Genetic Dereplication of Multiple Penicillium expansum Biosynthetic Gene Clusters Reveals Cryptic Penta-Cyclopeptide Production

Genetic dereplication by targeted deletion of major secondary metabolite biosynthetic pathways is an effective strategy to uncover novel compounds and cryptic biosynthetic gene clusters (BGCs). In this study, we engineered a “flatline” strain of Penicillium expansum by knocking out key biosynthetic genes responsible for producing citrinin, patulin, roquefortine C, andrastin A, and communesins. Comparative metabolomic profiles between the flatline and wild-type strains revealed a marked increase in the production of five-residue cyclopeptides featuring the nonproteinogenic amino acid pipecolic acid in the flatline strain when cultivated on a rice medium. Further analysis led to the identification of known cyclopeptide MBJ-0110 and its new analogues assigned as 3-hydroxy-MBJ-0110 (1) and Val-MBJ-0110 (2). Their associated BGC was predicted using antiSMASH, whereby deletion of the nonribosomal peptide synthetase (NRPS) mbjA abolished the production of these cyclopeptides, confirming the requirement of mbjA as the core biosynthetic gene. A phylogenetic search showed a high level of conservation of MbjA across 19 species, predominately in the taxon Eurotiomycetes. By linking biosynthetic genes to these rare cyclopeptides, our study provides support for the genetic dereplication strategy as an effective means to uncover cryptic fungal secondary metabolism.