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

Abstract

<i>Penicillium polonicum</i>, commonly found on food matrices, is a mycotoxigenic species able to produce a neurotoxin called verrucosidin. This methylated α-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 α-pyrones aurovertin (AurA) and citreoviridin (CtvA), 16 PKS genes for putative α-pyrones were identified in the <i>P. polonicum</i> genome. A single PKS gene, <i>verA</i>, was found to be transcribed under verrucosidin-producing growth conditions. The annotated functions of the genes neighboring <i>verA</i> correspond to those required for verrucosidin biosynthesis. To prove the involvement of <i>verA</i> in verrucosidin biosynthesis, the clustered regularly interspaced short palindrome repeats (CRISPR) technology was applied to <i>P. polonicum</i>. <i>In vitro</i> reconstituted CRISPR-Cas9 was used to induce targeted gene deletions in <i>P. polonicum</i>. This approach allowed identifying and characterizing the verrucosidin biosynthetic gene cluster. <i>VerA</i> 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 <i>P. polonicum</i> as a valuable source of novel compounds.