Reprogramming <i>Yarrowia lipolytica</i> metabolism for efficient synthesis of itaconic acid from flask to semipilot scale

Itaconic acid is an emerging platform chemical with extensive applications. Itaconic acid is currently produced by <i>Aspergillus terreus</i> through biological fermentation. However, <i>A. terreus</i> is a fungal pathogen that needs additional morphology controls, making itaconic acid production on industrial scale problematic. Here, we reprogrammed the Generally Recognized As Safe (GRAS) yeast <i>Yarrowia lipolytica</i> for competitive itaconic acid production. After preventing carbon sink into lipid accumulation, we evaluated itaconic acid production both inside and outside the mitochondria while fine-tuning its biosynthetic pathway. We then mimicked the regulation of nitrogen limitation in nitrogen-replete conditions by down-regulating NAD⁺-dependent isocitrate dehydrogenase through weak promoters, RNA interference, or CRISPR interference. Ultimately, we optimized fermentation parameters for fed-batch cultivations and produced itaconic acid titers of 130.1 grams per liter in 1-liter bioreactors and 94.8 grams per liter in a 50-liter bioreactor on semipilot scale. Our findings provide effective approaches to harness the GRAS microorganism <i>Y. lipolytica</i> for competitive industrial-scale production of itaconic acid.