Metabolic engineering of Yarrowia lipolytica for higher fatty alcohol production

Abstract Fatty alcohols are versatile chemicals with growing demand across a broad range of industries, yet conventional production methods pose environmental challenges. Microbial biosynthesis offers a promising alternative, if hosts can be engineered to achieve high product titers. The industrially relevant oleaginous yeast Yarrowia lipolytica is well suited to this challenge because of its natural capacity for lipid metabolism. Here, we engineered Y. lipolytica W29 for fatty alcohol production by disrupting pathways involved in lipogenesis and fatty alcohol oxidation, identifying fatty alcohol oxidation as a major constraint on product accumulation. Initial engineering yielded fatty alcohol titers of 2.5 g/L from 40 g/L glucose. Subsequent optimization of media supplements and nutrient concentrations, combined with deletion of the citrate transporter CEX1 and addition of 1% Tergitol to the extractive culture medium, increased titers to 10.9 g/L in shake flasks with 80 g/L glucose and a carbon-to-nitrogen ratio of 60. Scaling the process to fed-batch bioreactor cultivation further increased titers to 19.4 g/L. These results demonstrate that combining metabolic engineering with medium and process optimization enables high-titer fatty alcohol production in Y. lipolytica . Highlights Increased fatty alcohol titers by disrupting lipogenesis, fatty alcohol oxidation, and citrate export Dodecane and Tergitol addition increased flask fermentation titers to 10.7 g/L Fed-batch fermentation in bioreactors was performed with Tergitol to 19.4 g/L