Identification and functional characterization of the diterpene synthase family in Pogostemon cablin (Blanco) Benth.

Pogostemon cablin (Blanco) Benth (Patchouli) is an aromatic herb extensively used in pharmaceutical and cosmetic industries. Sesquiterpenes are the characteristic constitutes in patchouli which are synthesized in the glandular trichomes on leaves and stems. Gibberellic acid (GA), a tetracyclic diterpenoid, plays a crucial role in the formation of glandular trichome. However, the diterpene biosynthesis remains largely unknown in patchouli. Here we identified a small diterpene synthases (diTPSs) family comprising three class II diTPSs (PatCPS1-3) and three class I diTPSs (PatKSL1 and PatGLS1-2). These diTPSs are functionally characterized using a yeast heterologous expression system. PatCPS1 was identified as an ent-copalyl diphosphate synthase (ent-CPS), in combination with PatKSL1, yield ent-kaurene, the precursor of GA, indicating their involvement in primary metabolism. PatCPS2 converted GGPP into (+)-8, 13-copalyl diphosphate (CPP). No activity was detected for PatCPS3, PatGLS1 and PatGLS2. Three ohnologs of PatCPS1 were further characterized to explore the possible functional differentiation of ent-CPS during the evolution of tetraploid hybrid patchouli genome. GC-MS analysis showed all ohnologs are functional ent-CPSs, demonstrating the functional conservation of PatCPS1 during evolution. Expression profiling by qRT-PCR showed PatCPS1 and PatKSL1 are ubiquitously expressed in all tissues, consistent with their involvement in primary metabolism. Conversely, PatCPS2 and PatCPS3 were predominantly expressed in the above ground parts, indicating a role in specialized metabolism. In summary, these findings clarify the early stages of GA biosynthesis in patchouli and provide gene elements for further metabolic engineering of sesquiterpenes via diterpenoids.