Hairy root induction and benzylisoquinoline alkaloid production in Macleaya cordata

Peng Huang,Ruolan Jiang,Linlan Yu,Xiubin Liu,Jianguo Zeng,Liqiong Xia,Zhaoshan Tang,Qi Tang,Wei Liu,Min Xu

doi:10.1038/s41598-018-30560-0

Abstract

Sanguinarine is currently widely used to replace antibiotic growth promoters in animal feeding and has demonstrated useful anticancer activity. Currently, the main source of sanguinarine is from an important medicinal plant, Macleaya cordata. To obtain a new source of sanguinarine production, we established hairy root cultures of M. cordata by co-cultivating leaf and stem explants with Agrobacterium rhizogenes. Except the co-cultivation medium, all growth media contained 200 mg/L timentin to eliminate A. rhizogenes. Through comparing the metabolic profiles and gene expression of hairy roots and wild-type roots sampled at five time points, we found that the sanguinarine and dihydrosanguinarine contents of hairy roots were far higher than those of wild-type roots, and we revealed the molecular mechanism that causes these metabolites to increase. Consequently, this study demonstrated that the hairy root system has further potential for bioengineering and sustainable production of sanguinarine on a commercial scale. To the best of our knowledge, this is the first efficient protocol reported for the establishment of hairy root cultures in M. cordata using A. rhizogenes.

Highlights

Sanguinarine (SAN) is a quaternary benzylisoquinoline alkaloid (BIA)
We identified the protopine 6-hydroxylase (McP6H) and dihydrobenzophenanthridine oxidase (McDBOX) enzymes involved in the conversion of protopine (PROT) to SAN in M. cordata
The method we used for A. rhizogenes infection comes from an improvement on the previous transformation system of M. cordata[26]

Summary

Introduction

Sanguinarine (SAN) is a quaternary benzylisoquinoline alkaloid (BIA). SAN has been used for many years as a natural growth promoter (NGP) and alternative to antibiotics in livestock production[1,2]. Macleaya cordata (Chinese name “Bo-luo-hui”) is a traditional medicinal herb that belongs to the Papaveraceae family It is the most important commercial source of SAN3,15,16. In recent years, HR cultures have become a useful biological system to study the biosynthesis of alkaloids[18,19]. Our recent work includes functional characterizations of many metabolic genes involved in SAN biosynthesis and identification of the pattern of SAN synthesis in M. cordata[16]. In another previous study, we identified the protopine 6-hydroxylase (McP6H) and dihydrobenzophenanthridine oxidase (McDBOX) enzymes involved in the conversion of protopine (PROT) to SAN in M. cordata. We confirmed the functionality of the transgenic system and the integration of rol genes by molecular biological analysis

Methods

Results

Discussion

Conclusion