The unsaturated dicarboxylic acid itaconic acid is a bio-based chemical building block used in the industrial production of plastics, paints and cosmetics. Currently, itaconic acid is produced by fermentation of Aspergillus terreus. The phytopathogenic basidiomycete Ustilago maydis produces a variety of secondary metabolites e.g. itaconic acid under certain environmental conditions. The biosynthetic route of itaconate in this fungus, however, has not been elucidated, yet. The U. maydis genome contains a gene cluster comprising all the genes required for itaconic acid biosynthesis. This gene cluster is specifically regulated by the transcriptional regulator Ria1. Deletion analysis showed that U. maydis contains two genes essential for itaconate production coding for a PrpF-like enzyme and a CMLE-like enzyme. The activity of these enzymes has been identified by in vitro studies with the purified proteins. The PrpF-like enzyme aconitate-∆-isomerase (Adi1) catalyzes the first step of the itaconic acid biosynthesis pathway by isomerisation of cis-aconitate. The resulting trans-aconitate serves as a substrate for the trans-aconitate decarboxylase (Tad1), which catalyzes decarboxylation of trans-aconitate to form itaconate. A hypothetical pathway for itaconic acid biosynthesis in U. maydis has been modeled based on these data: cis-aconitate, which is a natural intermediate of the citric acid cycle, is probably exported from the mitochondria into the cytoplasm by the mitochondrial transporter Ctp1. In the cytoplasm cis-aconitate is converted to trans-aconitate by Adi1 and trans-aconitate is subsequently decarboxylated to itaconate by Tad1. Finally itaconate secretion into the surrounding medium is probably mediated by the MFS-transport protein Itp1. The itaconic acid gene cluster is highly expressed during pathogenic development of U. maydis. It has been demonstrated, however, that biosynthesis of itaconate is not essential for pathogenic development. Therefore, the biological role of itaconate for U. maydis is still unknown. The U. maydis genome contains a second gene cluster, which shows some similarities to the itaconic acid gene cluster. The PrpF-like enzyme Adi2, which is part of this gene cluster, is able to replace Adi1 in the itaconic acid pathway in vitro and also in vivo. Furthermore, growth assays showed that the small gene cluster of Adi2 is required for the metabolization of cis- and trans-aconitate. Trans-aconitate is known to be produced by the U. maydis host plant Zea mays. Therefore the ability to metabolize trans-aconitate might be an advantage for the fungus, even though pathogenicity tests showed that the Adi2 gene cluster is not required for the pathogenic development of U. maydis.