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

Endophytic fungi from 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 assembly revealed a genome of 32.8 Mb with 50% GC content, comprising 15 contigs (=50,000 bp; N50~7.3 Kb), and 11,817 protein-coding genes, including 570 encoding carbohydrate-active enzymes. AntiSMASH analysis predicted a total of 34 biosynthetic gene clusters , with 11 showing high similarity to known clusters. Comparative genomic analysis with six Cucurbitariaceae strains predicted its distinctive biosynthetic potential to produce compounds related to griseofulvin, usnic acid, hypothemycin, and phomasetin. Metabolomic analysis of the ethyl acetate extract 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, including cyclic hexapeptides,. We successfully isolated the main compound 1, a phomasetin analog with a decalin core and a tetramic acid moiety. Notably, compound 1 exhibited strong bactericidal effects against different methicillin-resistant and –sensitive S. aureus strains, with minimum inhibitory concentrations ranging from 0.5 to 1 µg/mL and minimum bactericidal concentrations ranging from 2 to 4 µg/mL, comparable to those of the clinically applied antibiotics vancomycin and daptomycin. Additionally, checkerboard assays revealed that the interactions between compound 1 and either daptomycin or vancomycin were mostly indifferent. These findings underscore the pharmaceutical potential of compound 1 and highlight Neocucurbitaria sp. VM-36 as a promising source for bioactive natural product discovery.