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

## TL;DR

This study explores the variety of terpenoids in Andrographis paniculata by identifying new enzymes involved in their production.

## Contribution

The study characterizes a novel ent-copalyl diphosphate synthase and six new sesquiterpene synthases in Andrographis paniculata.

## Key findings

- ApTPS4 converts GGPP to ent-CPP and ApTPS5 converts ent-CPP to kaurene.
- Six active sesquiterpene synthases were identified, indicating diverse terpenoid skeletons in A. paniculata.
- Site-directed mutagenesis revealed key amino acid sites in ApTPS16 and ApTPS17 affecting enzyme function.

## Abstract

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 Andrographis paniculata. To better study the diversity of terpenoids of A. paniculata, a total of 39 ApTPSs were screened, and 27 full-length genes encoding ApTPSs were obtained. The results showed that ApTPS4 could convert GGPP to ent-CPP and that ApTPS5 could convert ent-CPP to kaurene. This study first identified six sesquiterpene synthases with biological activity and also indicated the presence of sesquiterpenes with multiple skeletons in A. paniculata. The increase in the number of ent-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 A. paniculata compared with the metabolites produced by AtTPSs found in the Arabidopsis thaliana 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 ent-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 A. paniculata.