Metabolic engineering of morphinan alkaloids by over-expression and RNAi suppression of salutaridinol 7-O-acetyltransferase in opium poppy.

We demonstrate that both over-expression and suppression of the gene encoding the morphinan pathway enzyme salutaridinol 7-O-acetyltransferase (SalAT) in opium poppy affects the alkaloid products that accumulate. Over-expression of the gene in most of the transgenic events resulted in an increase in capsule morphine, codeine and thebaine on a dry-weight basis. The transgenic line with the highest alkaloid content had 41%, 37% and 42% greater total alkaloids than the control in three independent trials over 3 years. DNA-encoded hairpin RNA-mediated suppression of SalAT resulted in the novel accumulation of the alkaloid salutaridine at up to 23% of total alkaloid; this alkaloid is not detectable in the parental genotype. Salutaridine is not the substrate of SalAT but the substrate of the previous enzyme in the pathway, salutaridine reductase. RNA transcript analysis of 16 primary T0 transformants and their segregating T1 progeny revealed an average reduction in SalAT transcript to about 12% of the control. Reduction in SalAT transcript was evident in both leaves and latex. Reverse transcriptase PCR and high-performance liquid chromatography analyses confirmed cosegregation of the expressed transgene with the salutaridine accumulating phenotype.

Increasing morphinan alkaloid production by over-expressing codeinone reductase in transgenic Papaver somniferum.

Only plants of the Papaver genus (poppies) are able to synthesize morphinan alkaloids, and cultivation of P. somniferum, opium poppy, remains critical for the production and supply of morphine, codeine and various semi-synthetic analgesics. Opium poppy was transformed with constitutively expressed cDNA of codeinone reductase (PsCor1.1), the penultimate step in morphine synthesis. Most transgenic lines showed significant increases in capsule alkaloid content in replicated glasshouse and field trials over 4 years. The morphinan alkaloid contents on a dry weight basis were between 15% and 30% greater than those in control high-yielding genotypes and control non-transgenic segregants. Transgenic leaves had approximately 10-fold greater levels of Cor transcript compared with non-transgenic controls. Two cycles of crossing of the best transgenic line into an elite high-morphine genotype resulted in significant increases in morphine and total alkaloids relative to the elite recurrent parent. No significant changes in alkaloid profiles or quantities were observed in leaf, roots, pollen and seed.

RNAi-mediated replacement of morphine with the nonnarcotic alkaloid reticuline in opium poppy.

We report on the silencing of codeinone reductase (COR) in the opium poppy, Papaver somniferum, using a chimeric hairpin RNA construct designed to silence all members of the multigene COR family through RNA interference (RNAi). After gene silencing, the precursor alkaloid (S)-reticuline-seven enzymatic steps upstream of codeinone-accumulated in transgenic plants at the expense of morphine, codeine, oripavine and thebaine. Methylated derivatives of reticuline also accumulated. Analysis verified loss of Cor gene transcript, appearance of 22-mer degradation products and reduction of enzyme activity. The surprising accumulation of (S)-reticuline suggests a feedback mechanism preventing intermediates from general benzylisoquinoline synthesis entering the morphine-specific branch. However transcript levels for seven other enzymes in the pathway, both before and after (S)-reticuline, were unaffected. This is the first report of gene silencing in transgenic opium poppy and of metabolic engineering to cause the high-yield accumulation of the nonnarcotic alkaloid reticuline.

Analgesia: morphine-pathway block in top1 poppies.

The opium poppy is a source of the pharmaceuticals codeine, morphine and their derived analgesics. Here we describe the initial characterization of the poppy mutant known as top1 (for 'thebaine oripavine poppy 1'), which accumulates the morphine and codeine precursors thebaine and oripavine and does not complete their biosynthesis into morphine and codeine. The original discovery of top1 stimulated a re-engineering of the opioid industry in the island state of Tasmania, which grows over 40% of the world's licit opiates, in order to produce thebaine and oripavine efficiently from morphine-free poppy crops to provide precursors for highly effective analgesics and for treatment of opioid addiction.