Engineering Salidroside Biosynthetic Pathway in Hairy Root Cultures of Rhodiola crenulata Based on Metabolic Characterization of Tyrosine Decarboxylase

Xiaozhong Lan,Zhihua Liao,Lingjiang Zeng,Wanhong Liu,Wenlin Huang,Weilie Zheng,Kai Chang,Min Chen,Xiaoqiang Liu,Qiang Wang,Fei Qiu,Hong Quan,Rakesh K Srivastava

doi:10.1371/journal.pone.0075459

Abstract

Tyrosine decarboxylase initializes salidroside biosynthesis. Metabolic characterization of tyrosine decarboxylase gene from Rhodiola crenulata (RcTYDC) revealed that it played an important role in salidroside biosynthesis. Recombinant 53 kDa RcTYDC converted tyrosine into tyramine. RcTYDC gene expression was induced coordinately with the expression of RcUDPGT (the last gene involved in salidroside biosynthesis) in SA/MeJA treatment; the expression of RcTYDC and RcUDPGT was dramatically upregulated by SA, respectively 49 folds and 36 folds compared with control. MeJA also significantly increased the expression of RcTYDC and RcUDPGT in hairy root cultures. The tissue profile of RcTYDC and RcUDPGT was highly similar: highest expression levels found in stems, higher expression levels in leaves than in flowers and roots. The gene expressing levels were consistent with the salidroside accumulation levels. This strongly suggested that RcTYDC played an important role in salidroside biosynthesis in R. crenulata. Finally, RcTYDC was used to engineering salidroside biosynthetic pathway in R. crenulata hairy roots via metabolic engineering strategy of overexpression. All the transgenic lines showed much higher expression levels of RcTYDC than non-transgenic one. The transgenic lines produced tyramine, tyrosol and salidroside at higher levels, which were respectively 3.21–6.84, 1.50–2.19 and 1.27–3.47 folds compared with the corresponding compound in non-transgenic lines. In conclusion, RcTYDC overexpression promoted tyramine biosynthesis that facilitated more metabolic flux flowing toward the downstream pathway and as a result, the intermediate tyrosol was accumulated more that led to the increased production of the end-product salidroside.

Highlights

Rhodiola crenulata is a perennial herbaceous plant and mainly grows on the very high mountains of Tibet Plateau
The expression levels of RcTYDC in transgenic lines were 2.3–21.6 folds compared with that in nontransgenic lines (P,0.01). These results indicated that the cDNA of RcTYDC was introduced into R. crenulata and expressed in corresponding transgenic lines at significantly higher levels that reached the goal of breaking the tyrosine decarboxylase (TYDC)-defined step by overexpression and it would facilitate the metabolic flux flowing toward the downstream pathway
Overexpression of RcTYDC could remarkably promote biosynthesis of tyramine that facilitated more metabolic flux flowing toward the downstream pathway and as a result, the intermediate tyrosol was accumulated more that led to the increased production of the end-product salidroside

Summary

Introduction

Rhodiola crenulata is a perennial herbaceous plant and mainly grows on the very high mountains of Tibet Plateau. The stems of R. crenulata plants have been used as health food and herb more than 1000 years before by Chinese people especially the local Tibetan peoples because it is considered to be the best one of Rhodiola species [1]. R. crenulata belongs to the family of Crassulaceae and mostly grows in the very harsh environments with high altitude of 5000 meters and above, low oxygen concentration, strong ultraviolet radiation and poor soils [2]. R. crenulata is widely used as anti-depressive, and anti-fatigue and to reinforce immunity, improve memory and learning, scavenge active-oxygen species, and relieve altitude sickness because of its pharmaceutical natural products, salidroside [4] and tyrosol [5]. Metabolic engineering salidroside biosynthetic pathway might be the best method

Methods

Results

Discussion

Conclusion