Transcriptome assembly reveals putative genes involved in monoterpenoid indole alkaloid biosynthesis in Ochrosia elliptica.

Abstract

Monoterpenoid indole alkaloids (MIAs) are a class of plant-derived secondary metabolites which display valuable pharmacological properties, including the anticancer drug vinblastine from Catharanthus roseus and the antihypertensive reserpine from Rauvolfia serpentina. Decades of research employing methods such as radiolabelled feeding studies, protein purification, transcriptome sequencing, gene silencing, mutant screening, and cell culture have facilitated the characterization of dozens of MIA pathway genes in these two species. This knowledge is now facilitating the identification and characterization of biosynthetic pathways of other MIA-producing species through the ability to generate high-throughput sequencing databases and search for homologues of existing MIA biosynthetic genes. Ochrosia elliptica accumulates biologically active MIAs, including the antitumour ellipticine and the analgesics apparicine and conolidine. Little is known about the biosynthesis of these MIAs, however. In this study, the O. elliptica leaf transcriptome was generated using Illumina sequencing and assembled de novo into ~25 000 representative peptide sequences with the Trinity pipeline. The transcriptome was used to identify OeOMT, which showed activity towards 16-hydroxytabersonine and is expected to accept other endogenous and unidentified MIA substrates. Eleven CYP candidates were also identified from the transcriptome and cloned, but no activity was observed towards 11 MIA substrates tested here. Another CYP candidate, Oe10HL1, showed activity towards tabersonine, although its endogenous substrate remains unknown. Recombinant Oe10HL1 and OeOMT were biochemically characterized using their non-natural substrates tabersonine and 16-hydroxytabersonine, respectively, towards which they exhibited high KM values. The transcriptome will provide a valuable resouce for future ongoing research into MIA biosynthetic pathways.