(139b) Metabolic Engineering of Issatchenkia Orientalis for Succinic Acid Production

The nonconventional yeast Issatchenkia orientalis is an industrially relevant platform microorganism for production of organic acids thanks to its unusual ability to grow in highly acidic conditions and tolerance to high concentrations of several organic acids. Here we describe the metabolic engineering of I. orientalis to achieve robust production of succinic acid. A reductive TCA pathway was introduced into the yeast. Further genetic optimizations, including deletion of by-product pathways and heterologous expression of a dicarboxylic acid transporter, allowed 25 g/L of succinic acid to be produced from 50 g/L of glucose in shake flask fermentation. Nevertheless, pyruvic acid accumulated at titer of 20 g/L because of the lack of cytosolic NADH, which hindered further production of succinic acid. To increase cytosolic NADH availability, glycerol was used as co-substrate. Fermentation using 50 g/L of glucose and 20 g/L of glycerol allowed conversion of the accumulated pyruvic acid to succinic acid and attained a titer of 39 g/L of succinic acid. Knockouts of an external NADH dehydrogenase and a dicarboxylic acid importer further improved the titer of succinic acid to 47 g/L. However, glucose repression of glycerol utilization lowered the productivity. Current research efforts focus on two parallel strategies: (1) alleviating the catabolite repression on glycerol and (2) engineering an I. orientalis strain for succinic acid production using glycerol as sole carbon source. Overall, this work shows that I. orientalis can serve as a potential industrial platform for production of succinic acid.