Complete pathway elucidation and heterologous reconstitution of (+)‐nootkatone biosynthesis from Alpinia oxyphylla

Abstract

Summary (+)‐Nootkatone is a natural sesquiterpene ketone widely used in food, cosmetics, pharmaceuticals, and agriculture. It is also regarded as one of the most valuable terpenes used commercially. However, plants contain trace amounts of (+)‐nootkatone, and extraction from plants is insufficient to meet market demand. Alpinia oxyphylla is a well‐known medicinal plant in China, and (+)‐nootkatone is one of the main components within the fruits. By transcriptome mining and functional screening using a precursor‐providing yeast chassis, the complete (+)‐nootkatone biosynthetic pathway in Alpinia oxyphylla was identified. A (+)‐valencene synthase (AoVS) was identified as a novel monocot‐derived valencene synthase; three (+)‐valencene oxidases AoCYP6 (CYP71BB2), AoCYP9 (CYP71CX8), and AoCYP18 (CYP701A170) were identified by constructing a valencene‐providing yeast strain. With further characterisation of a cytochrome P450 reductase (AoCPR1) and three dehydrogenases (AoSDR1/2/3), we successfully reconstructed the (+)‐nootkatone biosynthetic pathway in Saccharomyces cerevisiae, representing a basis for its biotechnological production. Identifying the biosynthetic pathway of (+)‐nootkatone in A. oxyphylla unravelled the molecular mechanism underlying its formation in planta and also supported the bioengineering production of (+)‐nootkatone. The highly efficient yeast chassis screening method could be used to elucidate the complete biosynthetic pathway of other valuable plant natural products in future.