Genome mining of Trp-anthranilic acid cyclic dipeptides in Aspergillus driven by analysis of NRPS adenylation domains.

Aspergillus fungi are rich in secondary metabolites, yet their biosynthetic potential remains underexplored. Nonribosomal peptides (NRPs), an important class of natural products with diverse biological activities, are synthesized by nonribosomal peptide synthetases (NRPSs). In this study, we performed a large-scale bioinformatic analysis of NRPS adenylation (A) domains from 1,162 Aspergillus genomes to predict their substrate specificities. Using AdenylPred, we analyzed 40,684 A-domain sequences and found that large phenyl-derivative amino acids and small hydrophobic amino acids are the predominant substrates, accounting for 66% of all predictions. Focused genome mining of Aspergillus spinosus 2437 revealed a biosynthetic gene cluster (BGC 29.2) showing high similarity to known non-proteinogenic amino acid-containing cyclic dipeptide BGCs. Fermentation of this strain led to the isolation of three tryptophan-anthranilic acid cyclic dipeptides, namely asperdiazapinone G (1), 6-hydroxyaszonalenin (2), and aszonalenin (3), thereby providing support for the functional assignment of the identified gene cluster and suggesting the utility of A-domain substrate prediction for targeted genome mining. Enzyme similarity analysis further indicated that NRPSs responsible for cyclic dipeptide biosynthesis are predominantly distributed within the Aspergillus genus. This study indicates that combining A-domain substrate prediction with genome mining is an effective strategy for discovering NRPs in Aspergillus, expanding our understanding of their biosynthetic potential.