Profiles of Metabolic Genes in Uncaria rhynchophylla and Characterization of the Critical Enzyme Involved in the Biosynthesis of Bioactive Compounds-(iso)Rhynchophylline

Abstract

Rhynchophylline (RIN) and isorhynchophylline (IRN), two of the representative types of indole alkaloids, showed the unique spiroindole structures produced in Uncaria rhynchophylla. As the bioactive constituent of U. rhynchophylla, IRN has recently drawn extensive attention toward antihypertensive and neuroprotective activities. Despite their medicinal importance and unique chemical structure, the biosynthetic pathways of plant spiroindole alkaloids are still largely unknown. In this study, we used U. rhynchophylla, extensively used in traditional Chinese medicine (TCM), a widely cultivated plant of the Uncaria genus, to investigate the biosynthetic genes and characterize the functional enzymes in the spiroindole alkaloids. We aim to use the transcriptome platform to analyse the tissue-specific gene expression in spiroindole alkaloids-producing tissues, including root, bud, stem bark and leaf. The critical genes involved in the biosynthesis of precursors and scaffold formation of spiroindole alkaloids were discovered and characterized. The datasets from this work provide an essential resource for further investigating metabolic pathways in U. rhynchophylla and facilitate novel functional enzyme characterization and a good biopharming approach to spiroindole alkaloids.

Keywords: Uncaria rhynchophylla; biosynthetic pathway; rhynchophylline and isorhynchophylline; spiroindole alkaloids; transcriptome.

Figure 1

Proposed biosynthetic pathway of rhynchophylline and isorhynchophylline. (A) Metabolites and enzymes involved in the indole alkaloids biosynthetic pathway in U. rhynchophylla. (B) The homologs (P450 and FMO) for spiro-indole alkaloids formation in microbial natural product biosynthesis.

Figure 2

Specific indole alkaloids in U. rhynchophylla and LC-MS spectrum for indole alkaloids. Structures for rhynchophylline, corynoxeine and 3α-dihydrocadambine are shown in black, and their isomers (isorhynchophylline, isocorynoxeine, and 3β-dihydrocadambine) are shown in blue.

Figure 3

(A) the distribution of unigene length. (B) unigenes annotation by public databases. (C) Veen diagram of unigene annotation. (D) distribution of annotated species.

Figure 4

KEGG pathway annotation. (A) cellular processes; (B) environmental information processing; (C) genetic information processing; (D) metabolism; and (E) organismal systems.

Figure 5

Histogram of GO classifications. Upper: biological process; Lower: cellular component and molecular function.

Figure 6

Transcription Factor (TF) were predicted into 80 subgroups, and the histogram showed the abundance of the top 15 subgroups.

Figure 7

(A) tryptamine and analogues were proposed to be accepted by UrSTR for strictosidine and analogue formation. (B) biochemical characterization of UrSTR in vitro. (i) with tryptamine, (ii) with N-methyltryptamine and (iii) with N-dimethyltryptamine. (C) phylogenetic tree of STRs from indole alkaloids producing plants.

Figure 8

Docking results for tryptamine and analogues. (A) the structure of UrSTR in wheat and RsSTR in pale green and tryptamine in the active pocket. (B) the distance between the substrate (tryptamine, N-methyltryptamine and N,N-dimethyltryptamine) and the catalytic residue Glu309 (E309).

Figure 9

Candidate CYP450s and FMOs involved in spiroindole alkaloids. Phylogenetic analysis of CYP450s (A) and FMOs (B). (C) the co-expression analysis of candidate CYP450s and FMOs.