Integrated characterization of six sesquiterpene synthases unravels diversified terpene biosynthesis in the mint species Leucosceptrum canum

Introduction Sesquiterpenoids are important plant-derived compounds with diverse bioactivities and significant pharmaceutical potential. Leucosceptrum canum Smith, a monotypic species of the Lamiaceae family, accumulates a diverse array of terpenoids with important biological functions. However, the functional diversity and catalytic mechanisms of its sesquiterpene synthases (sesquiTPSs) remain poorly understood. Methods Six sesquiTPSs (LcTPS3-LcTPS8) from L. canum were functionally characterized through heterologous expression in Escherichia coli , followed by product identification using GC-MS and NMR analyses. Phylogenetic analysis, molecular docking, and gene expression profiling were performed to investigate evolutionary relationships, catalytic mechanisms, and expression patterns. Metabolic engineering strategies were applied to construct high-yield production systems. Results LcTPS3-LcTPS8 generated germacrene A, (+)-5-epi-aristolochene, γ-selinene, germacrene D, α-trans-bergamotene, and (+)-β-himachalene, respectively. Phylogenetic analysis clustered LcTPS3-6 and LcTPS8 in the TPS-a subfamily and LcTPS7 in TPS-b, reflecting their evolutionary divergence. Integrated structural and docking analyses revealed four distinct cyclization modes, with the spatial conformation of FPP determining product specificity. Expression analysis demonstrated tissue-specific patterns and stress responses, suggesting a regulated metabolic network. We established production systems for six sesquiterpenoids, with titers reaching up to 618.73 mg L⁻¹ in shake-flask cultures. Discussion These findings provide mechanistic insights into sesquiTPS function and terpene diversification in L. canum , and offer valuable enzymatic resources for the sustainable biosynthesis of high-value sesquiterpenoids.