Facchini, Peter J.Menendez Perdomo, Ivette M.2023-04-042022-08-29Menendez Perdomo, I. M. (2022). Benzylisoquinoline Alkaloid Biosynthesis in Sacred Lotus (Nelumbo nucifera) (Doctoral thesis, University of Calgary, Calgary, Canada). Retrieved from https://prism.ucalgary.ca.https://prism.ucalgary.ca/handle/1880/116030https://dx.doi.org/10.11575/PRISM/dspace/40876Benzylisoquinoline alkaloids (BIA) constitute a family of plant specialized metabolites comprising numerous bioactive compounds. BIA metabolism has been extensively studied in the opium poppy and related species in the Ranunculales order, but limited research has been conducted in non-model plants. Sacred lotus (Nelumbo nucifera), in the order Proteales, is an ancient aquatic plant rich in proaporphine, aporphine, and bisbenzylisoquinoline alkaloids, with a prevalence of unusual R-enantiomers. However, BIA biosynthetic genes and cognate enzymes in this plant remain unknown.Considering the proposed monophyletic origin of BIA biosynthesis in Angiosperms and sacred lotus’ reported alkaloid profile, opium poppy’s well-established pathways could be envisaged as a blueprint to predict the corresponding biosynthetic genes in N. nucifera. This thesis describes the isolation and characterization of several genes involved in Nelumbo’s BIA metabolism, including those purportedly encoding for norcoclaurine synthase, O- and N-methyltransferases, proaporphine synthase, bisbenzylisoquinoline synthase, and aporphine methylenedioxy bridge synthase. Through a combination of in vitro assays with recombinant enzymes and plant total protein extracts and organ-specific correlational analysis between transcript levels and alkaloid content, we demonstrate that NnOMT1 acts as the main 6-O-methyltransferase (favoring the conversion of (R)-norcoclaurine), whereas NnOMT5 and NnOMT7 are non-stereospecific 7-O-methyltransferases acting on several 1-benzylisoquinolines. Additionally, in vivo assays performed in engineered yeast strains allowed the preliminary characterization of three novel P450 catalysts, NnCYP80Q1 acting as a proaporphine synthase (R-stereospecific intramolecular C-C phenol coupling), NnCYP80Q2 as a bisbenzylisoquinoline synthase (R-stereospecific intermolecular C-O phenol coupling), and NnCYP719A22, the aporphine methylenedioxy bridge synthase. Furthermore, we describe how the absence of 3'-hydroxylase activity radically changes the alkaloid profile in Proteales versus Ranunculales.In addition, via in vivo deuterium labeling experiments, this work provides the first empirical evidence to support that L-tyrosine is the primary precursor for lotus BIA. Based on a consistent absence of norcoclaurine synthase activity for the recombinant enzyme candidates and plant total protein extracts, we suggest that a non-enzymatic spontaneous Pictet-Spengler condensation of dopamine and 4-hydroxyphenylacetaldehyde produces racemic norcoclaurine in lotus, in opposition to the enzyme-catalyzed (S)-norcoclaurine formation in the Ranunculales. These results support a possible convergent evolution of BIA biosynthesis in sacred lotus.EnglishNelumbo nuciferabenzylisoquinoline alkaloidsnorcoclaurine synthaseproaporphine synthasebisbenzylisoquinoline synthasemethylenedioxybridge synthasemethyltransferaseBiology--Moleculardoctoral thesis