Peroxisome Engineering of Yarrowia lipolytica for Fatty Alcohol Production

Fatty alcohols currently find use in areas such as surfactants, plasticizers, lubricants, fuels, and the cosmetics industry; however, traditional production methods rely on non-renewable petroleum-derived compounds or are harvested from non-sustainable oil-seed crops. Recently, conventional hosts including Escherichia coli and Saccharomyces cerevisiae have been used for fatty alcohol production with some success. Oleaginous yeasts, such as Yarrowia lipolytica, offer significant advantages to produce oleochemicals, as their native metabolism evolved for high-flux fatty acid biosynthesis. However, fatty alcohol production in the cytosol faces challenges, including toxicity, limited availability of acyl-CoA, and the presence of competing pathways. To overcome these limitations, we targeted fatty alcohol biosynthesis into the peroxisome, where fatty acyl-CoA flux is naturally directed toward beta-oxidation and with fewer competing pathways. Following media optimization, fatty acyl-CoA reductases (FAR) from bacterial and mammalian sources were screened using canonical peroxisome targeting sequences. Additionally, we implemented an enzyme fusion strategy to physically colocalize FAR next to the 3-ketoacyl-CoA thiolase (3KAT) enzyme in the peroxisome. 3KAT fusion resulted in nearly double the titer of fatty alcohols, irrespective of which FAR was overexpressed. We then systematically engineered the subcellular environment within peroxisomes by increasing peroxisome numbers and boosting localized NADPH availability via the peroxisomal malate pathway and NADH kinase. These strategies significantly improved the organelle capacity for fatty alcohol production. The highest titer we achieved in shake flask culture was over 1.6 g/L of fatty alcohols. Further, we scaled up the fatty alcohol production in a 2 L bioreactor, achieved 2.77 g/L of fatty alcohols, in which a peak production of 2.53 g/L of C16:0 hexadecanol was achieved.