(596c) Functional Reconstitution of a Pyruvate Hydrogenase in the Cytosol of Issatchenkia Orientalis for Overproduction of Organic Acids

Recent advances in metabolic engineering and synthetic biology have enabled microbial productions of value-added chemicals in an efficient and sustainable manner. Nevertheless, current engineering approaches rely mainly on pathway optimization and enzyme engineering with little regard to the role of cofactors and cofactor engineering. In particular, the production of succinic acid and citramalic acid in yeasts are challenging because of the insufficient supplies of NADH and acetyl-CoA in the cytosol, respectively. In the present study, we focus on enhancing the supply of these cofactors by reconstitution of a functional pyruvate dehydrogenase (PDH) in the cytosol for production of these organic acids in Issatchenkia orientalis, a non-model yeast with high tolerance to low pH conditions. We expressed the Escherichia coli PDH structural genes and localized the I. orientalis PDH complex to the cytosol through removal of mitochondrial targeting sequence. We found that I. orientalis PDH was more active than E. coli PDH, and its expression increased succinic acid yield by 32% and decreased pyruvate accumulation by 67% presumably through higher supply of cytosolic NADH in shake flask fermentation. Interestingly, the I. orientalis PDH was functional without expression of a lipoylation machinery. Furthermore, expression of cytosolic I. orientalis PDH increased citramalic acid yield by 40% in shake flask fermentation, indicating that higher level of acetyl-CoA was obtained in the cytosol. Our study has laid a solid foundation for the construction of an I. orientalis strain that has higher reducing power and overproduces acetyl-CoA for production of relevant organic acids.