Terpene synthases screening in agarwood tree Aquilaria yunnanensis leads to the discovery of agarospirol

Agarwood, renowned as one of the most valuable natural materials in the world of fragrance, is notoriously difficult and time-consuming to obtain, creating a critical bottleneck for its utilization and conservation. Sesquiterpenes are the key constituents of agarwood, with agarospirol recognized as one of its characteristic compounds. Nevertheless, the biosynthesis of agarospirol remains unreported. Here, we conducted a functional screening of terpene synthase (TPS) genes from the agarwood-producing tree Aquilaria yunnanensis. Using a heterologous expression system in Saccharomyces cerevisiae , we characterized 26 TPS genes, identifying 30 structurally diverse sesquiterpene products. Among them, AyTPS1 was identified as an agarospirol-producing sesquiterpene synthase, and the structure of the enzymatically produced agarospirol was unequivocally confirmed by NMR spectroscopy. Furthermore, model-guided mutagenesis of AyTPS1 revealed that residues L283 and G435 markedly influence the distribution of agarospirol and related eudesmane-type sesquiterpenes, suggesting their involvement in shaping the active-site environment for spiro-ring formation and hydroxylated product formation. The elucidation of the AyTPS1 and its catalytic mechanism lays a crucial foundation for the industrial-scale biotechnological production of this valuable sesquiterpene.