Identification and Functional Characterization of UbiA-Type Terpene Synthases in Inonotus obliquus

Inonotus obliquus (Chaga mushroom) is a medicinal basidiomycete rich in structurally diverse terpenoids, yet the biosynthetic enzymes responsible for sesquiterpene formation remain poorly understood. Here, we identified six UbiA-type terpene synthases (IoUbiA1-6) from the I. obliquus genome and functionally characterized them through heterologous expression in Aspergillus oryzae. GC-MS and NMR analyses demonstrated that IoUbiA1-4 catalyze the biosynthesis of bisabolane-type sesquiterpenes, including (+)-Z-α-bisabolene (1), (-)-β-curcumene (2), and (6R,7E)-α-bisabolene (3). Sequence alignment and mutational analyses revealed two highly conserved catalytic motifs, NQXXXXXXXED and QDXXDXXXD, that are essential for enzymatic activity, as alanine substitution of key residues completely abolished product formation. These results experimentally establish the catalytic significance of the conserved motifs in fungal UbiA-type terpene synthases. In addition, metabolic engineering of the mevalonate pathway in the A. oryzae expression host significantly increased 1 production from 3.99 to 61.13 mg/kg. This study expands the functional diversity of fungal UbiA-type terpene synthases, uncovers the molecular basis for bisabolane-type sesquiterpene biosynthesis in I. obliquus, and provides a foundation for the biotechnological production of valuable sesquiterpenoids.