Characterization of Terpene Synthases Reveals the Diversity of Terpenoids in Andrographis paniculata

Terpenoids have significant biological activity and good clinical efficacy and are important for defence and physiological regulation in plants. Andrographolide and similar labdane-related diterpenoids have been isolated and characterized as the main medicinal constituents of drugs from <i>Andrographis paniculata</i>. To better study the diversity of terpenoids of <i>A. paniculata</i>, a total of 39 <i>ApTPSs</i> were screened, and 27 full-length genes encoding ApTPSs were obtained. The results showed that ApTPS4 could convert GGPP to <i>ent</i>-CPP and that ApTPS5 could convert <i>ent</i>-CPP to kaurene. This study first identified six sesquiterpene synthases with biological activity and also indicated the presence of sesquiterpenes with multiple skeletons in <i>A. paniculata</i>. The increase in the number of <i>ent</i>-copalyl diphosphate synthases and the loss of biological function by most sesquiterpene synthases and monoterpene synthases may explain why diterpenoids are the main specific metabolites in <i>A. paniculata</i> compared with the metabolites produced by AtTPSs found in the <i>Arabidopsis thaliana</i> genome. As revealed by site-directed mutagenesis, 533Val of ApTPS16 is an important site for maintaining the single main product capability, and 534Tyr of ApTPS17 may also be more important. The ApTPS17 Y534V mutation caused it to lose its main biological function. This study characterized a novel <i>ent</i>-copalyl diphosphate synthase and six sesquiterpene synthases. This provided evidence for the existence of other terpenoids and revealed the diversity of chemical components, providing a reference for future pharmacological research for <i>A. paniculata</i>.