Selective Enzymatic Synthesis of the Grapefruit Flavor (+)-Nootkatone

(+)‐Nootkatone is a high‐value sesquiterpenoid known for its grapefruit‐odor impression. Its isolation from natural plant sources suffers from low yields, and chemical syntheses involve carcinogenic or hazardous compounds. Herein, a biocatalytic route for the synthesis of (+)‐nootkatone that combines two enzymes in one pot is presented. In the first step, a cytochrome P450 monooxygenase catalyzes the selective allylic hydroxylation of the sesquiterpene (+)‐valencene to the intermediate alcohol nootkatol. In the second step, nootkatol is further oxidized to (+)‐nootkatone by an alcohol dehydrogenase (ADH). The challenging task of finding a suitable cofactor regeneration system was solved by careful selection of an appropriate cosubstrate for the ADH, which works in a dual‐functional mode. After reaction optimization, involving cosolvent and cosubstrate screening, (+)‐nootkatone concentrations of up to 360 mg L−1 and a space‐time yield of 18 mg L−1 h−1 were achieved. Fruitful synthesis: A P450 monooxygenase and an alcohol dehydrogenase are combined in a one‐pot cascade to afford the high‐value grapefruit flavor (+)‐nootkatone by selective oxidation of (+)‐valencene.