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

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). To improve growth capacity, SA productivity, and reduce AA formation in Y. lipolytica SDH5 -deficient strains, namely Sdh5Δ and its evolved derivative Sdh5Δ-ALE, 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) , with the aim to enhance cytoplasmic NADPH turnover. The resulting strains ZWF1-GND1 and ZWF1-GND1-ALE were further engineered to increase NADH availability for the mitochondrial electron transport chain by overexpressing genes encoding either a mutated NADPH-dependent malate dehydrogenase ( mdH ) from Thermus flavus or the soluble transhydrogenase ( sthA ) from Escherichia coli , enabling indirect conversion of NADPH to NADH. During batch cultivation in bioreactor under non-optimized conditions, this strategy resulted in 1.4-fold and 1.7-fold increases in SA titer and productivity, respectively, in the EcStha-ALE strain, a ZWF1-GND1-ALE derivative overexpressing the sthA gene, compared with the Sdh5Δ-ALE strain. Moreover, AA accumulated for the EcStha-ALE strain similarly to the parental strain during the first 32 h. Subsequently, AA was reconsumed by EcStha-ALE, in contrast to Sdh5Δ-ALE, resulting in a two-fold difference in AA concentration after 60 h of culture upon glucose exhaustion. Furthermore, during fed-batch bioreactor cultivation, corn steep liquor was found an efficient low-cost nitrogen source compared with yeast extract and casein tryptone, with biomass reaching 48 and 56 gDCW·L⁻¹, respectively, while SA productivities were 0.36 and 0.44 g·L⁻¹·h⁻ 1 . The proposed engineering strategies, especially heterologous expression of sthA transhydrogenase, partly alleviated energy limitations in Y. lipolytica SDH5 -deficient strain, resulting in improved SA productivity and growth performance.