Discovery and characterization of four glycosyltransferases involved in anthraquinone glycoside biosynthesis inRubia yunnanensis

Glycosylation, which is catalyzed by UDP-glycosyltransferases, represents one of the most common modifications in plant secondary metabolites and plays significant roles in plant physiology. While the roots and rhizomes ofR. yunnanensisDiels are the well-known traditional Chinese medicine "Xiaohongshen" andR. yunnanensisis rich in different types of glycoside natural products, no glycosyltransferase has yet been molecular genetically and biochemically explored before. Here we report the discovery and characterization of four new glycosyltransferases (GTs), RyUGT3A, RyUGT3B, RyUGT11, and RyUGT12 that mediated anthraquinone glycoside biosynthesis, from the transcriptome ofR. yunnanensis. RyUGT3A and RyUGT12 are regioselective glycosyltransferases of beta-OH anthraquinones and showed broad substrate promiscuity toward at least 28 compounds to formO-,N-, andS-glycosides. Through the developed one-pot transglycosylation reaction, 18 glycoside congeners, including four new ones, were produced. Molecular modelling and site-directed mutagenesis further revealed the critical residues for substrate binding and glycosylation. The expression profiles of these four GTs with or without methyl jasmonate (MeJA) induced provided a reasonable genetic explanation for the phenomenon that the anthraquinone glycosides are majorly accumulated in the roots ofR. yunnanensis. Finally, the subcellular localization of RyUGT3A and RyUGT12 showed that both enzymes were located in the cytomembrane and nucleus. This study expands the growing toolbox of novel glycosyltransferases that could be exploited as practical biocatalysts and provides insights into the continued exploration of other family enzymes in this traditional Chinese medicine as well.