Utilization of ethanol for itaconic acid biosynthesis by engineered Saccharomyces cerevisiae

In Saccharomyces cerevisiae, ethanol can serve as both a carbon source and NADH donor for the production of acetyl-CoA derivatives. Here we investigated the metabolic regulation of ethanol utilization for itaconic acid production by S. cerevisiae. To understand the interconnection between the TCA cycle and the glyoxylate pathway, mitochondrial membrane transporter proteins SFC1, YHM2, CTP1, DIC1 and MPC1 were knocked out and results showed that SFC1 functions as an important entrance of the glyoxylate pathway into the TCA cycle, and YHM2 is helpful to IA production but not the primary pathway for citric acid supply. To decrease the accumulation of acetic acid, the major ADP/ATP carrier of the mitochondrial inner membrane, AAC2, was upregulated and determined to accelerate ethanol utilization and itaconic acid production. RNA sequencing results showed that AAC2 overexpression enhanced IA titer by upregulating the ethanol-acetyl-CoA pathway and NADH oxidase in the mitochondrial membrane. RNA-seq analysis also suggested that aconitase ACO1 may be a rate-limiting step of IA production. However, the expression of exogenous aconitase didn't increase IA production but enhanced the rate of ethanol utilization and decreased cell growth.