Ergothioneine is a naturally occurring antioxidant that has shown potential in ameliorating neurodegenerative and cardiovascular diseases. In this study, we investigated the potential of the Crabtree‐negative, oleaginous yeast Yarrowia lipolytica as an alternative host for ergothioneine production. We expressed the biosynthetic enzymes EGT1 from Neurospora crassa and EGT2 from Claviceps purpurea to obtain 158 mg·L−1 of ergothioneine in small‐scale cultivation, with an additional copy of each gene improving the titer to 205 mg·L−1. The effect of phosphate limitation on ergothioneine production was studied, and finally, a phosphate‐limited fed‐batch fermentation in 1 L bioreactors yielded 1.63 ± 0.04 g·L−1 ergothioneine in 220 h, corresponding to an overall volumetric productivity of 7.41 mg·L−1·h−1, showing that Y. lipolytica is a promising host for ergothioneine production.
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