Functional Characterization of UDP-Glycosyltransferases Involved in Anti-viral Lignan Glycosides Biosynthesis in Isatis indigotica

Abstract

Isatis indigotica is a popular herbal medicine with its noticeable antiviral properties, which are primarily due to its lignan glycosides such as lariciresinol-4-O-β-D-glucoside and lariciresinol-4,4'-bis-O-β-D-glucosides (also called clemastanin B). UDP-glucose-dependent glycosyltransferases are the key enzymes involved in the biosynthesis of these antiviral metabolites. In this study, we systematically characterized the UGT72 family gene IiUGT1 and two UGT71B family genes, IiUGT4 and IiUGT71B5a, with similar enzymatic functions. Kinetic analysis showed that IiUGT4 was more efficient than IiUGT1 or IiUGT71B5a for the glycosylation of lariciresinol. Further knock-down and overexpression of these IiUGTs in I. indigotica's hairy roots indicates that they play different roles in planta: IiUGT71B5a primarily participates in the biosynthesis of coniferin not pinoresinol diglucoside, and IiUGT1 primarily participates in the biosynthesis of pinoresinol diglucoside, while IiUGT4 is responsible for the glycosylation of lariciresinol and plays a dominant role in the biosynthesis of lariciresinol glycosides in I. indigotica. Analysis of the molecular docking and site-mutagenesis of IiUGT4 have found that key residues for its catalytic activity are H373, W376, E397, and that F151 could be associated with substrate preference. This study elucidates the biosynthetic route of anti-viral lignan glycosides in I. indigotica, and provides the foundation for the production of anti-viral lignan glycosides via synthetic biology under the heterologous model.

Keywords: Isatis indigotica; clemastanin B; diversification; glycosyltransferases; lignan biosynthesis; transgenic hairy roots.

FIGURE 1

Schematic for the putative biosynthetic pathway of O-glycosylation toward lignans in I. indigotica.

FIGURE 2

Phylogenetic relationships between candidate IiUGTs and their close relatives. IiUGT genes from this study were in red or blod in black. A Neighbor-Joining phylogenetic tree was constructed the tree with 1000 bootstrap replicates by MEGA7. The GenBank accession numbers of UGT proteins in the tree are in Supplementary Table 2. The key genes whose functions were characterized in this study were marked with *.

FIGURE 3

The hypothetical biosynthetic pathway for clemastanin B in I. indigotica and functional characterization of candidate IiUGT proteins. (A) UPLC/Q-TOF-MS analysis of candidate IiUGT enzymatic reaction products against lariciresinol. The enzyme reactions of crude proteins of E. coil carrying empty vector (EV), IiUGT1, IiUGT4, IiUGT71B5a were assayed with UDP-glucose as the sugar donor and Lar (lariciresinol) as the sugar acceptor. (1) lariciresinol; (1a) lariciresinol-4′-O-β-D-glucoside; (1b) lariciresinol-4-O-β-D-glucoside; (1c) clemastanin B. (B) MS spectra of the products in negative mode. (C) A proposed biosynthetic pathway for clemastanin B. The monoglycoside products were purified in this study and identified by NMR (Supplementary Table 4).

FIGURE 4

Subcellular localization of IiUGTs fused with GFP at the C-terminus in N. benthamiana. GFP, GFP channel; Bright, light microscopy image; Merged, merged image of the GFP and Bright channels. Scale bars are 50 μm.

FIGURE 5

qRT-PCR analysis of IiUGT1, IiUGT4, IiUGT71B5a expression patterns in I. indigotica and the accumulation of lignan glycosides in I. indigotica. Relative expression levels of IiUGT1 (A), IiUGT4 (B) and IiUGT71B5a (C) in I. indigotica were performed as described in the experimental procedures using total RNAs extracted from different organs. The tissue samples are listed as follows: R, roots; S, stems; L, leaves; HR, hairy roots cultivated in MS liquid medium for 2 weeks; HR30, hairy roots cultivated in 6,7-V liquid medium for 30 days. The expression of IiUGT in root tissue was defined as 1.0. (D) Quantities of lignan glycosides from different tissues of I. indigotica. The tissue samples are listed as follows as above. CF, coniferin; PD, pinoresinol diglucoside; L4G, lariciresinol-4-O-β-D-glucoside; L4′G, lariciresinol-4′-O-β-D-glucoside; CB, clemastanin B; n.d, not detected. All data represent the mean ± standard deviation (SD) of three biological replicates. Different letters above the error bars indicate significant differences (p < 0.05) according Tukey’s test.

FIGURE 6

RNA interference silencing of IiUGT1, IiUGT4 and IiUGT71B5a in I. indigotica hairy roots. (A) Relative mRNA levels of IiUGT in RNAi hairy roots was measured by monitoring relative transcript levels by qRT-PCR. Effect of IiUGT gene silencing on the levels of Pinoresinol diglucoside (B), Coniferin (C), Lariciresinol-4-O-β-D-glucoside (D), Lariciresinol-4′-O-β-D-glucoside (E), Clemastanin B (F). Standard errors were calculated from three biological replicates. Different letters above the error bars indicate significant differences (p < 0.05). Statistical sinificance levels between the variable groups and the control group were calculated using one-way ANOVA test and Tukey’s multiple comparisons test (*p < 0.05 and ^**p < 0.01). (G) Proposed pathway from coniferyl alcohol to lignan glycosides in I. indigotica.

FIGURE 7

Overexpression of IiUGTs in I. indigotica hairy roots. (A,C,E) Western-blot analysis showing IiUGT transcript expression. Effect of IiUGT1 (B), IiUGT4 (D) and IiUGT71B5a (F) overexpressing on the contents of coniferin (CF), pinoresinol diglucoside (PD), lariciresinol-4-O-β-D-glucoside (L4G), lariciresinol-4′-O-β-D-glucoside (L4′G), clemastanin B (CB). Standard errors were calculated from three biological replicates. Statistical sinificance levels between the variable groups and the control group were calculated using one-way ANOVA test and Tukey’s multiple comparisons test (*p < 0.05, ^**p < 0.01, and ^***p < 0.001).

FIGURE 8

Structure-based sequence alignment and site-directed mutagenesis revealed the essential residues of IiUGT4. (A) Structure-based sequence alignment of IiUGT4 using UGT76G1 structure as a reference. α-helices was presented as large squiggles, β-strands as arrows, and strict β-turns as TT letters. The conserved residues interacting with the substrate were labeled with star symbols. (B) site-directed mutagenesis to identification of the essential residues of IiUGT4.