Browsing by Author "Facchini, Peter J."
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Item Open Access Benzylisoquinoline Alkaloid Biosynthesis in Sacred Lotus (Nelumbo nucifera)(2022-08-29) Menendez Perdomo, Ivette M.; Facchini, Peter J.; Ng, Kenneth K.S.; Yeaman, Samuel J.; Back, Thomas G.; Ober, Dietrich, O.Benzylisoquinoline 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.Item Open Access Discovery of Novel Alkaloid Biosynthetic Genes Using Biochemical Genomics(2008-12) Liscombe, David Kenneth; Facchini, Peter J.The benzylisoquinoline alkaloids (BIAs) are a large and diverse group of plant specialized metabolites derived from the amino acid L-tyrosine via a complex array of biosynthetic enzymes. Many BIAs exhibit potent biological activities, such as the analgesic morphine and the antimicrobial sanguinarine, and are important therapeutic agents. However, plants remain the only commercial sources for many pharmaceutical alkaloids. Despite the occurrence of more than 2500 BIAs in plants, their biosynthesis is still poorly understood. This research aimed to isolate novel genes involved in BIA biosynthesis. Norcoclaurine synthase (NCS) catalyzes the first committed step in BIA biosynthesis in plants. NCS cDNAs were isolated from an opium poppy {Papaver somniferum) elicited cell culture expressed sequence tag (EST) collection and were functionally expressed in Escherichia coli. Recombinant forms of NCS homologs from other plants were shown to be devoid of NCS activity, and a virus-induced silencing of ncs expression significantly reduced the accumulation of morphinan alkaloids, suggesting that NCS is a unique enzyme responsible for norcoclaurine biosynthesis in plants. A cDNA encoding tetrahydroprotoberberine TV-methyl transferase (TNMT), an enzyme in sanguinarine biosynthesis, was also isolated from the opium poppy EST collection. Recombinant TNMT was purified, and characterized with respect to substrate specificity and kinetic parameters. TNMT activity and gene expression were detected in all opium poppy organs, which along with its ability to utilize canadine as a substrate, suggests that TNMT might be involved in other branches of BIA biosynthesis. An integrated comparative genomics approach employing transcript and mass spectrometry-based alkaloid profiling of cell cultures of Eschscholzia californica, Thalictrum flavum, and Papaver bracteatum, was used to discover additional novel BIA 7V-methyltransferase (NMT ) genes. Six novel NMT genes were identified and four were functionally characterized, including pavine N-methyltransferase from T. flavum, which represents the first characterized enzyme associated with pavine alkaloid biosynthesis. An integration of alkaloid profiling with the transcriptomic data generally facilitated the bidirectional prediction of both the substrate specificity of individual NMTs and the species-specific alkaloid content of different cellular systems, thereby demonstrating how this platform can be used to dissect the molecular basis of the chemical diversity generated in plant specialized metabolism.Item Open Access Elucidation of the noscapine biosynthetic pathway in opium poppy(2014-09-30) Dang, Thu Thuy T.; Facchini, Peter J.Noscapine is a phthalideisoquinoline alkaloid found in the latex of opium poppy (Papaver somniferum). Noscapine has long been used as a cough suppressant and extensively investigated for its potential anticancer effect. Thanks to its long of history of safe use as an antitussive, rapid absorption after oral administration, and apoptosis-inducing effect on a number of cancer cell lines, noscapine has an advantage over other tubulin-binding anticancer natural products such as the well-established taxanes. Despite its isolation from opium more than two centuries ago, the complete biosynthesis of noscapine has not been elucidated until very recently. My project aims at understanding the biochemical genetics that underlies noscapine biosynthesis in opium poppy by identifying and functional characterizing noscapine biosynthetic candidate genes using comparative transcriptomics and metabolomics. Transcriptomes of eight different opium poppy chemotypes that display different noscapine profiles were used to mine for gene candidates that are potentially involved in noscapine pathway. The search resulted in the discovery of three O-methyltransferases and four cytochrome P450s, all of which were fully characterized biochemically and confirmed to be involved in the noscapine pathway by virus-induced gene silencing approach. It has been shown that the noscapine pathway starts by the 9-O-methylation of scoulerine catalyzed by scoulerine 9-O-methyltransferase to yield tetrahydrocolumbamine, which is then oxidized by canadine synthase to form canadine. N-methylation of canadine catalysed by tetrahydroprotoberberine N-methyltransferase gives rise to N-methylcanadine, which is acted upon by CYP82Y1 to form hydroxy-N-methylcanadine. CYP82X2 further oxidizes hydroxy-N-methylcanadine to (S)-1,13-dihydroxy-N-methylcanadine, which then serves as a substrate for AT1, yielding (S)-1-hydroxy-13-O-acetyl-N-methylcanadine. CYP82X1 then oxidizes (S)-1-hydroxy-13-O-acetyl-N-methylcanadine to form 1,8-dihydroxy-3-O-acetyl-N-methylcanadine, which spontaneously rearranges to yield 4’-desmethyl-3-acetylpapaveroxine. Ester hydrolysis catalysed by CXE1 prompts the formation of a hemiacetal moiety that triggers the conversion to the phthalideisoquinoline scaffold. Noscapine synthase (NOS or SDR1) catalyzes the final step in the formation of noscapine from narcotine hemiacetal.Item Open Access Functional architecture of alkaloid biosynthetic gene promoters from opium poppy(1997) Johnson, Alison G.; Facchini, Peter J.Item Open Access Functional genomics reveals novel o-demethylases involved in the biosynthesis of codeine and morphine in opium poppy(2009) Hagel, Jillian M.; Facchini, Peter J.Opium poppy (Papaver somniferum) produces a diverse array of bioactive benzylisoquinoline alkaloids and has emerged as a versatile model system to study plant alkaloid metabolism. The biosynthesis of morphine and related alkaloids in opium poppy occurs via a complex, multistep pathway beginning with the amino acid tyrosine. Corresponding genes encoding many of the enzymes involved in morphine biosynthesis have been isolated. However, molecular clones are not yet available for some enzymes, and enzyme activity accounting for two key O-demethylation steps leading from thebaine to morphine has yet to be detected. As part of a functional genomics platform aimed at isolating new genes, 1H nuclear magnetic resonance (NMR) metabolite profiling was used to characterize six varieties of opium poppy exhibiting altered alkaloid accumulation profiles. Aqueous and chloroform extracts of six different opium poppy cultivars were subjected to chemometric analysis. Principal component analysis of the 1H NMR spectra for latex extracts clearly distinguished two varieties, including a low-alkaloid variety "P" and a high-thebaine, low-morphine cultivar "T." Loading plots confirmed that morphinan alkaloids contributed predominantly to the variance in latex extracts. Relatively few differences were found in the levels of other metabolites, indicating that the variation was specific for alkaloid metabolism. This finding provided a rational basis for a microarray-based, comparative transcriptomics approach, wherein the transcriptome of T poppy stem was compared with those of high-morphine cultivars. This study led to the isolation of thebaine 6-O-demethylase (T6ODM) and codeine O-demethylase (CODM), which together represented the first identified 0-demethylases in the 2-oxoglutarate/Fe(II)-dependent dioxygenase family. It was shown that gene-specific silencing of T6ODM and CODM dramatically alters morphinan alkaloid profiles of opium poppy.Item Open Access Genetic transformation and metabolic engineering of the papaveraceae(2002) Park, Sang-Un; Facchini, Peter J.Item Open Access Global characterization of the elicitation response in papaver somniferum cell cultures using transcriptomics, proteomics and metabolomics(2008) Zulak, Katherine Grace; Facchini, Peter J.Item Open Access A heat-activated protein expression system for biotechnology(2019-12) Fournier, Nicolas; Harrison, Joe J.; Chua, Gordon; Facchini, Peter J.The thermosensory diguanylate cyclase (TdcA) enzyme found in the Pseudomonas aeruginosa strain CF39S produces cyclic diguanylate (c-di-GMP) in response to increases in temperature. The application of TdcA in a heat-activated protein expression system was explored as an alternative to conventional induction systems. C-di-GMP-dependent gene expression measurements were conducted to investigate gene expression levels in Pseudomonas species in the presence of TdcA. An E. coli K12 mutant library was also used to investigate temperature-dependent gene expression levels for the development of a heat-activated protein expression system. Results demonstrated the thermostatted activity of TdcA in Pseudomonas species, while multiple molecular determinants were found to contribute to temperature-dependent biofilm formation and c-di-GMP-dependent gene expression levels in E. coli K12 species. Of these determinants, the c-di-GMP specific phosphodiesterase PdeO was identified as a significant contributor. Further investigation into the applicability of heat-activated expression systems would be of great benefit to the biotechnology sector.Item Open Access Immunolocalization and lc-ms/ms proteomics reveal in interplay between sieve elements and laticifers in the biosynthesis of morphine in opium poppy(2012) Onoyovwi, Akpevwe; Facchini, Peter J.Opium poppy produces a group of natural products called benzylisoquinoline alkaloids (BIAs) such as morphine and codeine. BIAs have been shown to be synthesized in the phloem specifically in sieve elements, and accumulate in the latex, which is the cytoplasm of specialized secretory cells called laticifers. This project aimed to identify the cell types involved morphine biosynthesis via immunolocalization of six biosynthetic enzymes and LC-MS/MS proteomics analysis on latex and whole stem proteins. This resulted in the localization of codeine 0-demethylase (CODM), the last enzyme involved in morphine biosynthesis in laticifers, different from the other biosynthetic enzymes. This indicates a biosynthetic role for the laticifers. The proteomes showed the presence of morphine biosynthetic enzymes and other BIA biosynthetic enzymes. A new model is proposed where both cell types function in synthesizing morphine, with the penultimate steps occurring predominantly in laticifers. It also suggests the need for intercellular transport of alkaloid.Item Open Access Integration of deep transcriptome and proteome analyses reveals the components of alkaloid metabolism in opium poppy cell cultures(BMC Plant Biology, 2010-11-18) Desgagné-Penix, Isabel; Khan, Morgan F.; Schriemer, David C.; Cram, Dustin; Nowak, Jacek; Facchini, Peter J.Papaver somniferum (opium poppy) is the source for several pharmaceutical benzylisoquinoline alkaloids including morphine, the codeine and sanguinarine. In response to treatment with a fungal elicitor, the biosynthesis and accumulation of sanguinarine is induced along with other plant defense responses in opium poppy cell cultures. The transcriptional induction of alkaloid metabolism in cultured cells provides an opportunity to identify components of this process via the integration of deep transcriptome and proteome databases generated using next-generation technologies.Item Open Access Localization of benzylisoquinoline alkaloid biosynthesis in papaver somniferum(2003) Bird, David Andrew; Facchini, Peter J.Item Open Access Metabolic engineering of hydroxycinnamic acid amide in nictoiana tabacum(2004) Hagel, Jillian M.; Facchini, Peter J.Feruloyltyramine and 4-coumaroyltyramine participate in the defense of plants against pathogens through their extracellular peroxidative polymerization, which is thought to reduce cell wall digestibility or otherwise inhibit fungal growth. HydroxycinnamoylCoA:tyramine N-(hydroxycinnamoyl)transferase (THT) and tyrosine decarboxylase (TYDC) are purported to play key roles in the stress-induced regulation of tyraminederived hydroxycinnamic acid amide (HCAAT) biosynthesis. Transgenic tobacco (Nicotiana tabacum cv Xanthi) was engineered to constitutively express tobacco THT. A T₁ plant over-expressing THT was crossbred with T₁ tobacco expressing opium poppy TYDC2 to produce a T₂ line with elevated THT and TYDC activities compared with wild type plants. The effects of an independent increase in TYDC or THT activity, or a dual increase in both TYDC and THT on the cellular pools of HCAAT pathway intermediates and the accumulation of soluble and cell wall-bound feruloyltyrarnine and 4-coumaroyltyramine were examined. In roots, THT and TYDC activities were not substantially altered in transgenic lines beyond their relatively high wild type levels; thus, no changes in HCAAT precursor or product levels were detected. In leaves, increased TYDC activity resulted in a larger cellular pool of tyramine, and lower levels of phenylalanine. In contrast, elevated THT activity reduced tyramine levels. HCAAT levels were low in healthy leaves, but were induced in response to wounding and accumulated around wound sites. Similarly, endogenous THT and TYDC activities were wound-induced. The initial rate of wound-induced HCAAT accumulation was highest in transgenic plants with elevated THT and TYDC activities. These results show that both THT and TYDC exert partial and synergistic control over the flux of intermediates involved in HCAAT biosynthesis under some conditions.Item Open Access Plant Defense Responses in Opium Poppy Cell Cultures Revealed by Liquid Chromatography-Tandem Mass Spectrometry Proteomics(Molecular & Cellular Proteomics, 2008-08-05) Zulak, Katherine G.; Khan, Morgan F.; Alcantara, Joenel; Schriemer, David; Facchini, Peter J.Opium poppy (Papaver somniferum) produces a diverse array of bioactive benzylisoquinoline alkaloids, including the narcotic analgesic morphine and the antimicrobial agent sanguinarine. In contrast to the plant, cell cultures of opium poppy do not accumulate alkaloids constitutively but produce sanguinarine in response to treatment with certain fungal-derived elicitors. The induction of sanguinarine biosynthesis provides a model platform to characterize the regulation of benzylisoquinoline alkaloid pathways and other defense responses. Proteome analysis of elicitor-treated opium poppy cell cultures by two-dimensional denaturing-polyacrylamide gel electrophoresis coupled with liquid chromatography-tandem mass spectrometry facilitated the identification of 219 of 340 protein spots based on peptide fragment fingerprint searches of a combination of databases. Of the 219 hits, 129 were identified through pre-existing plant proteome databases, 63 were identified by matching predicted translation products in opium poppy-expressed sequence tag databases, and the remainder shared evidence from both databases. Metabolic enzymes represented the largest category of proteins and included S-adenosylmethionine synthetase, several glycolytic, and a nearly complete set of tricarboxylic acid cycle enzymes, one alkaloid, and several other secondary metabolic enzymes. The abundance of chaperones, heat shock proteins, protein degradation factors, and pathogenesis-related proteins provided a comprehensive proteomics view on the coordination of plant defense responses. Qualitative comparison of protein abundance in control and elicitor-treated cell cultures allowed the separation of induced and constitutive or suppressed proteins. DNA microarrays were used to corroborate increases in protein abundance with a corresponding induction in cognate transcript levels.Item Open Access Purification and molecular cloning of norcoclaurine synthase and cellular localization of benzylisoquinoline alkaloid pathways in thalictrum flavum(2005) Samanani, Nailish; Facchini, Peter J.Item Open Access Regulation of benzylisoquinoline alkaloid biosynthesis in opium poppy(2006) MacLeod, Benjamin P.; Facchini, Peter J.Item Open Access Structural and Functional Diversity Among Alkaloid N-Methyltransferases(2019-07-02) Morris, Jeremy Stephane; Facchini, Peter J.; Harrison, Joe; Jez, Joseph; Ling, Chang-Chun; Ng, Kenneth K.-S.Alkaloids are a group of nitrogenous specialized metabolites occurring in approximately one fifth of all plant species and which includes over 20 000 individual molecules with diverse structures and potent biological activities. In particular, plants containing benzylisoquinoline alkaloids (BIAs; eg. morphine, noscapine, sanguinarine) and phenylalkylamine protoalkaloids (PAAs; eg. pseudoephedrine) have been integral elements of human medicinal practice for thousands of years. To produce these molecules, plants employ a number of enzyme-catalyzed reactions including methylation of the nitrogen atom which is, by definition, present in all alkaloids. With respect to BIA biosynthesis in opium poppy (Papaver somniferum) and related species, three homologous yet functionally distinct subtypes of N-methyltransferase (NMT) enzymes are implicated (Coclaurine, Tetrahydroprotoberberine, and Pavine NMTs). This thesis describes the isolation and functional characterization of Reticuline NMT (RNMT), which defines a BIA NMT subtype preferentially accepting tertiary 1-BIA and aporphine substrates. Gene silencing reveals that RNMT catalyzes the ultimate or penultimate step in the biosynthesis of taxonomically widespread alkaloid (S)-magnoflorine. Despite the classification of BIA NMTs into the aforementioned subtypes, homologs show substantial functional variation with important physiological and biotechnological consequences. This thesis describes seven additional NMTs which contribute to the unique BIA content reported in meadow rue (Thalictrum flavum) and yellow horned poppy (Glaucium flavum). In contrast to work on BIAs, PAA biosynthesis has received much less attention and no dedicated enzymes have been reported. This thesis describes the identification and functional characterization of a novel member of the BIA NMT-like enzyme family (Phenylkalkylamine NMT; PaNMT) which is implicated in (pseudo)ephedrine biosynthesis in Ephedra sinica. To highlight the biotechnological potential of PaNMT, a heterologous pathway yielding (pseudo)ephedrine was reconstituted in E. coli. To better understand the structural features underlying functional diversity in BIA NMTs, structure-guided mutagenesis of three representatives was carried out and the resulting insights into catalysis and substrate recognition are reported herein. Notably, structural variants tightly correlated with either Coclaurine or Reticuline NMT activities were identified, and reciprocal mutagenesis experiments showed that a single residue is responsible for the functional dichotomy (preference for secondary or tertiary amine substrates).Item Open Access Structure-Function Studies of the Methyltransferase Enzymes involved in Benzylisoquinoline Alkaloid Metabolism(2022-01-09) Lang, Dean; Facchini, Peter J.; Ng, Kenneth Kai Sing; Savchenko, Alexei; Palmer, David; Ro, Dae Kyun; Ling, ChangchunPlants represent a vast natural repository of unique and often potent compounds of pharmaceutical and industrial importance. Opium poppy (Papaver somniferum) is one of the first cultivated and most well studied species, particularly for its ability to produce an assortment of benzylisoquinoline alkaloids (BIAs) including the analgesic morphine and antitussive noscapine. A substantial body of functional and genomic studies have elucidated the dominant pathway and associated subset of enzymes responsible for BIA biosynthesis within opium poppy. The ability of many BIA enzymes to accept multiple substrates remains of particular interest, primarily for their potential to produce novel or rare BIAs as scaffolds for drug design. However, broad substrate promiscuity and lack of concerted mechanism of catalysis for homologous BIA enzymes prevents structure-function predictions by primary sequence alone. Tertiary structure determination of the BIA enzymes will help identify common motifs of substrate recognition and functionally important structure-activity relationships. Enzymatic methylation is one of the most frequent steps to the derivatization of BIAs in planta. Several methyltransferases have been identified which target the central nitrogen (NMTs) and peripheral oxygen atoms (OMTs) common amongst all BIAs. This thesis presents the crystal structure (at a dmin ranging from 1.6-2.5 Å) of Glaucium flavum tetrahydroprotoberberine N-methyltransferase (TNMT) in complex with a variety of preferred and alternative substrates. Structural determinants of substrate binding and catalysis are compared to the previously published structures of coclaurine and pavine NMT. The ability of the TNMT to only bind (S)-cis tetrahydroprotoberberine substrates is linked to the stereospecificity of the enzyme. Additional enzyme-substrate complexes highlight the role of specific residues in catalytic mechanism utilized by the BIA NMTs. Unlike the NMTs, the BIA OMTs possess broad evolutionary origins and distinct substrate binding poses. Homodimeric OMT structures highlight common motifs of BIA O-methylation, including a central dimerization domain which partially defines the substrate pocket. This thesis presents the first characterized heterodimeric BIA O-methyltransferase and resultant novel enzyme activity. Further characterization of the structural determinants required for heterodimerization and the effect of heterodimerization on differential substrate binding are presented. Homology modelling, activity assays, differential scanning fluorimetry, and crystallization trials provide a pathway for future structural studies by X-ray diffraction.Item Open Access Virus-induced gene silencing to investigate the regulation of benzylisoquinoline alkaloid metabolism in opium poppy(2012) Wijekoon, Champa P.; Facchini, Peter J.