Systematic knockdown of morphine pathway enzymes in opium poppy using virus‐induced gene silencing

Abstract

Summary Opium poppy (Papaver somniferum) remains the sole commercial source for several pharmaceutical alkaloids including the narcotic analgesics codeine and morphine, and the semi‐synthetic drugs oxycodone, buprenorphine and naltrexone. Although most of the biosynthetic genes have been identified, the post‐transcriptional regulation of the morphinan alkaloid pathway has not been determined. We have used virus‐induced gene silencing (VIGS) as a functional genomics tool to investigate the regulation of morphine biosynthesis via a systematic reduction in enzyme levels responsible for the final six steps in the pathway. Specific gene silencing was confirmed at the transcript level by real‐time quantitative PCR (polymerase chain reaction), and at the protein level by immunoblot analysis using antibodies raised against salutaridine synthase (SalSyn), salutaridine reductase (SalR), salutaridine 7‐O‐acetyltransferase (SalAT), thebaine 6‐O‐demethylase (T6ODM), codeinone reductase (COR), and codeine O‐demethylase (CODM). In some cases, silencing a specific biosynthetic gene resulted in a predictable accumulation of the substrate for the corresponding enzyme. Reduced SalSyn, SalR, T6ODM and CODM protein levels correlated with lower morphine levels and a substantial increase in the accumulation of reticuline, salutaridine, thebaine and codeine, respectively. In contrast, the silencing of genes encoding SalAT and COR resulted in the accumulation of salutaridine and reticuline, respectively, which are not the corresponding enzymatic substrates. The silencing of alkaloid biosynthetic genes using VIGS confirms the physiological function of enzymes previously characterized in vitro, provides insight into the biochemical regulation of morphine biosynthesis, and demonstrates the immense potential for metabolic engineering in opium poppy.

Dioxygenases Catalyze O-Demethylation and O,O-Demethylenation with Widespread Roles in Benzylisoquinoline Alkaloid Metabolism in Opium Poppy

“…The discovery of novel catalytic functions for recombinant T6ODM and CODM prompted an expanded investigation of their physiological roles using VIGS, which has proven effective for the characterization of numerous BIA biosynthetic genes in opium poppy (3,11,22,30). PODA was not targeted for VIGS analysis because of the lack of corresponding transcripts in the Bea's Choice cultivar.…”

“…Real time quantitative PCR was performed as described previously (22) using SYBR Green detection, 300 nm each of each genespecific forward and reverse primers (T6ODM, 5Ј-TTGAGG-CACAAATGAGAAAATTGA-3Ј and 5Ј-CACAACGCACTT-TCGAGAAATTAC-3Ј; CODM, 5Ј-TTGTGCTTAAATTTC-GTGGATGAC-3Ј and 5Ј-TGATTACATCACTTGACCCAA-ACAG-3Ј), and 1 l of cDNA. Relative transcript levels were determined using the 2…”

Characterization of a Flavoprotein Oxidase from Opium Poppy Catalyzing the Final Steps in Sanguinarine and Papaverine Biosynthesis

“…The use of VIGS as a functional genomics tool to investigate the physiological role of BIA biosynthetic genes is well established in opium poppy (3,16,48,49). The restriction of DBOX transcripts to roots ( Fig.…”

Metabolic Engineering of Microorganisms for the Production of Natural Compounds

“…[272] They noted that COR knockdown resulted in S-reticuline accumulation in plant studies. [278] Thus, COR-like enzymes were searched through various plant transcriptome databases and were put to test. In the E. coli system, in contrast with the previous reports, Nakagawa et al were able to produce both S and R forms of reticuline by the incorporation of only two methyltransferases-CNMT and 4′OMT-without expressing 6OMT.…”