<i>De\nNovo</i> Biosynthesis of 2‑Phenylethanol by Metabolic\nEngineering the Oleaginous Yeast Rhodotorula toruloides

Microbial production offers a sustainable\nroute to plant-\nand chemical-based\nmanufacturing. 2-Phenylethanol (2-PE) has been widely used in foods,\nflavors, and pharmaceuticals. Herein, <i>de novo</i> biosynthesis\nof 2-PE was achieved for the first time by rewiring the non-native\nproducer Rhodotorula toruloides. Our\nresults showed that the generation of phenylalanine (Phe) and the\nrecycling of the amino group could be bypassed since the Ehrlich pathway\noutperformed the phenylacetaldehyde-dependent one in <i>de novo</i> biosynthesis of 2-PE when the adenosine 5′-triphosphate (ATP)\ncitrate synthase (ACL) was inactivated. The 2-PE titer was enhanced\nto 151.7 mg/L in a shake flask by alleviating feedback inhibition,\nenhancing precursor availability and cofactor balance. Finally, the\nproduction of 2-PE was elevated to 1.06 g/L with a yield of 8.5 mg/g\nglucose and productivity of 8 mg/L/h in a 3 L bioreactor. Our results\nshould shed light on the microbial production of other aromatic derivatives\nwith R. toruloides.