The Biosynthetic Gene Cluster of Mushroom‐Derived Antrocin Encodes Two Dual‐Functional Haloacid Dehalogenase‐like Terpene Cyclases

(−)‐Antrocin (1), produced by the medicinal mushroom Antrodia cinnamomea, is a potent antiproliferative compound. The biosynthetic gene cluster of 1 was identified, and the pathway was characterized by heterologous expression. We characterized a haloacid dehalogenase‐like terpene cyclase AncC that biosynthesizes the drimane‐type sesquiterpene (+)‐albicanol (2) from farnesyl pyrophosphate (FPP). Biochemical characterization of AncC, including kinetic studies and mutagenesis, demonstrated the functions of two domains: a terpene cyclase (TC) and a pyrophosphatase (PPase). The TC domain first cyclizes FPP to albicanyl pyrophosphate, and the PPase domain then removes the pyrophosphate to form 2. Intriguingly, AncA (94 % sequence identity to AncC), in the same gene cluster, converts FPP into (R)‐trans‐γ‐monocyclofarnesol instead of 2. Notably, Y283/F375 in the TC domain of AncA serve as a gatekeeper in controlling the formation of a cyclofarnesoid rather than a drimane‐type scaffold. We uncovered the biosynthesis of (−)‐antrocin, from the medicinal mushroom Antrodia cinnamomea, using genetic and biochemical approaches. Furthermore, two unusual dual‐functional haloacid dehalogenase‐like terpene cyclases, AncA and AncC, which generate cyclofarnesoid‐ and drimane‐type products, respectively, were characterized. These findings lay the foundation for the biotechnological production of antrocins independent of the Antrodia mushroom.