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

(+)-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.

Automated summary

The complete biosynthetic pathway of (+)-nootkatone in Alpinia oxyphylla has been identified and successfully reconstructed in Saccharomyces cerevisiae. This study has important implications for understanding the molecular mechanism of (+)-nootkatone formation in plants and for bioengineering production. The highly efficient yeast chassis screening method can also be used to elucidate the biosynthetic pathways of other valuable plant natural products in the future.