Abstract

Vine tea (Nekemias grossedentata), a medicinal and edible plant, produces volatile sesquiterpenoids that remain underexplored despite their important roles in ecological adaptation and bioactive compound synthesis. While volatile sesquiterpenoids are well-established as key contributors to aroma, defense, and therapeutic properties in other plants, their biosynthetic pathways in vine tea are poorly characterized. To address this gap, we conducted a systematic investigation integrating multi-omics analyses and functional genomics. Metabolic profiling revealed a root-specific accumulation pattern for volatile sesquiterpenoids. Genome-wide identification uncovered 84 terpene synthase (NgTPS) genes, with family expansion primarily driven by tandem duplications. We functionally characterized ten NgTPS genes through heterologous expression in yeast and in vitro enzymatic assays using purified proteins. These enzymes collectively produced 23 sesquiterpenoids and 5 monoterpenes, several of which are known fragrance contributors. Furthermore, we achieved de novo synthesis of these volatile sesquiterpenoids in the high-terpene-yielding yeast chassis EPY300, with shake-flask titers reaching 3.00-21.51 mg L-1. This work establishes a foundation for elucidating the biosynthetic pathways of volatile sesquiterpenoids in vine tea and for harnessing these compounds in food, pharmaceutical, and agricultural applications.