Many fungal strains in the family Ustilaginaceae secrete itaconic acid (ITA) along with its derivatives, 2-hydroxyparaconate (2-HP) and itatartarate (ITT) under nitrogen limitation. While the antimicrobial properties of ITA are well documented, the ecological role of its derivatives remains largely unexplored. Notably, the bacterium Pseudomonas aeruginosa possesses a specialized ITA metabolic operon with genes not directly related to ITA degradation (PA0879-PA0881), but essential for tolerance to the ITA derivatives, suggesting a molecular arms race within their shared habitat (de Witt et al., 2023). This study aims to investigate how fungal secondary metabolites shape cross-kingdom communities and elucidate their effects on bacterial metabolism and tolerance, using the non-pathogenic strain P. putida KT2440, engineered to heterologously express the ITA metabolic operon from P. aeruginosa PAO1. Inhibition studies using Ustilago supernatants and purified 2-HP and ITT on different engineered Pseudomonas strains identified the uncharacterized ring-cleaving dioxygenase RdoPA (PA0880) as a key mediator of tolerance to ITA derivatives. Interestingly, the ITA synthesis clusters of several fungal strains, such as Ustilago maydis and Aspergillus terreus, contain genes with high sequence similarity to rdoPA (UMAG_12299, rdo1; ATEG_10557, rdoA). Complementation studies and biochemical characterization confirm that these evolutionarily distant enzymes share lactonase activity for the enantioselective conversion of (S)-2-HP to (S)-ITT. Combined with the finding that (S)-2-HP inhibits ITA degradation, this conversion clarifies RdoPA's physiological role in Pseudomonas, while its role in fungi, remains unclear. Preliminary results with eGfp-tagged Rdo variants in U. maydis revealed its localization to the mitochondria, and we currently hypothesize a self-protection mechanism against the ITA derivatives, potentially due to toxic side activities of mitochondrial enzymes. With a clearer understanding of the mode of action and tolerance mechanisms, future studies will employ rational genetic modifications to fine-tune fungal production and bacterial degradation of ITA and its derivatives, incorporating transporters to regulate metabolic flux (battle of rates). These targeted modifications aim to mimic the dynamic evolutionary interplay within the ecological niche in a synthetic co-culture, advancing our understanding about the rules of interaction.