Multidimensional Engineering of <i>Saccharomyces cerevisiae</i> for Efficient Production of Retinol

Retinol, the major active form of vitamin A, plays a crucial role in vision, immune function, and skin health. The industrial model yeast Saccharomyces cerevisiae (S. cerevisiae) inherently possesses the mevalonate pathway, which supplies precursors for retinol biosynthesis. However, the imbalanced distribution of metabolic flux between the retinol synthesis pathway and other competing pathways limits the retinol titer. To improve the efficiency of the retinol synthesis pathway, we first identified two key bottleneck enzymes, geranylgeranyl diphosphate synthase (CrtE) and β-carotene 15,15'-dioxygenase (Blh), and optimized their gene copy numbers, resulting in a 72.0% increase in retinol titer. Subsequently, to reduce the diversion of the metabolic flux toward squalene production, we employed a multidimensional manipulation strategy to regulate the expression of squalene synthase (ERG9). By replacing the native ERG9 promoter with P_SPI1 and using a decompartmentalization strategy, the retinol titer was further increased to 1.41 g/L. After auxotrophic marker gene complementation, the resulting retinol titer in a 5-L bioreactor was 7.19 g/L, which was the highest reported value in S. cerevisiae. This work establishes an effective engineering strategy for high-yield retinol production in S. cerevisiae, which can facilitate subsequent process development and scale-up.