The Peroxygenase Activity of the Caleosin, AfPXG, Modulates the Biosynthesis of Aflatoxins and Their Trafficking and Extracellular Secretion via Lipid Droplets.

Aflatoxins (AF) are highly detrimental to human and animal health. We recently demonstrated that the caleosin, AfPXG, had peroxygenase activity and mediated fungal development and AF accumulation. We now report the characterization of an -deficient line using reference strain NRRL3357. The resulting fungal phenotype included a severe decrease in mycelium growth, failure to sporulate, and reduced AF production. Increasing cellular oxidative status by administration of hydrogen peroxide and cumene hydroperoxide did not restore the -deficient phenotype, which suggests that -deficiency is not directly related to oxidative stress. To investigate possible alternative roles of , a gain of function approach was used to overexpress , with the reporter gene , in an -deficient line, termed . The resulting phenotype included elevated numbers of stable lipid droplets (LDs) plus enhanced AF production. Highly purified LDs from cultures sequestered AF and this ability was positively correlated with overall LD number. Site-specific mutagenesis of to delete Histidine 85 (AfPXG), a residue essential for its catalytic activity, or deletion of the putative LD targeting domain (AfPXG), showed that AfPXG-peroxygenase activity was required for AF biosynthesis and that integration of AF into LDs was required for their export via a LD-dependent pathway. Ectopic expression in fungal cells of the plant LD-associated protein, oleosin, also resulted in both additional LD accumulation and enhanced AF secretion. These results suggest that both fungal LDs and their associated caleosin proteins are intimately involved in the biosynthesis, trafficking, and secretion of AF.