The palmitoyl-CoA ligase Fum16 is part of a <i>Fusarium verticillioides</i> fumonisin subcluster involved in self-protection

<i>Fusarium verticillioides</i> produces the mycotoxin fumonisin B₁ (FB₁), which disrupts sphingolipid biosynthesis by inhibiting ceramide synthase and affects the health of plants, animals, and humans. The means by which <i>F. verticillioides</i> protects itself from its own mycotoxin are not completely understood. Some fumonisin (<i>FUM</i>) cluster genes do not contribute to the biosynthesis of the compound, but their function has remained enigmatic. Recently, we showed that <i>FUM17</i>, <i>FUM18,</i> and <i>FUM19</i> encode two ceramide synthases and an ATP-binding cassette transporter, respectively, which play a role in antagonizing the toxicity mediated by FB₁. In the present work, we uncovered functions of two adjacent genes, <i>FUM15</i> and <i>FUM16</i>. Using homologous and heterologous expression systems, in <i>F. verticillioides</i> and <i>Saccharomyces cerevisiae</i>, respectively, we provide evidence that both contribute to protection against FB₁. Our data indicate a potential role for the P450 monooxygenase Fum15 in the modification and detoxification of FB₁ since the deletion and overexpression of the respective gene affected extracellular FB₁ levels in both hosts. Furthermore, relative quantification of ceramide intermediates and an <i>in vitro</i> enzyme assay revealed that Fum16 is a functional palmitoyl-CoA ligase. It co-localizes together with the ceramide synthase Fum18 to the endoplasmic reticulum, where they contribute to sphingolipid biosynthesis. Thereby, <i>FUM15-19</i> constitute a subcluster within the <i>FUM</i> biosynthetic gene cluster dedicated to the fungal self-protection against FB₁.IMPORTANCEThe study identifies a five-gene <i>FUM</i> subcluster (<i>FUM15-19</i>) in <i>Fusarium verticillioides</i> involved in self-protection against FB₁. <i>FUM16</i> (palmitoyl-CoA ligase), <i>FUM17,</i> and <i>FUM18</i> (ceramide synthases) enzymatically supplement ceramide biosynthesis, while <i>FUM19</i> (ATP-binding cassette transporter) acts as a repressor of the <i>FUM</i> cluster. The evolutionary conservation of <i>FUM15</i> (P450 monooxygenase) in <i>Fusarium</i> and <i>Aspergillus FUM</i> clusters is discussed, and its effect on extracellular FB₁ levels in both native (<i>F. verticillioides</i>) and heterologous (<i>Saccharomyces cerevisiae</i>) hosts is highlighted. These findings enhance our understanding of mycotoxin self-protection mechanisms and could inform strategies for predicting biological activity of unknown secondary metabolites, managing mycotoxin contamination, and developing resistant crop cultivars.