The palmitoyl-CoA ligase Fum16 is part of a fumonisin subcluster involved in self-protection.

produces the mycotoxin fumonisin B (FB), which disrupts sphingolipid biosynthesis by inhibiting ceramide synthase and affects the health of plants, animals, and humans. The means by which protects itself from its own mycotoxin are not completely understood. Some fumonisin () cluster genes do not contribute to the biosynthesis of the compound, but their function has remained enigmatic. Recently, we showed that , and encode two ceramide synthases and an ATP-binding cassette transporter, respectively, which play a role in antagonizing the toxicity mediated by FB. In the present work, we uncovered functions of two adjacent genes, and . Using homologous and heterologous expression systems, in and , respectively, we provide evidence that both contribute to protection against FB. Our data indicate a potential role for the P450 monooxygenase Fum15 in the modification and detoxification of FB since the deletion and overexpression of the respective gene affected extracellular FB levels in both hosts. Furthermore, relative quantification of ceramide intermediates and an 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, constitute a subcluster within the biosynthetic gene cluster dedicated to the fungal self-protection against FB.IMPORTANCEThe study identifies a five-gene subcluster () in involved in self-protection against FB. (palmitoyl-CoA ligase), and (ceramide synthases) enzymatically supplement ceramide biosynthesis, while (ATP-binding cassette transporter) acts as a repressor of the cluster. The evolutionary conservation of (P450 monooxygenase) in and clusters is discussed, and its effect on extracellular FB levels in both native () and heterologous () 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.