Functional Characterization of a Sesquiterpene Synthase Involved in Stereospecific Biosynthesis of β-Bisabolol

β-Bisabolol, an odor-active monocyclic sesquiterpene alcohol, is the primary constituent of essential oils from various agriculturally and medically important plants. Despite its attractive potential for applications in pest control, flavoring agents, medicine, and cosmetics, the terpene synthase responsible for the biosynthesis of β-bisabolol in plants remains to be elucidated. Here, we report the characterization of a sesquiterpene synthase encoded by <i>VjBisS</i> in the medicinal plant <i>Valeriana jatamansi</i> Jones ex Roxb. <i>In vitro</i> and <i>in vivo</i> enzyme assays showed that VjBisS could catalyze the stereospecific biosynthesis of (1<i>R</i>,10<i>R</i>)-β-bisabolol. Subsequently, the potential catalytic mechanism of VjBisS was investigated by protein modeling, molecular docking, and site-directed mutagenesis. As a result, residues critical for the catalytic activity and catalytic specificity of VjBisS were determined. Together, our work enables the enzyme-catalyzed efficient stereoselective synthesis of (1<i>R</i>,10<i>R</i>)-β-bisabolol, laying a foundation for understanding and optimizing plant essential oil constituents for applications in agriculture, food, medicine, and cosmetics.