Fungal–Fungal Cocultivation Unveils Bioactive Metabolites, Including Rare Terpene–Amino Acid Hybrids, with Antibacterial Activity

Background The growing threat of drug-resistant bacterial infections demands the discovery of bioactive compounds from natural sources. This study explored the chemical diversity generated through the cocultivation of two fungal strains, Aspergillus pseudocaelatus and Trichoderma gamsii , isolated from the rhizospheres of medicinal plants. Methods Co-culture fermentation on rice medium was employed to induce unique hybrid metabolites alongside other bioactive metabolites. LC-QTOF/MS metabolomic profiling combined with molecular network-based dereplication identified terpene–amino acid conjugates, while genome mining revealed biosynthetic potential. Results Time-course experiments demonstrated that prolonged incubation enhanced metabolite yields, with coculture significantly increasing secondary metabolite production versus monocultures. Chromatographic isolation and spectroscopic characterization yielded two terpene–amino acid hybrids ( 1 and 2 ), one organic acid ( 3 ), and one polyketide ( 4 ), with structures elucidated through 1D/2D NMR and HRMS analysis. Compound 1 exhibited antibacterial inhibition of 48.9%, 48.1%, and 27.9% against Staphylococcus aureus , Bacillus subtilis , and E. coli , respectively. Compound 2 showed 44.2% and 33.7% inhibition against S. aureus and B. subtilis , while compound 4 demonstrated 43.1%, 29.8%, and 22.8% inhibition against the same pathogens at 100 μg/mL. Conclusion These findings underscore the value of microbial interactions in activating cryptic biosynthetic pathways and expanding the chemical space accessible from microbial sources. This work contributes to the growing field of microbial natural product discovery by demonstrating that fungal‒fungal interactions can be strategically leveraged to access active molecules with promising bioactivities, supporting ongoing efforts to address the global challenge of antibiotic resistance.