Neofunctionalization underlies the evolutionary origin of sclareol biosynthesis in the mint family

Plant specialized metabolites play essential ecological roles, yet the mechanisms underlying their diversification remain poorly understood. Here, we investigate the biosynthesis of sclareol, a potent antifungal diterpene produced by Salvia sclarea (clary sage). A complete telomere-to-telomere genome assembly of clary sage, compared with genomes of related Lamiaceae species that do not produce sclareol, reveals a recent tandem duplication of a class II diterpene synthase gene (SsLPPS). This duplicated enzyme acquires a specific catalytic activity, synthesizing labda-13-en-8-ol diphosphate (LPP), the direct precursor of sclareol. Structural modeling and site-directed mutagenesis identify key amino acid substitutions responsible for this neofunctionalization. Integrative genome, chromatin, and transcriptome analyses show that SsLPPS and additional diterpenoid biosynthetic genes are organized in a trichome-specific, co-regulated gene cluster. Together, our findings illustrate how enzyme innovation and regulatory rewiring can give rise to unique metabolic pathways and may inform future strategies for engineering valuable plant terpenoids.