Metabolic Engineering of the Yeast Saccharomyces Cerevisiae for Itaconic Acid Production

A current pathway for itaconic acid production utilizes the carbon metabolism of a fungus, Aspergillus terreus, which converts the cis-aconitate intermediate of the TCA cycle to itaconic acid through the enzyme, cis-aconitic decarboxylase (CAD).  In 2008, Kanamasa et. al, successfully cloned a novel gene, CAD1, that coded for the CAD enzyme in S. cerevisiae.  The ability to synthesize itaconic acid in a microorganism suggests an alternative method of production that is economically competitive to current fungal fermentation pathways in A. terreus.  To increase itaconic acid production in S. cerevisiae, the CAD1 gene was imported into the genome and several enzymes that regulate production away from cis-aconitate were selectively knocked out of the genome.  A double knockout of the ADE3 and BNA2 genes of S. cerevisiae raised itaconic acid yield to 120 mg/L.  Moving the TCA enzymes upstream of cis-aconitate production out of the mitochondria increased yield to 160 mg/L.  Additionally, inserting multiple genes for overexpression the cis-aconitate precursors, oxaloacetate and citrate, did not noticeably improve yields.  Relocalizing the CAD1 gene to the mitochondria decreased itaconic acid yields, and was directly proportional to the number of CAD enzymes expressed in the cell.  Therefore, it is suggested that the CAD enzyme destabilized the mitochondria.