Enhancing selective itaconic acid synthesis in Yarrowia lipolytica through targeted metabolite transport reprogramming

Abstract
Background: Itaconic acid is a valuable platform chemical with applications in polymer synthesis and other industrial sectors. Microbial fermentation offers a sustainable production route, involving two fungi such as Aspergillus terreus and Ustilago maydis. However, their employment in industrial bioprocesses for itaconic acid production is characterized by several challenges. Yarrowia lipolytica is a non-conventional yeast that shows suitability for industrial production and it is widely employed as heterologous host to obtain relevant metabolites. This study aimed to engineer Y. lipolytica for the selective production of itaconic acid by optimising intracellular metabolic fluxes and transport mechanisms.

Results: A metabolic engineering strategy was developed to prevent the secretion of citric and isocitric acids by blocking their transport at both mitochondrial and plasma membrane levels in Y. lipolytica strains. Specifically, the inactivation of YlYHM2 and YlCEX1 genes reduced secretion of citric and isocitric acid, enabling their accumulation in the mitochondria. Additionally, heterologous transporters from Aspergillus terreus (mttA and mfsA) and Ustilago maydis (mtt1 and itp1) were introduced to enhance the mitochondrial export of cis-aconitate and the extracellular secretion of itaconic acid. For the first time, complete gene set of the itaconate biosynthetic pathways from both fungal species were functionally expressed and compared in a yeast system with a similar genetic background. A synergistic increase in itaconic acid production was observed when both pathways were co-expressed, combined with the inactivation of native citric and isocitric transport. In contrast to previously engineered Y. lipolytica strains for itaconic acid production, the optimised strain obtained in this study does not require complex or nutrient-rich media, while achieving the highest product yield (0.343 mol IA/mol glucose) and productivity (0.256 g/L/h) reported in yeast, with minimal by-product formation.

Conclusions: By integrating transporter engineering and pathway diversification, this study demonstrates an effective strategy to enhance itaconic acid production in Y. lipolytica while minimising by-product formation. The findings provide new insights into organic acid transport in yeast and open avenues for further optimization of microbial cell factories for sustainable biochemical production.