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dc.contributor.advisorFacchini, Peter James
dc.contributor.authorBeaudoin, Guillaume Arthur Welch
dc.date.accessioned2015-03-16T18:35:23Z
dc.date.issued2015-03-16
dc.date.submitted2015en
dc.identifier.citationBeaudoin, G. A. (2015). Characterization of Oxidative Enzymes Involved in the Biosynthesis of Benzylisoquinoline Alkaloids in Opium Poppy (Papaver somniferum) (Unpublished doctoral thesis). University of Calgary, Calgary, AB. doi:10.11575/PRISM/25284en_US
dc.identifier.urihttp://hdl.handle.net/11023/2115
dc.description.abstractBenzylisoquinoline alkaloids (BIAs) are a large group of nitrogen-containing specialized metabolites. Opium poppy (Papaver somniferum) is an important pharmaceutical plant and has been cultivated for thousands of years for its analgesic constituents: the morphinan BIAs codeine and morphine. In addition, opium poppy produces other BIAs with biological activities, such as the vasodilator papaverine, the potential anti-cancer drug noscapine and the antimicrobial agent sanguinarine. The objective of this work was to identify and characterize oxidative enzymes involved in BIA biosynthesis, with a specific focus on cytochromes P450 (P450s). The biosynthesis of sanguinarine from (S)-reticuline requires four P450s. Our first objective was to identify these unidentified P450s in opium poppy. Our approach was based on the coordinated induction of all sanguinarine biosynthetic enzyme transcripts and proteins in elicited opium poppy cell suspension cultures. Using data from a previous microarray study, we identified 12 uncharacterized inducible P450s. Recombinant expression in Saccharomyces cerevisiae revealed that 4 of these enzymes (CYP719A20, CYP719A25, CYP82N3 and CYP82N4) were involved in sanguinarine biosynthesis. Unexpectedly, in planta silencing using virus-induced gene silencing showed that plants suppressed in these P450s transcripts have significantly increased levels of sanguinarine and dihydrosanguinarine. Many changes in the accumulation of sanguinarine biosynthetic intermediates were also detectable. In CYP82N3- and CYP82N4-silenced plants, we detected a significant accumulation of some alkaloids in roots and a significant reduction of some of these in latex. Previous silencing of the penultimate step in morphine biosynthesis, codeinone reductase (COR), showed a dramatic decrease in morphinan alkaloids, as well as a significant increase in reticuline, the central BIA branch point intermediate. A search for similar enzymes that may be co-silenced revealed a P450 reductase fusion. Further in vitro and in planta characterization showed that this enzyme is responsible for the final unidentified and first committed steps in morphine biosynthesis, the epimerization of (S)-reticuline. Papaver rhoeas, which does not accumulate morphinan alkaloids, possesses this enzyme as two polypeptides. The significance of this difference remains to be studied. We were also able to identify a paralog of COR which can catalyze the isomerization of many morphinans, potentially opening new routes to manufacture novel opiates.en_US
dc.language.isoeng
dc.rightsUniversity of Calgary graduate students retain copyright ownership and moral rights for their thesis. You may use this material in any way that is permitted by the Copyright Act or through licensing that has been assigned to the document. For uses that are not allowable under copyright legislation or licensing, you are required to seek permission.
dc.subjectBiology--Molecular
dc.subjectPlant Physiology
dc.subjectBiochemistry
dc.subject.classificationAlkaloiden_US
dc.subject.classificationSpecialized metabolismen_US
dc.subject.classificationOpium poppyen_US
dc.subject.classificationCytochrome P450en_US
dc.subject.classificationAldo-keto reductaseen_US
dc.subject.classificationFusion proteinen_US
dc.subject.classificationVirus-induced gene silencingen_US
dc.titleCharacterization of Oxidative Enzymes Involved in the Biosynthesis of Benzylisoquinoline Alkaloids in Opium Poppy (Papaver somniferum)
dc.typedoctoral thesis
dc.publisher.facultyGraduate Studies
dc.description.embargoterms2 yearsen_US
dc.date.embargolift2017-03-15T18:35:23Z
dc.publisher.institutionUniversity of Calgaryen
dc.identifier.doihttp://dx.doi.org/10.11575/PRISM/25284
thesis.degree.nameDoctor of Philosophy
thesis.degree.namePhD
thesis.degree.disciplineBiological Sciences
thesis.degree.grantorUniversity of Calgary
atmire.migration.oldid3016
dc.publisher.placeCalgaryen
ucalgary.item.requestcopytrue


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University of Calgary graduate students retain copyright ownership and moral rights for their thesis. You may use this material in any way that is permitted by the Copyright Act or through licensing that has been assigned to the document. For uses that are not allowable under copyright legislation or licensing, you are required to seek permission.