Enhancement of lipid production using formate-driven NADPH regeneration in Yarrowia lipolytica

The global transition toward a sustainable bioeconomy requires efficient microbial platforms for converting renewable feedstocks into energy-dense chemicals. Yarrowia lipolytica is a well-established oleaginous yeast used in lipid biotechnology because of its acetyl-CoA metabolism and genetic tractability. However, lipid biosynthesis is frequently limited by restricted intracellular NADPH availability, creating a common redox bottleneck. To address this, we investigated the use of formate as an auxiliary electron donor in combination with heterologous NADP + -dependent formate dehydrogenase (FDH) and lipid-pathway engineering. We overexpressed the highly NADP⁺-specific formate dehydrogenase (FDH) from Pseudomonas sp. 101 and observed an increase of up to 47.8% in the relative NADPH/NADP⁺ under the tested conditions. This redox module was integrated into systematic pathway engineering, including suppression of β-oxidation (∆ PEX10 ) and overexpression of the triacylglycerol assembly gene ( DGA1 ). Specifically, the genes encoding mitochondrial enoyl-[acyl-carrier-protein] reductase ( ETR1 ) and 3-ketoacyl-CoA reductase ( KAR ) were overexpressed to examine whether their combination with the FDH module could further support lipid production. Under glucose-formate co-feeding, the engineered strain (YXRF5-3) achieved a FAME titer of 1.12 g/L and volumetric productivity of 11.69 mg/L·h, representing an 88.5% increase in productivity compared to the engineered control strain (YXR-1). A similar enhancement pattern was observed under xylose-based cultivation, where YXRF5-3 achieved a FAME titer of 0.90 g/L and volumetric productivity of 4.91 mg/L·h. This study establishes a coordinated metabolic engineering strategy in Y. lipolytica that combines formate-driven NADPH regeneration with systematic lipid pathway optimization. Under the tested conditions, the resulting engineered strain showed enhanced lipid production from both glucose and xylose. These findings provide proof of concept suggesting that formate can serve as an effective auxiliary electron donor in engineered oleaginous yeast, successfully enhancing lipid production.