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.

Benzylisoquinoline alkaloid biosynthesis in opium poppy

“…In contrast, overexpression of COR (Larkin et al 2007) and SalAT (Allen et al 2008) specifically resulted in increased morphinan alkaloid accumulation. Surprisingly, the RNAi-mediated silencing of COR (Allen et al 2004) did not result in the accumulation of codeine, but rather caused a substantial accumulation of (S)-reticuline and a concomitant decrease in the levels of morphine pathway intermediates downstream of salutaridine. The biochemical basis for the accumulation of an intermediate several enzymatic steps upstream of the silenced gene is not known, but suggestions include the possible occurrence of (1) feedback regulation and (2) metabolic channels (Allen et al 2004).…”

“…Surprisingly, the RNAi-mediated silencing of COR (Allen et al 2004) did not result in the accumulation of codeine, but rather caused a substantial accumulation of (S)-reticuline and a concomitant decrease in the levels of morphine pathway intermediates downstream of salutaridine. The biochemical basis for the accumulation of an intermediate several enzymatic steps upstream of the silenced gene is not known, but suggestions include the possible occurrence of (1) feedback regulation and (2) metabolic channels (Allen et al 2004). The occurrence of multienzyme complexes in morphine biosynthesis is supported by the suppression of SalAT in using RNAi (Allen et al 2008).…”

“…The RNAi-mediated suppression of COR transcript levels resulted in the accumulation of reticuline and methylated 1-benzylisoquinoline derivatives with a corresponding decrease in morphinan alkaloid content (Allen et al 2004). In support of a role for COR in the modulation of morphine biosynthesis, over-expression of COR resulted in a significant increase in morphinan alkaloid levels (Larkin et al 2007).…”

Removal of Substrate Inhibition and Increase in Maximal Velocity in the Short Chain Dehydrogenase/Reductase Salutaridine Reductase Involved in Morphine Biosynthesis

“…Pentacyclic morphinan alkaloids possess several chiral centers, which preclude chemical synthesis as an option for the efficient production of these widely used pharmaceuticals. Therefore, the worldwide supply of these narcotic compounds is still achieved by their isolation mainly from the opium poppy, Papaver somniferum L. With the availability of an increasing number of isolated genes encoding several pathway enzymes, recent interest has focused on the qualitative and quantitative modulation of the alkaloid profile in transgenic opium poppy plants (2)(3)(4)(5)(6), the production of BIAs in microbes (7,8), and de novo synthesis by a combination of chemical and biochemical conversions. BIA biosynthesis begins with the condensation of the tyrosinederived precursors dopamine and p-hydroxyphenylacetaldehyde to (S)-norcoclaurine (see Fig.…”

An Efficient Fungal RNA-Silencing System Using the DsRed Reporter Gene

“…This is advantageous when the information about genomic sequences is restricted. Beyond this, it is possible to silence different genes or gene families simultaneously (1,9,19).…”