Integration of Genome Mining and HiTES Reveals Secondary Metabolic Potential in Marine-Derived Aspergillus sp. WHUF0304

Aims Marine-derived Aspergillus species are prolific producers of bioactive secondary metabolites, yet the majority of their biosynthetic gene clusters (BGCs) remain silent. This study aimed to integrate genome mining with High-throughput Elicitor Screening (HiTES) to unlock the metabolic potential of Aspergillus sp. WHUF0304 and identify elicitors that promote the accumulation of previously undetected metabolites. Methods and Results A high-quality genome of Aspergillus sp. WHUF0304 was assembled and annotated using multiple functional databases, revealing substantial secondary metabolic potential. antiSMASH analysis identified diverse BGCs, including NRPS/indole-related clusters potentially associated with indole diketopiperazine biosynthesis. A HiTES-inspired elicitor screening strategy was then applied to evaluate 42 small molecules for their ability to alter the metabolite profile of this strain. Among the tested elicitors, fluconazole was identified as the optimal inducer, triggering the production of several indole diketopiperazine-related differential metabolites. Subsequent activity-guided isolation led to the identification of a bioactive indole diketopiperazine dimer, cristatumin E, which exhibited antibacterial activity against Escherichia coli and Bacillus subtilis with minimum inhibitory concentrations (MICs) of 32 µg mL−1 and 256 µg mL−1, respectively. Conclusions These findings demonstrate that integrating genomic and functional approaches effectively activates silent BGCs in marine fungi. The fluconazole-associated accumulation and subsequent isolation of cristatumin E, a bioactive indole diketopiperazine dimer, highlight the potential of elicitor-mediated activation to expand the detectable metabolite profile of Aspergillus sp. WHUF0304.