Transcriptome dissection of the Zeb2-associated network reveals a role for NRPS15 in zearalenone biosynthesis in Fusarium graminearum

Fungal secondary metabolites, including mycotoxins, play important roles in fungal biology and represent major concerns for food and feed safety. Zearalenone (ZEA) is an estrogenic polyketide mycotoxin produced by several Fusarium species, yet the genetic network governing its production remains poorly characterized. Here, we investigated genes associated with ZEA production in Fusarium graminearum using two complementary transcriptomic approaches. Time-resolved transcriptome analysis revealed transcriptional changes preceding detectable ZEA accumulation. Temporal clustering further revealed coordinated changes in primary metabolism, transcriptional regulation, carbohydrate utilization, and stress- and transport-associated pathways during ZEA-inducing cultivation. Genotype-based transcriptome analysis of ZEB2 deletion and overexpression strains subsequently identified Zeb2-responsive genes. Functional screening identified NRPS15, encoding a previously uncharacterized nonribosomal peptide synthetase-like protein adjacent to the ZEA biosynthetic cluster, as an important contributor to ZEA production. Deletion of NRPS15 markedly reduced ZEA production and expression of PKS4, PKS13, ZEB1, and ZEB2. NRPS15 expression did not restore ZEA production in the absence of Zeb2. NRPS15 orthologs were conserved in several ZEA-producing Fusarium species, and deletion of NRPS15 substantially reduced ZEA production in Fusarium culmorum. These findings expand the functional genetic context of ZEA production beyond the canonical four-gene biosynthetic cluster.