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

## TL;DR

By deleting genes responsible for known compounds in Penicillium expansum, researchers discovered new cyclopeptides and confirmed a key biosynthetic gene.

## Contribution

A novel penta-cyclopeptide and its analogues were identified using genetic dereplication in Penicillium expansum.

## Key findings

- A flatline strain of Penicillium expansum produced increased levels of five-residue cyclopeptides containing pipecolic acid.
- Two new cyclopeptide analogues, 3-hydroxy-MBJ-0110 and Val-MBJ-0110, were identified.
- The NRPS gene mbjA was confirmed as essential for the biosynthesis of these cyclopeptides.

## Abstract

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.