Elucidating the itaconic acid pathway dynamics in Saccharomyces cerevisiae

Itaconic acid, a versatile platform chemical, has garnered significant attention due to its broad use in polymers, resins, and bio-based materials. Although fungi, especially Aspergillus and Ustilago, are the main producers of itaconic acid, reprogramming yeast species like Saccharomyces cerevisiae and Yarrowia lipolytica as alternative production platforms offers advantages for industrial bioproduction, including rapid growth, accessible genetic tools, and well-established fermentation methods. In this study, we systematically investigated the fungal itaconic acid pathway dynamics in S. cerevisiae, identifying the Ustilago metabolic route and mitochondrial transport mechanism as the most efficient. Notably, the heterologous produced itaconic acid was predominantly secreted by the yeast transformants. With in silico methods, we confirmed the role of Aqr1p, Dtr1p, and Qdr3p in itaconic acid transport in S. cerevisiae. Significant increases in itaconic acid biosynthesis were obtained when the main promiscuous itaconic acid transporter Dtr1p was swapped with the specialized Ustilago itaconic acid transporter Itp1, reaching titers of up to 1.3 g/L in shake flask cultivations. Transferring to 3.8 L bioreactor fermentations achieved a final titer of 4.7 g/L, the highest itaconic acid titer reported in S. cerevisiae to date. Although current yeast production levels are still below those of natural fungal producers, the molecular and mechanistic insights gained here are useful for improving itaconic acid biosynthesis, both in yeast and in the existing fungal production systems.