Rapid screening of glycosyltransferases in plants using a linear DNA expression template based cell-free transcription-translation system. (January 2022)

• Record Type: Journal Article
• Authors: Guo, Shaobin; Wang, Mingdi; Xu, Wen; Zou, Fuxian; Lin, Jingjing; Peng, Qin; Xu, Wei; Xu, Shaohua; Shi, Xianai
• Is Part Of: Phytochemistry. Volume 193(2022)
• Publication Date: 2022-01
• Subjects: Anoectochilus roxburghii -- Orchidaceae -- Linear DNA expression Template (LET) -- Cell-free transcription-translation system (TX-TL) -- Isoquercitrin -- UDP-Glycosyltransferases; Botanical chemistry -- Periodicals; Biochemistry -- Periodicals; Botany -- Periodicals; Chimie végétale -- Périodiques; 572.2
• DOI: 10.1016/j.phytochem.2021.113007
• Languages: English
• ISSNs: 0031-9422

Abstract

Abstract: Plants have an extensively large number of enzymes including glycosyltransferases that are important in the biosynthesis of natural products. However, it is time-consuming and challenging to study these enzymes and only a small percentage of them have been well-characterized. Here, we report a rapid method to screen plant glycosyltransferases using a linear DNA expression template (LET) based cell-free transcription-translation system (TX-TL). As a proof of concept, we amplified and tested glycosyltransferases from Arabidopsis thaliana and showed that the catalytic activity results of these glycosyltransferases from LET-based-TX-TL were consistent with previous studies. We then chose a local medicinal plant Anoectochilus roxburghii, acquired its transcriptome sequences, and applied this method to study its glycosyltransferases. We rapidly expressed all the putative UDP-glucose glycosyltransferases using LET-based-TX-TL and discovered 6 unreported active glycosyltransferases which can catalyze the glycosylation of quercetin into isoquercitrin. Thus, LET-based-TX-TL was shown to be a powerful tool for researchers to rapidly screen plant glycosyltransferases for the first time. Graphical abstract: Image 1 Highlights: Plant glycosyltransferases were rapidly screened using LET-based-TX-TL. Activities of AtUGTs tested in this work were consistent with previous studies. Six unreported ArUGTs with glycosyltransferase activity were discovered. Researchers could use this tool to rapidly screen novel plant enzymes.