Functional characterization of NES and GES responsible for the biosynthesis of (E)-nerolidol and (E,E)-geranyllinalool in Tripterygium wilfordii

Ping Su,Tianyuan Hu,Yifeng Zhang,Wei Gao,Hongyu Guan,Yuru Tong,Luqi Huang,Jiawei Zhou,Yujia Liu

doi:10.1038/srep40851

Abstract

Triptolide and celastrol, two principal bioactive compounds in Tripterygium wilfordii, are produced from geranylgeranyl diphosphate (GGPP) and farnesyl diphosphate ((E,E)-FPP) through terpenoid biosynthesis pathway. However, little is known about T. wilfordii terpene synthases which could competitively utilize GGPP and (E,E)-FPP as substrates, producing C15 and C20 tertiary alcohols. Here we firstly cloned the genes encoding nerolidol synthase (NES) and geranyllinalool synthases (GES1, GES2), which are responsible for the biosynthesis of (E)-nerolidol and (E,E)-geranyllinalool. In vitro characterization of recombinant TwNES and TwGES1 revealed both were functional enzymes that could catalyze the conversion of (E,E)-FPP and GGPP to (E)-nerolidol and (E,E)-geranyllinalool, which were consistent with the results of yeast fermentation. Biochemical characterization revealed TwNES and TwGES1 had strong dependency for Mg2+, Km and Kcat/Km values of TwNES for (E,E)-FPP were 12.700 μM and 0.029 s−1/μM, and TwGES1 for GGPP were 2.039 μM and 0.019 s−1/μM. Real-time PCR analysis showed the expression levels of NES and GES1 increased by several fold in the suspension cells treated with alamethicin, indicating TwNES and TwGES1 are likely to utilize GGPP and (E,E)-FPP to generate tertiary alcohols as precursor of plant volatiles, which play important roles in the ecological interactions between T. wilfordii and other organisms.

Highlights

Tripterygium wilfordii Hook. f., known as lei gong teng, is widely used in the treatment of rheumatoid arthritis and other inflammatory diseases in Traditional Chinese Medicine[1,2]
Previous reports indicated that nerolidol synthase (NES) and geranyllinalool synthase (GES) utilize geranylgeranyl diphosphate (GGPP), (E,E)-FPP or geranyl diphosphate (GPP) as substrates[15,16], which were two competitors of the triptolide and celastrol biosynthetic pathway in T. wilfordii
We have identified three enzymes (TwNES, TwGES1 and TwGES2), involved in the biosynthesis of the C15 and C20 tertiary alcohols in T. wilfordii

Summary

Introduction

Tripterygium wilfordii Hook. f., known as lei gong teng, is widely used in the treatment of rheumatoid arthritis and other inflammatory diseases in Traditional Chinese Medicine[1,2]. The biosynthesis pathway leading to (E)-nerolidol and (E,E)-geranyllinalool, same as that of other terpenoids in higher plants, starts from the condensation of isopentenyl diphosphate (IPP) and dimethylallyl diphosphate (DMAPP), which formed through either the cytoplasmic mevalonate (MVA) or the plastidic 2-C-methyl-D-erythritol-4-phosphate (MEP) pathway[12] Sequential condensations of these two C5-units, result in the formation of linear elongated prenyldiphosphates, including GPP, (E,E)-FPP and GGPP13–15. None of enzymes, impacting the contents of triptolide and celastrol by consuming their precursor compounds competitively or affecting the plant growth through regulating the production of defense compounds, have been reported In this manuscript, we firstly clone the full-length genes encoding NES and GES enzymes in T. wilfordii suspension cells and identify the functions of the enzymes both in vitro and in yeast. Functional characterization of TwNES and TwGES provide two gene regulatory elements for further regulating the biosynthesis of the bioactive compounds (e.g. triptolide and celastrol) in T. wilfordii

Methods

Results

Conclusion