Integrated metabolomic and transcriptomic analyses of Dendrobium chrysotoxum and D. thyrsiflorum reveal the biosynthetic pathway from gigantol to erianin

Abstract

Erianin is one of the most representative bibenzyls with significant inhibitory activity against a wide range of tumor cells. However, the low erianin level in natural materials has severely inhibited its further development in health care. Our aim was to uncover the erianin biosynthetic pathway to lay the foundation for promoting its production. Firstly, we screened and obtained two Dendrobium species (Dendrobium thyrsiflorum stems with lower erianin content and D. chrysotoxum stems with higher erianin content), belonging to the same Dendrobium section (Chrysotoxae). A systematic analysis of bibenzyl structure and content in two stems revealed that gigantol might be an erianin biosynthetic intermediate, which was verified by introducing deuterium-labeled gigantol. Chemical structure analyses indicated that gigantol was modified by two kinds of enzymes (hydroxylases and O-methyltransferases), leading to erianin synthesis. Up-regulated hydroxylases and O-methyltransferases (OMTs) were screened out and were performed by molecular docking simulation experiments. We propose a rational biosynthetic pathway from gigantol to erianin, as well as relevant enzymes involved in the process. Our findings should significantly contribute to comprehensive resolution of the erianin biosynthetic pathway, promote its large-scale industrial production as well as contribute to biosynthesis studies of other bibenzyls.

Keywords: biosynthetic pathway; dendrobium; erainin; metabolomics; transcriptomics.

Figure 1

Erianin content in different wild Dendrobium species (A); Top 10 differential secondary metabolites in DT and DC stems (B).

Figure 2

Bibenzyl compounds identified in the metabolome. (A) Structure of identified bibenzyl compounds; (B) Difference in the relative content of identified compounds in the stems of DT and DC.

Figure 3

Chromatographic profiles of treated DC stems (A), deuterium-labeled gigantol (B) and erianin (C).

Figure 4

Analysis of differentially expressed genes (DEGs) between DC and DT. (A) Heat map of DEGs based on hierarchical clustering analysis; (B) Volcano plots of DEGs. The red dots represent the significantly upregulated genes, and the green dots represent the significantly downregulated genes; (C) GO enrichment analysis of DEGs in the three functional groups; (D) Scatter plots of top 20 KEGG pathways with the most significant enrichment.

Figure 5

Pathways and genes involved in the biosynthesis of erianin in DT and DC. The pathway was built using published literatures and KEGG pathway analysis. The colored grids on the top or right of each gene is the expression heat map of key enzyme genes for the biosynthesis of erianin between DT and DC. The red solid circles represent Log₂ fold change (Log₂FC) of metabolites (DT vs DC). PAL, phenylalanine ammonia lyase; C4H, cinnamate 4-hydroxylase; 4CL, 4-coumadin CoA ligase; BBS, bibenzyl synthase (or bibenzyl synthase-like); DBR, double bond reductase; OMTs, O-methyltransferases.

Figure 6

The comprehensive analysis of differentially expressed hydroxylases and OMTs. All the differentially expressed hydroxylases (A), and OMTs (B) between DT and DC. *, 0.01 < P ≤ 0.05; **, P ≤ 0.01; NS, not significant. Phylogenetic analysis of hydroxylases (C) and OMTs (D). Numbers on branches indicate the bootstrap percentage values calculated from 1000 bootstrap replicates. Genes marked with red circles show that it was significantly up-regulated in DC stems (Log2FC>1, p<0.05). Sequence accession numbers are given in Supplementary Table S4 .

Figure 7

Results of molecular docking between substrates (compound 1, gigantol and compound 2) and key enzymes. (A) binding energy between the individual compounds and target proteins predicted by AutoDock Vina. The global diagrams and the details and the 2D diagrams of the docking between compound 1 and LOC110095638 (B), gigantol and LOC110106749 (C), compound 2 and LOC110108303 (D).

Figure 8

Hypothesized biosynthetic pathway from gigantol to erianin. Solid lines represent more likely biosynthetic pathways. Candidate genes were predicted according to molecular docking results.