Gene cloning, functional identification, structural and expression analysis of sucrose synthase from <italic>Cistanche tubulosa</italic>

Wei-sheng Tian; Ya-ru Yan; Xiao-xue Cui; Ying-xia Wang; Wen-qian Huang; Sai-jing Zhao; Jun LI; She-po Shi; Peng-fei TU; Xiao Liu

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Gene cloning, functional identification, structural and expression analysis of sucrose synthase from Cistanche tubulosa

VernacularTitle:管花肉苁蓉蔗糖合酶基因的克隆鉴定、蛋白结构及表达分析
Author: Wei-sheng TIAN ^{1
,

2} ; Ya-ru YAN ^{1
,

3} ; Xiao-xue CUI ^{1
,

2} ; Ying-xia WANG ^{1
,

2} ; Wen-qian HUANG ^{1
,

2} ; Sai-jing ZHAO ^{1
,

2} ; Jun LI ^{1
,

2} ; She-po SHI ¹ ; Peng-fei TU ^{1
,

4} ; Xiao LIU ¹
Author Information

1. Modern Research Center for Traditional Chinese Medicine, Beijing Research Institute of Chinese Medicine, Beijing University of Chinese Medicine, Beijing
2. School of Chinese Materia Medica, Beijing University of Chinese Medicine, Beijing
3. Institute of Taihang Materia Medica, Shanxi University of Chinese Medicine, Jinzhong
4. State Key Laboratory of Natural and Biomimetic Drugs, School of Pharmaceutical Sciences, Peking University, Beijing
Publication Type:Research Article
Keywords: italic>Cistanche tubulosa; sucrose synthase; glycosides; UDP-glucose; enzymatic catalysis
From: Acta Pharmaceutica Sinica 2024;59(11):3153-3163
CountryChina
Language:Chinese
Abstract: Sucrose synthase plays a crucial role in the plant sugar metabolism pathway by catalyzing the production of uridine diphosphate (UDP)-glucose, which serves as a bioactive glycosyl donor for various metabolic processes. In this study, a sucrose synthase gene named CtSus was cloned from Cistanche tubulosa, a traditional Chinese medicine known for its rich content of diverse glycosides, based on transcriptome analysis results. The open reading frame of CtSus was 2 418 bp long encoding 805 amino acids. Sequence analysis revealed conserved domains associated with the plant sucrose synthase family at both the N-terminal and C-terminal regions of CtSus protein. Phylogenetic analysis demonstrated that CtSus shares a close evolutionary relationship with sucrose synthases from other plants within the same order. Functional identification of CtSus was performed through whole-cell catalysis coupling with UGT71BD1, a characterized glycosyltransferase enzyme. The results showed that the introduction of CtSus significantly enhanced the conversion rate of glycosylation catalyzed by UGT71BD1. The soluble expression of CtSus in Escherichia coli was further achieved using the pCold^TM TF expression vector. In vitro enzymatic assay indicated the activity of CtSus to catalyze the formation of UDP-glucose in the presence of sucrose and UDP. Real-time quantitative-polymerase chain reaction results showed that the expression patterns of CtSus gene in different parts of C. tubulosa and the suspension cell cultures of C. tubulosa under drought stress correlated with the accumulation patterns observed for phenylethanol glycosides, respectively. Furthermore, key amino acids and their interactions between enzyme and substrate were explored based on protein structure prediction and molecular docking results. Overall, our findings identify a sucrose synthase CtSus responsible for the supply of the active glycosyl donor UDP-glucose during the biosynthesis of glycoside products in C. tubulosa and have also provided a gene element that can be utilized in engineering strain construction for glycoside products production.