CRISPR-Cas9-Based Discovery of the Verrucosidin Biosynthesis Gene Cluster in Penicillium polonicum

Penicillium polonicum, commonly found on food matrices, is a mycotoxigenic species able to produce a neurotoxin called verrucosidin. This methylated alpha-pyrone polyketide inhibits oxidative phosphorylation in mitochondria and thereby causes neurological diseases. Despite the importance of verrucosidin as a toxin, its biosynthetic genes have not been characterized yet. By similarity analysis with the polyketide synthase (PKS) genes for the alpha-pyrones aurovertin (AurA) and citreoviridin (CtvA), 16 PKS genes for putative alpha-pyrones were identified in the P. polonicum genome. A single PKS gene, verA, was found to be transcribed under verrucosidin-producing growth conditions. The annotated functions of the genes neighboring verA correspond to those required for verrucosidin biosynthesis. To prove the involvement of verA in verrucosidin biosynthesis, the clustered regularly interspaced short palindrome repeats (CRISPR) technology was applied to P. polonicum. In vitro reconstituted CRISPR-Cas9 was used to induce targeted gene deletions in P. polonicum. This approach allowed identifying and characterizing the verrucosidin biosynthetic gene cluster. VerA deletion mutants were no longer able to produce verrucosidin, whereas they were displaying morphological characteristics comparable with the wild-type strain. The available CRISPR-Cas9 technology allows characterizing the biosynthetic potential of P. polonicum as a valuable source of novel compounds.

Automated summary

Penicillium polonicum, a common mold found on food, produces the neurotoxin verrucosidin. Through genetic analysis, the biosynthetic gene verA has been identified and confirmed to be involved in the production of verrucosidin. The use of CRISPR-Cas9 technology has allowed further characterization of the biosynthetic potential of P. polonicum for novel compounds.