Reprogramming Yarrowia lipolytica metabolism for efficient synthesis of itaconic acid from flask to semi-pilot scale

Itaconic acid is an emerging platform chemical with extensive applications. It is currently produced by Aspergillus terreus through biological fermentation. However, A. terreus is a fungal pathogen and needs additional morphology controls, and therefore the production remains problematic. Here, we reprogrammed the GRAS yeast Yarrowia lipolytica metabolism for competitive itaconic acid production. After redirecting the flux of lipid accumulation as carbon sink, we evaluated itaconic acid production both inside and outside the mitochondria, and fine modulated its synthetic pathway. We then mimicked the regulation of nitrogen limitation in nitrogen replete conditions through down regulation of IDH by weak promoter changing, RNAi, or CRISPRi. Ultimately, we optimized fermentation parameters for fed-batch cultivations, and produced itaconic acid with titres of 130.1 g/L in 1L bioreactors and 94.8 g/L in a 50L bioreactor on semi-pilot scale. Our finds provide effective approaches for harnessing GRAS microorganism for competitive industrial itaconic acid production.