(553f) Pathway Localization and Redox Engineering of Yarrowia Lipolytica for Fatty Alcohol Production

Fatty alcohols currently find its 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 the low-yield harvest from plants. Recently, conventional hosts including E. coli and S. cerevisiae have been used for fatty alcohol production with some success. Oleaginous yeast, such as Yarrowia lipolytica, offer significant advantages for the production of oleochemicals, as its native metabolism evolved to high flux in fatty acid biosynthetic pathway. Fatty alcohol production in the cytosolic space is hindered by low availability of acyl-CoA and presence of competing metabolic pathways. As a result, we have targeted fatty alcohol production in the peroxisome, where acyl-CoA flux is directed during beta-oxidation, and potential fewer competing pathways. Several Fatty Acyl-CoA Reductases (FAR) from bacterial and mammalian sources were screened using canonical peroxisome targeting sequences, as well as used enzymes fusion strategy to physically localize FAR next to 3-ketoacyl-CoA thiolase (3KAT) in peroxisome. 3KAT fusion resulted in nearly double the titer of fatty alcohol irrespective of any FAR’s overexpression. The nature of the linker attached between 3KAT and FAR had a significant impact on the titer. Engineering of peroxisome physiology had moderately improvement of titer. In addition, we addressed the cofactor availability in the peroxisome by overexpression of auxiliary enzyme IDP3 and NADH kinase to provide an additional co-factors supply for FAR, which had a large impact on fatty alcohol titer as well as free fatty acid concentration. The highest titer achieved was over 1.6 g/L of fatty alcohol in shake flask culture.