Ustilago maydis produces itaconic acid via the unusual intermediate trans ‐aconitate

Summary Itaconic acid is an important biomass‐derived chemical building block but has also recently been identified as a metabolite produced in mammals, which has antimicrobial activity. The biosynthetic pathway of itaconic acid has been elucidated in the ascomycetous fungus A spergillus terreus and in human macrophages. In both organisms itaconic acid is generated by decarboxylation of the tricarboxylic acid ( TCA ) cycle intermediate cis ‐aconitate. Here, we show that the basidiomycetous fungus U stilago maydis uses an alternative pathway and produces itaconic acid via trans ‐aconitate, the thermodynamically favoured isomer of cis ‐aconitate. We have identified a gene cluster that contains all genes involved in itaconic acid formation. Trans ‐aconitate is generated from cis ‐aconitate by a cytosolic aconitate‐Δ‐isomerase ( A di1) that belongs to the PrpF family of proteins involved in bacterial propionate degradation. Decarboxylation of trans ‐aconitate is catalyzed by a novel enzyme, trans ‐aconitate decarboxylase ( T ad1). Tad1 displays significant sequence similarity with bacterial 3‐carboxy‐ cis , cis ‐muconate lactonizing enzymes ( CMLE ). This suggests that U . maydis has evolved an alternative biosynthetic pathway for itaconate production using the toxic intermediate trans ‐aconitate. Overexpression of a pathway‐specific transcription factor ( R ia1) or a mitochondrial tricarboxylic acid transporter ( M tt1) resulted in a twofold increase in itaconate yield. Therefore, our findings offer new strategies for biotechnological production of this valuable biomass‐derived chemical.