Metabolic Engineering of an Acid-tolerant Yeast Strain for Itaconic Acid Production

Itaconic acid (IA), or 2-methylenesuccinic acid, has a broad spectrum of applications in the biopolymer industry owing to the presence of one vinyl bond and two acid groups in the structure. Its polymerization can follow a similar mechanism as acrylic acid but additional functionality can be incorporated into the extra beta acid group. Currently, the bio-based production of IA in industry relies on the fermentation of the filamentous fungus . However, the difficulties associated with the fermentation undertaken by filamentous fungi together with the pathogenic potential of pose a serious challenge for industrial-scale production. In recent years, there has been increasing interest in developing alternative production hosts for fermentation processes that are more homogenous in the production of organic acids. is a non-conventional yeast with high acid tolerance to organic acids at low pH, which is a highly desirable trait by easing downstream processing. We introduced -aconitic acid decarboxylase gene () from (designated ) into this yeast and established the initial titer of IA at 135 ​± ​5 ​mg/L. Subsequent overexpression of a native mitochondrial tricarboxylate transporter (herein designated ) presumably delivered -aconitate efficiently to the cytosol and doubled the IA production. By introducing the newly invented CRISPR-Cas9 system into . , we successfully knocked out both copies of the gene encoding isocitrate dehydrogenase (), aiming to increase the availability of -aconitate. The resulting strain, devoid of and overexpressing and on its genome produced IA at 505 ​± ​17.7 ​mg/L in shake flasks, and 1232 ​± ​64 ​mg/L in fed-batch fermentation. Because the usage of an acid-tolerant species does not require pH adjustment during fermentation, this work demonstrates the great potential of engineering . as an industrial chassis for the production of organic acid.