De Novo Biosynthesis of 2-Phenylethanol by Metabolic Engineering the Oleaginous Yeast Rhodotorula toruloides

Microbial production offers a sustainable route to plant- and chemical-based manufacturing. 2-Phenylethanol (2-PE) has been widely used in foods, flavors, and pharmaceuticals. Herein, de novo biosynthesis of 2-PE was achieved for the first time by rewiring the non-native producer Rhodotorula toruloides. Our results showed that the generation of phenylalanine (Phe) and the recycling of the amino group could be bypassed since the Ehrlich pathway outperformed the phenylacetaldehyde-dependent one in de novo biosynthesis of 2-PE when the adenosine 5'-triphosphate (ATP) citrate synthase (ACL) was inactivated. The 2-PE titer was enhanced to 151.7 mg/L in a shake flask by alleviating feedback inhibition, enhancing precursor availability and cofactor balance. Finally, the production of 2-PE was elevated to 1.06 g/L with a yield of 8.5 mg/g glucose and productivity of 8 mg/L/h in a 3 L bioreactor. Our results should shed light on the microbial production of other aromatic derivatives with R. toruloides.