Engineering reduced nicotinamide cofactor metabolism for enhanced cell growth and succinic acid production in a succinate dehydrogenase deficient Yarrowia lipolytica strain

Background Succinic acid (SA) is a four-carbon dicarboxylic acid of considerable industrial relevance, with applications spanning the food, chemical, and pharmaceutical sectors. The remarkable acid tolerance of the yeast Yarrowia lipolytica makes it a promising microbial cell factory for SA production. Numerous metabolic engineering strategies have focused on disrupting genes encoding the succinate dehydrogenase (SDH) complex to enhance SA accumulation. However, such a modification is associated with impaired growth and the accumulation of by-products, notably acetic acid (AA). Results To improve growth capacity, SA productivity, and reduce AA formation in Y. lipolytica SDH5-deficient strains ( Sdh5Δ ), carbon flux from glycolysis was partially redirected toward the pentose phosphate pathway by overexpression of the native genes encoding glucose-6-phosphate dehydrogenase ( ZWF1) and 6-phosphogluconate dehydrogenase ( GND1) , thereby enhancing NADPH generation. The resulting strain was further engineered to increase NADH availability for the mitochondrial electron transport chain by overexpressing genes encoding either a mutated NADPH-dependent malate dehydrogenase (TfMdh) from Thermus flavus or the soluble transhydrogenase (EcSthA) from Escherichia coli , enabling indirect conversion of NADPH to NADH. This strategy resulted in 2-fold and 2.2-fold increase in SA productivity and titre, respectively, compared to the Sdh5Δ-ALE strain during bioreactor cultivation on glucose-based media. Moreover, AA accumulation was reduced 1.2-fold, while growth rates were significantly improved. Conclusions The proposed engineering strategies, especially heterologous expression of EcSthA, partly alleviated energy limitations in Y. lipolytica Sdh5Δ strain, resulting in improved SA productivity and growth performance.