Molecular identification and functional characterization of two glycosyltransferases genes from Fallopia multiflora

Qizhong Cai; Changzheng Liu; Lu Liu; Yuewei Ge; Xuanxuan Cheng; Bi Luo; Liangyun Zhou; Quan Yang

doi:10.3389/fpls.2022.1017122

Molecular identification and functional characterization of two glycosyltransferases genes from Fallopia multiflora

Front Plant Sci. 2022 Dec 6:13:1017122. doi: 10.3389/fpls.2022.1017122. eCollection 2022.

Authors

Qizhong Cai^{1

2}, Changzheng Liu³, Lu Liu^{1

2}, Yuewei Ge⁴, Xuanxuan Cheng^{1

2}, Bi Luo^{1

2}, Liangyun Zhou^{1

2}, Quan Yang^{1

2}

Affiliations

¹ School of Traditional Chinese Medicine, Guangdong Pharmaceutical University, Guangdong Provincial Research Center on Good AgriculturalPractice and Comprehensive Agricultural Development Engineering Technology of Cantonese Medicinal Materials, Comprehensive Experimental Station of Guangzhou, Chinese Material Medica, China Agriculture Research System (CARS-21-16), Guangzhou, China.
² Key Laboratory of State Administration of Traditional Chinese Medicine for Production & Development of Cantonese Medicinal Materials, Guangzhou, China.
³ State Key Laboratory Breeding Base of Dao-di Herbs, National Resource Center for Chinese Materia Medica, China Academy of Chinese Medical Sciences, Beijing, China.
⁴ School of Traditional Chinese Medicine, Guangdong Pharmaceutical University, Guangzhou, China.

Abstract

The traditional Chinese medicine plant Fallopia multiflora (Thunb.) Harald. contains various pharmacodynamically active glycosides, such as stilbene glycosides, anthraquinone (AQ) glycosides, and flavonoid glycosides. Glycosylation is an important reaction in plant metabolism that is generally completed by glycosyltransferase in the last step of the secondary metabolite biosynthesis pathway, and it can improve the beneficial properties of many natural products. In this study, based on the transcriptome data of F. multiflora, we cloned two Uridine-diphosphate-dependent glycosyltransferases (UGTs) from the cDNA of F. multiflora (FmUGT1 and FmUGT2). Their full-length sequences were 1602 and 1449 bp, encoding 533 and 482 amino acids, respectively. In vitro enzymatic reaction results showed that FmUGT1 and FmUGT2 were promiscuous and could catalyze the glycosylation of 12 compounds, including stilbenes, anthraquinones, flavonoids, phloretin, and curcumin, and we also obtained and structurally identified 13 glycosylated products from both of them. Further experiments on the in vivo function of FmUGT1 and FmUGT2 showed that 2, 3, 5, 4'- tetrahydroxy stilbene-2-O-β-d-glucoside (THSG) content in hairy roots was elevated significantly when FmUGT1 and FmUGT2 were overexpressed and decreased accordingly in the RNA interference (RNAi) groups. These results indicate that FmUGT1 and FmUGT2 were able to glycosylate a total of 12 structurally diverse types of acceptors and to generate O-glycosides. In addition, FmUGT1 and FmUGT2 efficiently catalyzed the biosynthesis of THSG, and promoted the production of AQs in transgenic hairy roots.

Keywords: Fallopia multiflora (Thunb.) Harald.; glycosylation; metabolic engineering; transgenic hairy roots; uridine-diphosphate glycosyltransferase.