 -carbon signals less than phomasetin, indicating the absence of one double bond in compound 1 compared to phomasetin 40 , 41 . View this table: View inline View popup Download powerpoint Table 1. 1 H NMR (600 MHz) and 13 C NMR (150 MHz) data of compounds 1 (in DMSO-d6) This feature matches that of CJ-21,058 and its conformational isomers, the hyalodendrins A and B 41 , 42 . Compared to the CJ-21,058 NMR data 41 , 42 , compound 1 has similar signals except for slight differences in the chemical shifts ( Table 1 ) 41 , 42 . Most notably, the 13 C chemical shift for C-1 in compound 1 is 203.14 ppm instead of 199 ppm, for C-2’ 170.82 ppm instead of 177 ppm, for C-3’ 107.16 ppm instead of 100 ppm, and for C-4’ 194.78 ppm instead of 190 ppm 41 . Also, the 2D NMR correlations ( 1 H- 1 H COSY and 1 H- 13 C HMBC) of compound 1 indicate high structural similarity with the hyalodendrins A and B 41 ( Figure 6A and S9-S12). 1 H- 1 H NOESY correlations ( Figure 6B ), including H6/H7a, H7b/H9a, H7b/H11, H9a/H10b, and H9a/H17, indicate that proton H-6 is on the opposite face of H-11 and H17, forming a trans-decalin structure. To determine the absolute configuration of 1 and to pinpoint the functional groups at the C-1 and C-2’ positions, we collected experimental ECD spectra and compared them to calculated ECD spectra ( Figure 6D and S6B). The experimental ECD spectrum indicates the presence of a mixture of two isomers, with different spectral contributions across wavelength regions. In the 210-225 nm range, the experimental peak shape aligns well with both computed spectra, suggesting overlapping contributions. From 225-250 nm, the experimental peak closely matches that of isomer B (Figure S6B) in both position and shape, though with higher intensity, while isomer A ( Figure 6D ) shows a weaker, red-shifted response. Within the 270-300 nm region, the experimental ECD spectrum displays a pronounced positive peak. A similar feature is present in the computed spectrum of isomer A ( Figure 6D ), albeit with reduced intensity. In contrast, isomer B (Figure S6B) shows only a weak signal, remaining near the baseline. Beyond 300 nm, both the experimental and calculated spectra exhibit a negative peak. However, the intensity is significantly stronger in the experimental spectrum. The lack of solvent treatment in the final calculations, due to the high computational cost and frequent convergence difficulties, combined with limited conformer sampling and a restricted number of excited states considered, likely contributes to the discrepancies observed between experiment and theory. Overall, the data support a mixed-isomer system with varying contributions across the ECD spectrum. Based on this analysis, we assigned the (2 R , 3 S , 6 R , 8 S , 11 S , 5’ S ) configuration and propose that interconversion occurs at the C-1 and C-2’ positions in solution, resulting in the isolated compound existing as a mixture of the two tautomers shown in Figures 6A and S6B. 3.5. Proposed Biosynthetic Pathway for Compound 1 The cluster in region 13.1, classified as a hybrid T1PKS/NRPS BGC, shares partial similarity with the phomasetin biosynthetic gene ( phm ) cluster from Pyrenochaetopsis sp. RK10-F058 (BGC0001738) and is therefore likely to give rise to compound 1 ( Figure 7 ). In particular, the core enzyme NeoA shares 72% amino acid identity with the PKS-NRPS hybrid Phm1 in the phm cluster 43 . It comprises one highly-reducing PKS module and one NRPS module with a terminal reductase (TD) domain. The PKS module is likely complemented by a stand-alone enoyl reductase (NeoB), thus producing the linear polyene intermediate via 7 cycles of polyketide chain elongation, reduction, and methylation. Subsequently, L-serine is incorporated by the NRPS module and upon release from the assembly line, the tetramic acid moiety is formed by the Diekmann cyclase function of the terminal reductase (TD) domain. Download figure Open in new tab Figure 7. Gene cluster (A) and proposed biosynthetic pathway (B) of compound 1 . β-ketosynthase (KS), acyl-transferase (AT), dehydratase (DH), carbon-methyltransferase (cMT), β-ketoreductase (KR), and acyl-carrier protein (ACP), condensation (C), adenylation (A), peptidyl carrier protein (PCP), and terminal reductase (TD). NeoC, displaying 57% amino acid identity with Phm7 in the phm cluster 43 – 45 , is predicted to be a Diels-Alderase (DAse) 40 – 42 . DAses can catalyze intramolecular Diels-Alder cycloadditions between a conjugated diene and substituted alkene via C-C bond formation, yielding a cyclohexene with up to four chiral centers 44 . Evolutionary analysis of NeoC with some representative [4+2]-cyclases shows that NeoC and Phm7 are in the same clade, with a close relationship to homologs gNR600, PvhB, and Fsa2 ( Figure 8 ). Although DAses can theoretically form four stereoisomers of trans- and cis- decalin, most of these enzymes, including Phm7, Fsa2, and gNR600, tend to stereo-selectively produce the trans- decalin structure in conjunction with a fungal PKS 43 – 48 . PvhB, involved in the biosynthesis of varicidin A in Penicillium variabile , is an exception of a fungal DAse to form cis-decalin, most likely driven by the presence of an electron-withdrawing carboxylate on the diene, which kinetically favors the exo-transition state in the DA reaction 46 , 49 . Among the trans-decalins, equisetin and phomasetin produced by Fsa2 and Phm7, respectively, represent the two possible enantiomers with (2 S , 3 R , 8 S , 11 R ) and (2 R , 3 S , 8 R , 11 S ) configurations, respectively 44 , 45 . Computational modeling, including molecular dynamics simulations and quantum chemical calculations, demonstrated that the reactions proceed through synergetic conformational constraints assuring transition state-like substrate folds and their stabilization by specific protein-substrate interactions 44 . Furthermore, the flexibility of bound substrates is largely different in two enzymes, suggesting the distinct mechanism of dynamic regulation behind these stereoselective reactions 44 . Download figure Open in new tab Figure 8. Evolutionary analysis of NeoC with other decalin-forming [4+2]-cyclases with Pyrl4 as an outgroup. Enzymes marked with stars have reported crystal structures, and the remaining protein structures were predicted by AlphaFold3. The substructure resulting from the enzyme-catalyzed cyclo-addition is highlighted in red in the respective compound. Enzyme selection and figure layout were adapted from Xu and Yang (2021) 50 . The dominant compound in the crude EtOAc extract of Neocucurbitaria sp. VM-36, compound 1, has a trans-decalin scaffold with the same configuration as phomasetin, but its side chain at position C3 is shorter than that of phomasetin. We also detected trace amounts of phomasetin itself (compound 21 ) and other structurally related compounds ( 20, 22 - 26 ) in our untargeted metabolomics analysis, indicating that the same BGC may give rise to a group of related compounds with compound 1 being the favored one under the chosen culture conditions. This suggests that the PKS-NRPS domain has some flexibility in the number of polyketide chain elongation cycles with a preference for the compound 1 precursor and/or that the preference of the DA enzyme for the compound 1 precursor leads to the dominance of compound 1 . When comparing the amino acid sequences of NeoC and Phm7 (Figure S13), we note that most of the active amino acid residues are conserved 44 , 45 , except for residue 258, where isoleucine replaces threonine. Whether this amino acid change influences the substrate or product scope of the enzyme, remains to be investigated. Lastly, NeoD, annotated as methyltransferase, shares 71% amino acid identity with the methyltransferase Phm5 in the phm cluster. We hypothesize that the -NH group in the tetramic acid moiety of an N-demethylated precursor (putatively compound 23 in the molecular network) can be methylated by NeoD to generate the N-methylated product compound 1 . In addition to the enzymes described above, there are still many other functional enzymes encoded in this gene cluster. Whether they contribute to further structural complexity remains to be explored. 3.6. Bioactivity Evaluation of Compound 1 As the crude extract of Neocucurbitaria sp VM-36 exerted a strong antibacterial effect, we were curious to see whether compound 1 was responsible for or contributed to this effect. Thus, we screened the compound against ESKAPE pathogens and found that it exerts strong inhibitory effects against several Gram-positive bacterial strains, including E. faecalis ATCC 29212 (MIC = 4 µg/mL), S. aureus ATCC 29213, S. aureus NCTC 8325, S. aureus Newman (MIC = 1 µg/mL), and S. aureus HG001 (MIC = 2 µg/mL), while it was inactive against the Gram-negative bacterial strains (MICs > 64 µg/mL) (Table S6). We further determined the MIC and MBC values of compound 1 against four MRSA strains, including S. aureus USA300, two clinical isolates S. aureus D15 and D17, and the transposon mutant S. aureus NE1688 (Sle1), as well as the MSSA reference strain S. aureus ATCC 29213 ( Table 2 ). Compound 1 shows strong antibacterial activity against these MRSA and MSSA strains (MIC = 0.5 - 1 µg/mL), similar to the positive controls vancomycin (MIC = 1 µg/mL) and daptomycin (MIC = 0.5 - 2 µg/mL). The MBC values of compound 1 (2 - 4 µg/mL) were slightly higher than those of vancomycin and daptomycin (MBC = 1 - 2 µg/mL). With MBC/ MIC ratios ≤ 4, we conclude that compound 1 shows bactericidal effects on S. aureus USA300, D15 and D17, and ATCC 29213. Strain S. aureus NE1688 (Sle1) appears to be more sensitive to growth inhibition by low concentrations of compound 1 but it is similarly sensitive to lethal doses as the other strains. View this table: View inline View popup Download powerpoint Table 2. Antibacterial activity evaluation of compound 1 against S. aureus We also analysed the bacterial growth curves of the strains in the presence of compound 1 at MBC levels (2, 4, and 8 µg/mL) to monitor the dose-dependent change of optical density ( Figure 9 ). After the exposure to compound 1 , the bacteria stopped growing and the OD 600 values declined. Also, the maximum OD 600 values of all bacterial cultures were significantly reduced in the presence of compound 1 compared to the vehicle controls (DMSO). This shows that compound 1 not only has a dose-dependent bactericidal activity but also that it induces bacterial lysis, with the strongest effects observed at 8 µg/mL. To further corroborate this observation, we assessed the bacterial viability before and after 40 h treatment by counting CFUs per mL liquid culture (Figure S14). Compared with the positive controls, compound 1 exerts stronger bactericidal activity against S. aureus ATCC 29213, USA300, D15 and D17 at 8 µg/mL and 4 µg/mL, with little to no bacterial colonies observed. Against S. aureus NE1688 (Sle1), compound 1 showed greater bactericidal activity than both controls at 8 µg/mL, but was less effective than daptomycin at 4 µg/mL. As expected, at the lowest tested concentration (2 µg/mL), none of the compounds exerts a significant bactericidal effect. Download figure Open in new tab Figure 9. Growth curves of 5 S. aureus strains in the presence of compound 1 , or the positive controls vancomycin and daptomycin at 2, 4 and 8 µg/mL. (A) S. aureus ATCC 29213 (MSSA); (B) S. aureus USA300 (MRSA); (C) S. aureus D15 clinical isolate (MRSA); (D) S. aureus D17 clinical isolate (MRSA); (E) S. aureus NE1688 (MRSA). The scattered line (---) in the x-axis indicates the time point at which the compound was added (t = 8 h). Results are expressed as mean OD 600nm values and error bars represent standard deviation (n = 3). Lastly, we also assessed whether a combination treatment of three MRSA strains ( S. aureus ATCC 29213, D15, and D17) with compound 1 and daptomycin or vancomycin had any synergistic or antagonistic effects ( Figure 11 ). Among 22 treatment combinations in the checkerboard assay, none exhibited antagonistic effects. Most interactions were indifferent, with a minimum fractional inhibitory concentration index (FICImin) of 1.25, except for the combination of compound 1 and daptomycin, which showed an additive effect against the clinical isolate D17 from a hospital-acquired infection (FICImin = 0.625). These findings indicate that compound 1 holds potential for combination therapy with established antibiotics, particularly daptomycin, to improve efficacy against particular MRSA strains, as exemplified with the D17 strain. The lack of antagonistic interactions suggests a favourable safety profile for such combination treatments. Download figure Open in new tab Figure 11. Effects of combination treatment with compound 1 and daptomycin or vancomycin on clinically relevant MRSA strains. Heatmaps depict the outputs of the checkerboard assays used for evaluating antibacterial drug interaction. Each square represents quantitative data expressed as the mean value of optical density (OD 600 nm , n = 3). Dark blue regions and white regions represent highest and lowest bacterial growth densities, respectively. (A-B) clinical isolate D15 (CA-MRSA); (C-D) clinical isolate D17 (HA-MRSA); (E-F) reference MRSA strain USA300. The FIC index (FICI) was calculated as the sum of the individual FICs of the antibacterial compounds under test in each well. FICI values were interpreted as follows: synergistic (Syn = FICI ≤ 0.5); additive (Add = 0.5 < FICI ≤1); indifferent (Ind = 1 4). Lastly, we performed a preliminary toxicological assessment of compound 1 on human red blood cells (hRBCs) (Figure S15). The concentration causing 50% haemolysis (HEC□□) was 34.54 μg/mL, while daptomycin and vancomycin showed no hemolysis up to 200 μg/mL. Notably, the HEC□□ of compound 1 is about four times higher than its MBC against MRSA and MSSA strains, indicating moderate hemolysis (∼23%) at the bactericidal concentration of 8 μg/mL. Compared to other antibacterial secondary metabolites, the haemolytic activity of compound 1 at this concentration is considered moderate 51 . 4. Discussion Decalin-containing tetramic acids (DTAs), such as compound 1 , have attracted widespread attention due to their potent antibacterial activity against various bacterial strains, such as B. subtilis , MRSA, vancomycin-resistant Enterococcus , and multidrug-resistant Mycobacterium tuberculosis 46 , 52 , 53 . Recent research has explored the structure-activity relationship of the antibacterial activity of DTA derivatives against MRSA, identifying key chemical features associated with antimicrobial potency 46 . For instance, dehydrogenation of the tyrosine sidechain of the tetramic acid substructure in conipyridoins, and the configuration at the C5’ position in 5’-epi-equisetin are crucial for antimicrobial activity against MRSA 46 , 48 . For several DTAs, such as N-demethylophiosetin, paecilosetin, and 8-epi-equisetin, the absence of oxygen groups as well as the stereochemistry of the methyl groups at the C8 position on the decalin skeleton are important determinants of the antibacterial activity of DTAs 46 , 48 . In cordysetin A, the D configuration of serine and N-methylation of the tetramic acid are crucial for its anti-tuberculosis activity 53 . Conversely, the antifungal activity of fusaramin appears to be more dependent on the identity of the amino acid than its cyclization to tetramic acid 54 . DTAs with planar structures similar to compound 1 , such as CJ-21058 and the hyalodendrins A and B, have been relatively underexplored, with most studies focusing primarily on their chemical structures and larvicidal activities, while their antibacterial properties remain largely uninvestigated 41 , 42 . Therefore, our in-depth bioactivity investigation of compound 1 provides interesting new information. Our observation that it exerts strong antibacterial effects on Gram-positive bacteria, while showing no inhibitory effects against Gram-negative bacteria is consistent with previously reported DTAs 46 . Compared to the MIC values of other DTAs against MRSA (0.002-42 µg/mL) 46 , compound 1 also shows excellent antibacterial effects, with MIC values of 0.5-1 µg/mL. In comparison to equisetin (MIC = 1.25 µg/mL) 52 , the presence or absence of the methyl group at the C4 position, as well as differences in stereo-centres, do not appear to significantly affect the antibacterial activity against the tested MRSA strains. Our findings further show that compound 1 has significant bactericidal effect on S. aureus , as exemplified for the three MRSA strains USA300, D15 and D17, and the MSSA strain ATCC 29213. Notably, compound 1 exerts stronger bactericidal activity against these strains compared to the positive control antibiotics daptomycin and vancomycin at 8 µg/mL, and it reveals additive or indifferent effects in combination treatment with daptomycin or vancomycin against the three tested MRSA strains. The structurally related compound equisetin has been demonstrated to effectively eradicate MRSA or VRE with low-level antibiotic resistance and to synergize with colistin, although it also has an indifferent effect with other antibiotics, including daptomycin 55 . Furthermore, equisetin has been shown to efficiently eliminate intracellular S. aureus by enhancing the host autophagy and inducing mitochondria-mediated reactive oxygen species production to clear the infection, demonstrating remarkable anti-infective activity in a peritonitis mouse infection model 56 . Although the bactericidal mechanism of compound 1 requires further investigation, these findings suggest its potential as a valuable antibacterial agent. In addition to their antimicrobial activity, DTAs have been shown to possess a variety of other biological activities, for instance, antifungal 54 , anticancer 52 , 57 – 59 , anti-HIV 60 , pesticidal activity 61 , anti-obesity 62 , and anti-atherosclerosis effects 63 . These diverse biological activities open avenues for further exploration of the pharmacological potential of compound 1 . In conclusion, our findings imply that compound 1 is a promising antibacterial agent that may help to overcome antibiotic resistance. Altogether, this study not only presents a comprehensive genomic and metabolomic characterization of an underexplored fungus that has led to the identification of a potent antibacterial agent. Funding K.H. is grateful for funding from the Federation of European Biochemical Societies through the FEBS Excellence Award 2021 and from the Gratama Foundation (project number 2024-07). X.L. and T.H. are funded by the scholarships 202106550001 and 202006550001 from the China Scholarship Council, respectively. RdCFV acknowledges funding from CONACyT-Mexico (grant No. 773955) for her PhD studies. Supplementary Information Supplementary Information file with Supplementary Methods and Results. Table S1. Staphylococcus aureus strains used for MBC tests in this study. Table S2. Reads and assembly statistics of Neocucurbitaria Lsp. VM-36. Table S3. Biosynthetic gene clusters of Neocucurbitaria sp. VM-36 predicted by antiSMASH version 7.0.1. Table S4. Compounds reported to be isolated from the Cucurbitariaceae family. Table S5. Molecular networking-based identification of secondary metabolites in Neocucurbitaria sp. VM-36. Table S6. MIC values of compound 1 against ESKAPE strains. Figure S1. Contigs of Neocucurbitaria sp . VM-36 visualized by Bandage. Figure S2. Functional genome annotation of Neocucurbitaria sp. VM-36. Figure S3. Structures of compound predicted to be produced by Neocucurbitaria sp. VM-36 based on antiSMASH analysis. Figure S4. Structures of compounds reported to be produced by fungi from the Cucurbitariaceae family. Figure S5. Total ion chromatograms recorded in positive ion mode of EtOAc extracts of Neocucurbitaria sp. VM-36 grown on PDA solid and DPY liquid medium. Figure S6. Calculated ECD spectra of the compound 1 isomer with C-1 being a carbonyl group and C-2’ being a hydroxyl group. Figure S7. 1 H NMR spectrum of compound 1 in DMSO-d6 (600 MHz). Figure S8. 13 C NMR spectrum of compound 1 in DMSO-d6 (150 MHz). Figure S9. HSQC spectrum of compound 1 in DMSO-d6. Figure S10. HMBC spectrum of compound 1 in DMSO-d6. Figure S11. 1 H- 1 H COSY spectrum of compound 1 in DMSO-d6. Figure S12. NOESY spectrum of compound 1 in DMSO-d6. Figure S13. Amino acid sequence alignment between NeoC in Neocucurbitaria sp. VM-36 and Phm7 in Pyrenochaetopsis sp. RK10-F058. Figure S14. Effect of combination treatment with compound 1 and daptomycin or vancomycin on clinically relevant MRSA strains. Figure S15. In vitro hemolytic activity of compound 1 towards human red blood cells as compared to vancomycin and daptomycin. Data Availability statement The sequencing data and genome assembly for this study have been deposited in the European Nucleotide Archive (ENA) at EMBL-EBI under the accession number PRJEB97715. The mass spectrometry data and the Cytoscape file of molecular networking have been deposited on GNPS under the accession number MassIVE ID: MSV000098902. The raw NMR data of compounds 1 is listed in the Supplementary Information. Antibacterial activity of compound 1 . Author Contributions XL, TH, and KH designed the study and developed the workflow. XL and TH performed the genomic and metabolomic experiments. XL performed the purification and structural analysis of compound 1. RdCFV and JMvD designed and RdCFV performed and analyzed the antibacterial assays and in vitro human toxicology assays. KH supervised the project. XL wrote the manuscript with input from all authors. All authors have read and approved the final version of this manuscript. Conflicts of Interest The authors declare no conflicts of interest. Acknowledgments The authors are grateful for technical assistance by Pieter Tepper, Rita Setroikromo, the staff at the interfaculty mass spectrometry center of the University Medical Center RUG/UMCG, bioinformatic support by Dr. Thomas Hackl (Groningen Institute for Evolutionary Life Sciences, RUG), NMR analysis support by Dr. Peter Fodran (Chemical and Pharmaceutical Biology, RUG), and ECD measurement support by Dr. Alexander Ryabchun (Faculty of Science and Engineering, Synthetic Organic Chemistry, RUG). We are grateful for Xiaofang Li in the Department of Medical Microbiology and Infection Prevention in UMCG for providing the MRSA strains. Funder Information Declared Federation of European Biochemical Societies, https://ror.org/0100zbm77 , FEBS Excellence Award 2021 Gratama Foundation , 2024-07 China Scholarship Council, https://ror.org/04atp4p48 , 202106550001 , 202006550001 CONACyT-Mexico , 773955 Footnotes xiao.li{at}rug.nl ; t.he{at}rug.nl ; r.c.flores.vallejo{at}umcg.nl ; j.m.van.dijl01{at}umcg.nl Abbreviations A domain adenylation domain Aas auxiliary activities ACP acyl-carrier protein AT acyl-transferase ATCC American type culture collection BGCs biosynthetic gene clusters BLAST basic local alignment search tool C domain condensation domain CAZyme carbohydrate-active enzyme CBMs carbohydrate-binding modules CD circular dichroism CEs carbohydrate esterases CFUs colony-forming units cMT carbon-methyltransferase DAse diels-alderases DH dehydratase DMSO dimethyl sulfoxide DPY dextrose peptone yeast DTAs decalin-containing tetramic acids ECD electronic circular dichroism EtOAc ethyl acetate FICI fractional inhibitory concentration index GenSAS genome sequence annotation server GHs glycoside hydrolases GNPS global natural products social molecular networking GO gene ontology GTs glycosyl transferases hRBCs human red blood cells LC-MS/MS high-resolution, high-performance liquid chromatography-coupled tandem mass spectrometry HR-PKS highly-reducing polyketide synthase ITS internal transcribed spacer KR ketoreductase KS ketosynthase LSU large ribosomal subunit MBC minimum bactericidal concentration MEA malt extract agar MICs minimum inhibitory concentrations ML Maximum Likelihood MRSA methicillin-resistant S. aureus MSSA methicillin-sensitive S. aureus NCBI national center for biotechnology information NCTC national collection of type cultures NMR nuclear magnetic resonance spectroscopy NR-PKS non-reducing polyketide synthase NRPS non-ribosomal peptide synthetase PCP peptidyl carrier protein PCR polymerase chain reaction PDA potato dextrose agar PKS polyketide synthase PLs polysaccharide lyases RiPP post-translationally modified peptide Rpb2 RNA polymerase II subunits 2 SAGM Saline adenine glucose mannitol SDA Sabouraud dextrose agar SMs secondary metabolites SNA synthetically nutrient-poor agar SSU small ribosomal subunit TD terminal reductase TE thioesterase Tef1 translation elongation factor 1α. 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