Study on the Functional Characterization of 9-epi-Caryophyllene Synthase from <i>Lavandula x intermedia</i> (Lavandin)

Lavender species hold considerable economic importance and are widely grown across the globe for their essential oils (EOs), which are rich in terpenes vital for the cosmetic, personal care, and pharmaceutical sectors. EOs consist mainly of monoterpenes, though certain species may also include small quantities of sesquiterpenes, depending on genetic and environmental factors. The sesquiterpene 9-epi-caryophyllene, produced by <i>Lavandula x intermedia</i> 9-epi-caryophyllene synthase (LiCPS), constitutes up to 8% of lavender EOs. However, the mechanistic basis of LiCPS activity is still unclear. Herein, we determined the hydrodynamic radius of LiCPS to be 5.7 ± 0.2 nm. Molecular docking simulations were conducted using a structural model predicted using AlphaFold2, followed by site-specific mutagenesis. Mutations D292A, D296A, R433A, D436A, and E444A reduced enzyme activity by 100- to 200-fold. Deletions of residues 1-15 or 513-539 (Δ1-15 or Δ513-539) significantly enhanced activity, whereas deletions of residues 246-257 or 448-464 (Δ246-257 or Δ448-464) caused a dramatic loss of activity. The highest expression level of LiCPS was observed in flowers under white light. These discoveries improve our grasp of the LiCPS function in lavender, enabling new genetic strategies to optimize essential oil biosynthesis.