Unravelling the metabolism of itaconate and related branched C5-dicarboxylates for CO₂-based production in Cupriavidus necator H16

Branched C5-dicarboxylates (C5-DCAs), such as itaconate, are high-value platform chemicals with diverse applications in the synthesis of bio-based polymers. In addition to their industrial relevance, itaconate has attracted attention as a key immunomodulatory metabolite. In this study, we demonstrated that Cupriavidus necator can grow on itaconate as its sole carbon source and identified key genes involved in its degradation through targeted deletions. These include multiple functional homologs of itaconate CoA-transferase, itaconyl-CoA hydratase, and (S)-citramalyl-CoA lyase, which collectively mediate itaconate catabolism. We also identified ItcR1, a LysR-type transcriptional regulator, as a key mediator of itaconate-responsive gene expression. Transcriptomic analysis revealed broader transcriptional responses to itaconate, methylsuccinate, and mesaconate, suggesting an expanded role of C5-DCA metabolism. Finally, by expressing a heterologous cis-aconitate decarboxylase, we achieved the autotrophic production of a C5-DCA mixture of methylsuccinate, citramalate, mesaconate, and itaconate from CO₂ and hydrogen. These findings provide new insight into branched C5-DCA metabolism in C. necator and advance its development as a chassis for the sustainable production of these industrially relevant compounds from inorganic feedstocks.