Functional genomics of monoterpenoid indole alkaloid biosynthesis in Rauvolfia serpentina

Abstract

Monoterpenoid indole alkaloids (MIAs) are a large and heterogeneous group of nitrogen-containing specialized metabolites produced by plants belonging to the Apocynaceae, Loganiaceae and Rubiaceae families. Many of these MIAs exhibit interesting biological activities and are currently used as pharmaceutical drugs to treat several medical conditions. Thus, the biosynthetic pathways responsible for their production have been extensively investigated. Recent advancements in large-scale DNA-sequencing technologies have provided access to a vast collection of genes. Here we have used bioinformatics guided screen to identify candidate genes involved in MIA biosynthesis in Rauvolfia serpentina. We utilized our large annotated transcriptome databases (www.phytometasyn.ca) as a source to mine genes. The identification of a Catharanthus roseus enzyme responsible for indole nitrogen methylation of an MIA intermediate of the vindoline pathway provided us with a query sequence to mine candidate genes responsible for N-methylation of other MIAs in R. serpentina. This led to the identification, molecular cloning and biochemical characterization of four enzymes catalyzing N-methylation. Two separate genes cloned from R. serpentina and V. minor, both encoded enzymes displaying high affinity and specificity for picrinine converting it to N-methyl-picrinine (ervincine) in the presence of S-adenosyl-L-methionine. The other two genes, cloned from R. serpentina, encoded enzymes involved in final steps of ajmaline biosynthesis. Norajmaline N-methyltransferase catalyzed the indoline N-methylation of norajmaline to generate ajmaline, while ajmaline Nβ-methyltransferase catalyzed the side chain N-methylation of ajmaline to generate Nβ-methylajmaline, an unusual positively charge MIA molecule found in Rauvolfia.