LAUSR

Abstract Renewable alternatives for petroleum-derived chemicals are achievable through biosynthetic production. In this work, Pichia stipitis was engineered for itaconate production from xylose. The heterologous cis -aconitate decarboxylase from Aspergillus terreus was overexpressed with codon optimization, and 52 mg/L itaconate was produced by the initial engineered strain TJUIA01. To improve itaconate production, optimization of medium composition was performed and the availability of the critical substrate cis -aconitate was discovered as the limited factor for higher itaconate production. Then native cytoplasmic and mitochondrial aconitase were overexpressed to overcome the limitation, respectively. Furthermore, an efficient strain TJUIA03 with cytoplasmic aconitase was obtained, and the batch and fed-batch fermentations in a bioreactor were implemented, respectively. Finally, the itaconate titer reached to 1.52 g/L in a 3 L bioreactor, with an improvement of 28-fold than that of the strain TJUIA01, which also highlighted areas for future strain improvement compared to the glucose-based fermentation. Nevertheless, the results suggest that P. stipitis is a promising platform for itaconate production from xylose.