(86b) Metabolic Engineering of an Acid-Tolerant Strain Pichia Kudriavzevii for Itaconic Acid Production

Itaconic acid is an unsaturated dicarboxylic acid with a methylene group. As a replacement for petroleum-derived chemicals that are used as polymer precursors, Itaconic acid has a broad application spectrum in biopolymer industry owing to the remaining double bond for incorporating additional functionalities. Currently itaconic acid is produced by the fermentation of the filamentous fungus Aspergillus terreus. Due to the low robustness of fungus cultivation, researchers have been working on engineering other microbes for itaconic acid production.

Pichia kudriavzevii is an acid-tolerant nonconventional yeast that has been engineered to produce succinic acid in industry. By introducing the cis-aconitic acid decarboxylase (cad) gene from A. terreus, the initial titer of itaconic acid reached 105 mg/L. Considering that the substrate cis-aconitate is produced in the mitochondrial whereas itaconic acid is produced in the cytosol, a transporter is needed to transport cis-aconitate to the cytosol. To do so, we overexpressed a native mitochondrial transporter, which led to doubled production of itaconic acid. By introducing the newly inverted CRISPR-Cas9 system into P. kudriavzevii, we successfully knocked out the isocitrate dehydrogenase (icd) gene to increase the availability of cis-aconitate. Finally, we integrated cad and the mitochondrial transporter gene to the genome of the P. kudriavzevii icd knockout strain. The production increased to 521mg/L in shake-flask culture, and XX g/L in fed-batch fermentation. Our work demonstrates the great potential of engineering P. kudriavzevii as an industrial platform for organic acid production.