Coupling deep transcriptome analysis with untargeted metabolic profiling in Ophiorrhiza pumila to further the understanding of the biosynthesis of the anti-cancer alkaloid camptothecin and anthraquinones

Abstract

The Rubiaceae species, Ophiorrhiza pumila, accumulates camptothecin, an anti-cancer alkaloid with a potent DNA topoisomerase I inhibitory activity, as well as anthraquinones that are derived from the combination of the isochorismate and hemiterpenoid pathways. The biosynthesis of these secondary products is active in O. pumila hairy roots yet very low in cell suspension culture. Deep transcriptome analysis was conducted in O. pumila hairy roots and cell suspension cultures using the Illumina platform, yielding a total of 2 Gb of sequence for each sample. We generated a hybrid transcriptome assembly of O. pumila using the Illumina-derived short read sequences and conventional Sanger-derived expressed sequence tag clones derived from a full-length cDNA library constructed using RNA from hairy roots. Among 35,608 non-redundant unigenes, 3,649 were preferentially expressed in hairy roots compared with cell suspension culture. Candidate genes involved in the biosynthetic pathway for the monoterpenoid indole alkaloid camptothecin were identified; specifically, genes involved in post-strictosamide biosynthetic events and genes involved in the biosynthesis of anthraquinones and chlorogenic acid. Untargeted metabolomic analysis by Fourier transform ion cyclotron resonance mass spectrometry (FT-ICR-MS) indicated that most of the proposed intermediates in the camptothecin biosynthetic pathway accumulated in hairy roots in a preferential manner compared with cell suspension culture. In addition, a number of anthraquinones and chlorogenic acid preferentially accumulated in hairy roots compared with cell suspension culture. These results suggest that deep transcriptome and metabolome data sets can facilitate the identification of genes and intermediates involved in the biosynthesis of secondary products including camptothecin in O. pumila.

Keywords: Anthraquinone; Camptothecin; Hairy root; Metabolome; Ophiorrhiza pumila; Transcriptome.

Fig. 1

Possible biosynthetic pathway of camptothecin and related alkaloids.

Fig. 2

Deep transcriptome analysis of hairy roots and cell suspension cultures of O. pumila. (A) Expression profile of all unigenes plotted by log10 FPKM in hairy roots (HR) (y-axis) and cell suspension culture (CSC) (x-axis). The unigenes differentially expressed with a threshold of an FDR-corrected P-value <0.05 are shown in color: red, up-regulated inHR; green, up-regulated in CSC. (B) The differentially expressed unigenes are shown in a heatmap diagram sorted by the magnitude of fold change (log2). The 3,649 and 1,777 unigenes from HR and CSC are preferentially expressed in the respective samples.

Fig. 3

Plausible biosynthetic pathway and expression of unigenes involved in the biosynthesis of anthraquinones in O. pumila. The expression levels of unigenes encoding the enzymes of each step are shown. Separated proteins of MenD, MenH and MenC in bacteria are known to be fused to a single protein in Arabidopsis (Gross et al. 2006). HR, hairy roots; CSC, cell suspension culture; MenF, menaquinone-specific isochorismate synthase; MenD, 2-succinyl-5-enolpyruvyl-6-hydroxy-3-cyclohexene-1-carboxylate synthase; MenH, 2-succinyl-6-hydroxy-2,4-cyclohexadiene-1-carboxylate synthase; MenC, o-succinylbenzoate synthase; MenE, o-succinylbenzoic acid-CoA ligase; MenB, naphthoate synthase; DXPS, 1-deoxy-d-xylulose-5-phosphate synthase; CLA, cloroplastos alterados; DXR, 1-deoxy-d-xylulose 5-phosphate reductoisomerase; ISPD, 2-C-methyl-d-erythritol 4-phosphate cytidylyltransferase; CDPMEK, 4-diphosphocytidyl-2-C-methyl-d-erythritol kinase; ISPF, 2-C-methyl-d-erythritol 2,4-cyclodiphosphate synthase; HDS, (E)-4-hydroxy-3-methylbut-2-enyl diphosphate synthase; HDR, 4-hydroxy-3-methylbut-2-enyl diphosphate reductase. The expression levels of unigenes are represented with a black–blue color code and FPKM values.