Discovery of two diterpene cyclases involved in the scopadulane-type diterpene biosynthesis in Scoparia dulcis

The tropical medicinal plant, sweet-broom (Scoparia dulcis L.), produces unique tetracyclic diterpenes such as scopadulcic acid B (SDB) and scopadulciol in young leaf. SDB exhibits various pharmacological activities including antivirus and antitumor. SDB production in S. dulcis leaf tissue is rapidly and transiently stimulated by methyl jasmonate (MJ) as an elicitor. It has been proposed that SDB biosynthesis is initiated by cyclization of geranylgeranyl diphosphate (GGPP) to syn-copalyl diphosphate (CPP) by a syn-CPP synthase (syn-CPS) and subsequent further cyclization to scopadulane by a kaurene synthase-like cyclase (KSL). Despite the medicinal importance of S. dulcis, the enzyme genes involved in the SDB biosynthesis are not fully understood.

In previous work, we performed RNA-sequencing and de-novo assembly of Scoparia young leaf and root, and identified putative key diterpene cyclase genes, SdSCPS and SdKSL1 [1]. Here, we report the characterization of these diterpene cyclases from S. dulcis, which has led to the identification of unique biosynthetic pathway. Using a bacterial expression system, we clarified the biological function of the SdSCPS as syn-CPS. Moreover, from the enzymatic analysis and in silico structural analysis of the deletion mutants of SdSCPS, we predicted several key residues those maintain enzymatic activity. The recombinant SdKSL1 was also suggested to be a type 1 diterpene cyclase, because it was found that the novel diterpene hydrocarbon alcohol was produced by recombinant E. coli with pACYC-SdGGPPS-SdSCPS and pET-SdKSL1. On the other hand, qPCR study revealed that the level of expression of the SdSCPS and SdKSL1 transcripts in Scoparia leaves increased drastically in response to MJ treatment, whereas expression of the Scoparia ent-CPP synthase and kaurene synthase transcripts were not induced by MJ. Our results strongly suggest that SdSCPS and SdKSL1 are responsible for the biosynthesis of SDB.

This study will provides a valuable resource for conducting future diterpene biosynthesis researches in other important medicinal plants as well as this medicinal plant.

Acknowledgements: This work was supported by JSPS KAKENHI Grant Number 15K07991 (Grant-in-Aid for Scientific Research (C)).

Keywords: Diterpene cyclase, secondary metabolism, RNA-seq.

References:

[1] Yamamura Y and Lee J.-B. Prediction of genes involved in terpenoid diversity in Scoparia dulcis L. 11th International Congress of Plant Molecular Biology (IPMB), Iguazú Falls, Brazil; 2015