Engineering <i>Yarrowia lipolytica</i> for Efficient Production of Erythritol by Decoding and Harnessing Osmotic‐Stress‐Responsive Genetic Targets

Metabolic engineering has proven effective for enhancing erythritol production in Yarrowia lipolytica, while current approaches remain limited by their focus on laboratory strains and labor-intensive genetic modifications. To address these challenges, this study developed a streamlined engineering strategy by systematically investigating the osmotic stress response mechanisms of Yarrowia lipolytica CA20 during erythritol fermentation and identifying key genetic determinants of cell growth and erythritol biosynthesis. Integrated transcriptomics and metabolomics analyses revealed that nucleotide, lipid, amino acid, cofactor, and vitamin metabolism were essential for osmotic adaptation, which was experimentally validated through targeted gene overexpression. Interestingly, while these modifications improved cell viability, they did not consistently enhance erythritol production. Further, genes A000169 and A004467, coding for a hypothetical protein and a GPR1/FUN34/YaaH family protein, respectively, were shown to be closely associated with erythritol production. Overexpression of A000169 combined with deletion of A004467 and EYD1 generated a robust strain, Yarrowia lipolytica EG01, capable of producing erythritol with a maximum titer of 218.70 g/L and a productivity of 2.27 g/L/h, with an increase of 62.96% relative to the original strain CA20, exceeding previously reported production levels of erythritol in Y. lipolytica using batch fermentation. Finally, two-stage fed-batch fermentation could achieve erythritol titer and yield up to 276.70 g/L and 0.69 g/g within 132 h. Overall, these findings provide valuable clues to the engineering of Y. lipolytica capable of efficient production of erythritol.