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

Abstract

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.

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Figure 1: A summary of the biosynthetic pathway leading to morphine, showing also the methylated derivatives of reticuline; enzymes indicated are those for which cDNAs have been previously cloned and that are investigated in this study.
Figure 2: The T-DNA from the transformation vector COR 1.1/2 hpRNA designed to produce hpRNA and initiate silencing of all members of the Cor family in P. somniferum.
Figure 3: Thin layer chromatography of poppy latex alkaloids.
Figure 4: HPLC chromatograms.
Figure 5: HPLC quantification of the alkaloid contents of 18 T0 hpRNA transgenics and a set of nine independent, nontransgenic control CO58-34 plants (cntls 1–9).
Figure 6: Analysis of T1 family segregants derived from hpCOR parent 220-2-2.

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Acknowledgements

We would like to thank Neil Smith and Peter Waterhouse for helpful discussions and 22-mer detection; Pat Hallam, Peta Dolan and Noel Davies for HPLC and LC/MS work; Stephen Pyne and Alison Ung for chiral determinations; Toni Kutchan, Meinhart Zenk and Susanne Frick for helpful discussions.

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Correspondence to Philip J Larkin.

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Supplementary Fig. 1

RT-PCR analysis of eight T1 family segregants from hpCOR parent 220-2-2; these are the same individuals as shown in Figure 6. (PDF 341 kb)

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Allen, R., Millgate, A., Chitty, J. et al. RNAi-mediated replacement of morphine with the nonnarcotic alkaloid reticuline in opium poppy. Nat Biotechnol 22, 1559–1566 (2004). https://doi.org/10.1038/nbt1033

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