Functional genomics reveals novel o-demethylases involved in the biosynthesis of codeine and morphine in opium poppy

Abstract

Opium poppy (Papaver somniferum) produces a diverse array of bioactive benzylisoquinoline alkaloids and has emerged as a versatile model system to study plant alkaloid metabolism. The biosynthesis of morphine and related alkaloids in opium poppy occurs via a complex, multistep pathway beginning with the amino acid tyrosine. Corresponding genes encoding many of the enzymes involved in morphine biosynthesis have been isolated. However, molecular clones are not yet available for some enzymes, and enzyme activity accounting for two key O-demethylation steps leading from thebaine to morphine has yet to be detected. As part of a functional genomics platform aimed at isolating new genes, 1H nuclear magnetic resonance (NMR) metabolite profiling was used to characterize six varieties of opium poppy exhibiting altered alkaloid accumulation profiles. Aqueous and chloroform extracts of six different opium poppy cultivars were subjected to chemometric analysis. Principal component analysis of the 1H NMR spectra for latex extracts clearly distinguished two varieties, including a low-alkaloid variety "P" and a high-thebaine, low-morphine cultivar "T." Loading plots confirmed that morphinan alkaloids contributed predominantly to the variance in latex extracts. Relatively few differences were found in the levels of other metabolites, indicating that the variation was specific for alkaloid metabolism. This finding provided a rational basis for a microarray-based, comparative transcriptomics approach, wherein the transcriptome of T poppy stem was compared with those of high-morphine cultivars. This study led to the isolation of thebaine 6-O-demethylase (T6ODM) and codeine O-demethylase (CODM), which together represented the first identified 0-demethylases in the 2-oxoglutarate/Fe(II)-dependent dioxygenase family. It was shown that gene-specific silencing of T6ODM and CODM dramatically alters morphinan alkaloid profiles of opium poppy.