Functional characterization of grammicin biosynthesis genes in Xylaria grammica and strategies for enhanced metabolite production

Fungi produce diverse secondary metabolites with agricultural and medical potential. Xylaria grammica EL000614 synthesizes grammicin, an isomer of patulin, which shows nematicidal activity against root-knot nematodes with low cytotoxicity. However, the genetic basis of grammicin biosynthesis remains unclear. Here, we characterized two genes, xgpatK and xgpatL, encoding a 6-methylsalicylic acid synthase XgPatK and a Zn(II)2Cys6 transcription factor XgPatL, respectively, within a putative grammicin biosynthetic cluster. Comparative genomic analysis identified 11 cluster genes in X. grammica that are also conserved in patulin-producing fungi, including Aspergillus clavatus and Penicillium expansum. Deletion of xgpatK or xgpatL abolished grammicin production, while complementation restored it, confirming their essential roles. Furthermore, constitutive overexpression of xgpatL increased grammicin production three-fold compared with the wild-type. These findings provide the first genetic and functional evidence of grammicin biosynthesis in X. grammica and demonstrate the regulatory role of XgPatL. Our study advances the understanding of fungal secondary metabolism and highlights grammicin as a potential agent for sustainable biological control of plant-parasitic nematodes.