Benzophenanthridine Alkaloid Biosynthesis Research Articles

Engineering strategies for the fermentative production of plant alkaloids in yeast

Microbial hosts engineered for the biosynthesis of plant natural products offer enormous potential as powerful discovery and production platforms. However, the reconstruction of these complex biosynthetic schemes faces numerous challenges due to the number of enzymatic steps and challenging enzyme classes associated with these pathways, which can lead to issues in metabolic load, pathway specificity, and maintaining flux to desired products. Cytochrome P450 enzymes are prevalent in plant specialized metabolism and are particularly difficult to express heterologously. Here, we describe the reconstruction of the sanguinarine branch of the benzylisoquinoline alkaloid pathway in Saccharomyces cerevisiae, resulting in microbial biosynthesis of protoberberine, protopine, and benzophenanthridine alkaloids through to the end-product sanguinarine, which we demonstrate can be efficiently produced in yeast in the absence of the associated biosynthetic enzyme. We achieved titers of 676μg/L stylopine, 548μg/L cis-N-methylstylopine, 252μg/L protopine, and 80μg/L sanguinarine from the engineered yeast strains. Through our optimization efforts, we describe genetic and culture strategies supporting the functional expression of multiple plant cytochrome P450 enzymes in the context of a large multi-step pathway. Our results also provided insight into relationships between cytochrome P450 activity and yeast ER physiology. We were able to improve the production of critical intermediates by 32-fold through genetic techniques and an additional 45-fold through culture optimization.

Metabolic Diversification of Benzylisoquinoline Alkaloid Biosynthesis Through the Introduction of a Branch Pathway in Eschscholzia californica

Higher plants produce a diverse array of secondary metabolites. These chemicals are synthesized from simple precursors through multistep reactions. To understand how plant cells developed such a complicated metabolism, we examined the plasticity of benzyl isoquinoline alkaloid biosynthesis in transgenic Eschscholzia californica cells with the ectopic expression of Coptis japonica scoulerine-9-O-methyltransferase (CjSMT). CjSMT catalyzes the O-methylation of scoulerine to produce tetrahydrocolumbamine (THC) in berberine biosynthesis and is not involved in benzophenanthridine alkaloid biosynthesis in E. californica. While a preliminary characterization confirmed that columbamine (oxidized product of THC) was produced in transgenic E. californica cells, many newly found peaks were not identified. Here, we report the identification of novel products, including allocryptopine and 10-hydroxychelerythrine. This result indicates that CjSMT reaction products were further converted by endogenous enzymes to produce double O-methylated compounds instead of a methylenedioxy ring at the 7,8-position of the original benzophenanthridine alkaloids. Further metabolite profiling revealed the enhanced diversification of the alkaloid profile in transgenic cells. Metabolic plasticity and the enzymes involved in metabolic diversity are discussed.

Molecular Cloning and Characterization of Tetrahydroprotoberberine cis-N-Methyltransferase, an Enzyme Involved in Alkaloid Biosynthesis in Opium Poppy

S-Adenosyl-l-methionine:tetrahydroprotoberberine cis-N-methyltransferase (EC 2.1.1.122) catalyzes the conversion of (S)-stylopine to the quaternary ammonium alkaloid, (S)-cis-N-methylstylopine, as a key step in the biosynthesis of protopine and benzophenanthridine alkaloids in plants. A full-length cDNA encoding a protein exhibiting 45 and 48% amino acid identity with coclaurine N-methyltransferase from Papaver somniferum (opium poppy) and Coptis japonica, respectively, was identified in an elicitor-treated opium poppy cell culture expressed sequence tag data base. Phylogenetic analysis showed that the protein belongs to a unique clade of enzymes that includes coclaurine N-methyltransferase, the predicated translation products of the Arabidopsis thaliana genes, At4g33110 and At4g33120, and bacterial S-adenosyl-L-methionine-dependent cyclopropane fatty acid synthases. Expression of the cDNA in Escherichia coli produced a recombinant enzyme able to convert the protoberberine alkaloids stylopine, canadine, and tetrahydropalmatine to their corresponding N-methylated derivatives. However, the protoberberine alkaloids tetrahydroxyberbine and scoulerine, and simple isoquinoline, benzylisoquinoline, and pavine alkaloids were not accepted as substrates, demonstrating the strict specificity of the enzyme. The apparent K(m) values for (R,S)-stylopine and S-adenosyl-L-methionine were 0.6 and 11.5 microm, respectively. TNMT gene transcripts and enzyme activity were detected in opium poppy seedlings and all mature plant organs and were induced in cultured opium poppy cells after treatment with a fungal elicitor. The enzyme was detected in cell cultures of other members of the Papaveraceae but not in species of related plant families that do not accumulate protopine and benzophenanthridine alkaloids.

Molecular cloning and characterization of methylenedioxy bridge‐forming enzymes involved in stylopine biosynthesis in Eschscholzia californica

(S)-stylopine is an important intermediate in the biosynthesis of benzophenanthridine alkaloids, such as sanguinarine. Stylopine biosynthesis involves the sequential formation of two methylenedioxy bridges. Although the methylenedioxy bridge-forming P450 (CYP719) involved in berberine biosynthesis has been cloned from Coptis japonica[Ikezawa N, Tanaka M, Nagayoshi M, Shinkyo R, Sakaki T, Inouye K & Sato F (2003) J Biol Chem278, 38557-38565], no information is available regarding the genes for methylenedioxy bridge-forming enzymes in stylopine biosynthesis. Two cytochrome P450 cDNAs involved in stylopine biosynthesis were isolated using degenerate primers designed for C. japonica CYP719 from cultured Eschscholzia californica cells. Heterologous expression in Saccharomyces cerevisiae showed that both CYP719A2 and CYP719A3 had stylopine synthase activity to catalyze methylenedioxy bridge-formation from cheilanthifoline to stylopine, but not cheilanthifoline synthase activity to convert scoulerine to cheilanthifoline. Functional differences and expression patterns of CYP719A2 and CYP719A3 were examined to investigate their physiological roles in stylopine biosynthesis. Enzymatic analysis showed that CYP719A2 had high substrate affinity only toward (R,S)-cheilanthifoline, whereas CYP719A3 had high affinity toward three similar substrates (R,S)-cheilanthifoline, (S)-scoulerine, and (S)-tetrahydrocolumbamine. An expression analysis in E. californica plant tissues showed that CYP719A2 and CYP719A3 exhibited expression patterns similar to those of three stylopine biosynthetic genes (CYP80B1, berberine bridge enzyme, and S-adenosyl-l-methionine : 3'-hydroxy-N-methylcoclaurine 4'-O-methyltransferase), whereas the specific expression of CYP719A3 in root was notable. Treatment of E. californica seedlings with methyl jasmonate resulted in the coordinated induction of CYP719A2 and CYP719A3 genes. The physiological roles of CYP719A2 and CYP719A3 in stylopine biosynthesis are discussed.

Biochemical Evidence That Berberine Bridge Enzyme Belongs to a Novel Family of Flavoproteins Containing a Bi-covalently Attached FAD Cofactor

Berberine bridge enzyme (BBE) is involved in the transformation of (S)-reticuline to (S)-scoulerine in benzophenanthridine alkaloid biosynthesis of plants. In this report, we describe the high level expression of BBE encoded by the gene from Eschscholzia californica (California poppy) in the methylotrophic yeast Pichia pastoris employing the secretory pathway of the host organism. Using a two-step chromatographic purification protocol, 120 mg of BBE could be obtained from 1 liter of fermentation culture. The purified protein exhibits a turnover number for substrate conversion of 8.2 s(-1). The recombinant enzyme is glycosylated and carries a covalently attached FAD cofactor. In addition to the previously known covalent attachment of the 8alpha-position of the flavin ring system to a histidine (His-104), we could also demonstrate that a covalent linkage between the 6-position and a thiol group of a cysteine residue (Cys-166) is present in BBE. The major evidence for the occurrence of a bi-covalently attached FAD cofactor is provided by N-terminal amino acid sequencing and mass spectrometric analysis of the isolated flavin-containing peptide. Furthermore, it could be shown that anaerobic photoirradiation leads to cleavage of the linkage between the 6-cysteinyl group yielding 6-mercaptoflavin and a peptide with the cysteine residue replaced by alanine due to breakage of the C-S bond. Overall, BBE is shown to exhibit typical flavoprotein oxidase properties as exemplified by the occurrence of an anionic flavin semiquinone species and formation of a flavin N(5)-sulfite adduct.

Selective desensitization of jasmonate- and pH-dependent signaling in the induction of benzophenanthridine biosynthesis in cells of Eschscholzia californica

The biosynthesis of benzophenanthridine alkaloids, phytoalexins of Eschscholzia californica, in cultured cells can be induced by a glycoprotein preparation from yeast, methyljasmonate, artificial acidification with permeant acids, or mild osmotic stress. Each of these stimuli strongly attenuated the subsequent response to the same stimulus (homologous desensitization). Elicitor contact and artificial acidification mutually desensitized the cells for either signal. In contrast, elicitor-treated cells maintained their responsiveness to methyljasmonate or hyperosmolarity (sorbitol). Elicitor concentrations that nearly saturated the alkaloid response did not cause a detectable increase of jasmonate content. Transient acidification of the cytoplasm is a necessary step of signaling by low elicitor concentrations but was not detectable after jasmonate treatment. Seen together, the data indicate the existence of a jasmonate-dependent and jasmonate-independent (ΔpH controlled) signal pathway towards the expression of benzophenanthridine biosynthesis. Selective desensitization allows either stimulus to activate a distinct share of the biosynthetic capacity of the cell and limits the accumulation of toxic defense metabolites.

Modulation of berberine bridge enzyme levels in transgenic root cultures of California poppy alters the accumulation of benzophenanthridine alkaloids.

California poppy (Eschscholzia californica Cham.) root cultures produce a variety of benzophenanthridine alkaloids, such as sanguinarine, chelirubine and macarpine, with potent biological activity. Sense and antisense constructs of genes encoding the berberine bridge enzyme (BBE) were introduced into California poppy root cultures. Transgenic roots expressing BBE from opium poppy (Papaver somniferum L.) displayed higher levels of BBE mRNA, protein and enzyme activity, and increased accumulation of benzophenanthridine alkaloids compared to control roots transformed with a beta-glucuronidase gene. In contrast, roots transformed with an antisense-BBE construct from California poppy had lower levels of BBE mRNA and enzyme activity, and reduced benzophenanthridine alkaloid accumulation, relative to controls. Pathway intermediates were not detected in any transgenic root lines. Suppression of benzophenanthridine alkaloid biosynthesis using antisense-BBE also reduced the growth rate of the root cultures. Two-dimensional 1H-NMR spectroscopy showed no difference in the abundance of carbohydrate metabolites in the various transgenic roots lines. However, transformed roots with low levels of benzophenanthridine alkaloids contained larger cellular pools of certain amino acids compared to controls. In contrast, cellular pools of several amino acids were reduced in transgenic roots with elevated benzophenanthridine alkaloid levels relative to controls. The relative abundance of tyrosine, from which benzophenanthridine alkaloids are derived, was only marginally altered in all transgenic root lines; thus, altering metabolic flux through benzophenanthridine alkaloid pathways can affect cellular pools of specific amino acids. Consideration of such interactions is important for the design of metabolic engineering strategies that target benzophenanthridine alkaloid biosynthesis.

Antisense RNA-Mediated Suppression of Benzophenanthridine Alkaloid Biosynthesis in Transgenic Cell Cultures of California Poppy

California poppy (Eschscholzia californicaCham.) cell cultures produce several benzophenanthridine alkaloids, such as sanguinarine, chelirubine, and macarpine, with potent pharmacological activity. Antisense constructs of genes encoding two enzymes involved in benzophenanthridine alkaloid biosynthesis, the berberine bridge enzyme (BBE) and N-methylcoclaurine 3′-hydroxylase (CYP80B1), were introduced separately into California poppy cell cultures. Transformed cell lines expressing antisenseBBE or antisense CYP80B1 constructs and displaying low levels of BBE or CYP80B1 mRNAs, respectively, showed reduced accumulation of benzophenanthridine alkaloids compared with control cultures transformed with a β-glucuronidase gene. Pathway intermediates were not detected in any of the transformed cell lines. The suppression of benzophenanthridine alkaloid biosynthesis using BBE or CYP80B1 antisense RNA constructs also reduced the growth rate of the cultures. Two-dimensional 1H-nuclear magnetic resonance and in vivo 15N-nuclear magnetic resonance spectroscopy showed no difference in the abundance of carbohydrate metabolites in the various transgenic cell lines. However, transformed cells with reduced benzophenanthridine alkaloid levels contained larger cellular pools of several amino acids including alanine, leucine, phenylalanine, threonine, and valine compared with controls. The relative abundance of tyrosine, from which benzophenanthridine alkaloids are derived, was less than 2-fold higher in antisense-suppressed cells relative to controls. These results show that alterations in the metabolic flux through benzophenanthridine alkaloid biosynthesis can affect the regulation of amino acid pools. These data provide new insight into the metabolic engineering of benzophenanthridine alkaloid pathways.

Antisense RNA-mediated suppression of benzophenanthridine alkaloid biosynthesis in transgenic cell cultures of California poppy.

California poppy (Eschscholzia californica Cham.) cell cultures produce several benzophenanthridine alkaloids, such as sanguinarine, chelirubine, and macarpine, with potent pharmacological activity. Antisense constructs of genes encoding two enzymes involved in benzophenanthridine alkaloid biosynthesis, the berberine bridge enzyme (BBE) and N-methylcoclaurine 3'-hydroxylase (CYP80B1), were introduced separately into California poppy cell cultures. Transformed cell lines expressing antisense BBE or antisense CYP80B1 constructs and displaying low levels of BBE or CYP80B1 mRNAs, respectively, showed reduced accumulation of benzophenanthridine alkaloids compared with control cultures transformed with a beta-glucuronidase gene. Pathway intermediates were not detected in any of the transformed cell lines. The suppression of benzophenanthridine alkaloid biosynthesis using BBE or CYP80B1 antisense RNA constructs also reduced the growth rate of the cultures. Two-dimensional (1)H-nuclear magnetic resonance and in vivo (15)N-nuclear magnetic resonance spectroscopy showed no difference in the abundance of carbohydrate metabolites in the various transgenic cell lines. However, transformed cells with reduced benzophenanthridine alkaloid levels contained larger cellular pools of several amino acids including alanine, leucine, phenylalanine, threonine, and valine compared with controls. The relative abundance of tyrosine, from which benzophenanthridine alkaloids are derived, was less than 2-fold higher in antisense-suppressed cells relative to controls. These results show that alterations in the metabolic flux through benzophenanthridine alkaloid biosynthesis can affect the regulation of amino acid pools. These data provide new insight into the metabolic engineering of benzophenanthridine alkaloid pathways.

Shifts of intracellular pH distribution as a part of the signal mechanism leading to the elicitation of benzophenanthridine alkaloids . Phytoalexin biosynthesis in cultured cells of eschscholtzia californica

Cultured cells of Eschscholtzia californica (Californian poppy) respond to a yeast elicitor preparation or Penicillium cyclopium spores with the production of benzophenanthridine alkaloids, which are potent phytoalexins. Confocal pH mapping with the probe carboxy-seminaphthorhodafluor-1-acetoxymethylester revealed characteristic shifts of the pH distribution in challenged cells: within a few minutes after elicitor contact a transient acidification of cytoplasmic and nuclear areas occurred in parallel with an increase of the vacuolar pH. The change of proton concentration in the vacuole and in the extravacuolar area showed a nearly constant relation, indicating an efflux of vacuolar protons into the cytosol. A 10-min treatment with 2 mM butyric or pivalic acid caused a transient acidification of the cytoplasm comparable to that observed after elicitor contact and also induced alkaloid biosynthesis. Experimental depletion of the vacuolar proton pool reversibly prevented both the elicitor-triggered pH shifts and the induction of alkaloid biosynthesis. pH shifts and induction of alkaloid biosynthesis showed a similar dependence on the elicitor concentration. Net efflux of K+, alkalinization of the outer medium, and browning of the cells were evoked only at higher elicitor concentrations. We suggest that transient acidification of the cytoplasm via efflux of vacuolar protons is both a necessary and sufficient step in the signal path toward biosynthesis of benzophenanthridine alkaloids in Californian poppy cells.

P3A33 - Scope of antioxidants in the management of Epidemic Dropsy caused by argemone oil

Tetrahydrobenzylisoquinoline alkaloids comprise a diverse class of secondary metabolites with many pharmacologically active members. The biosynthesis at the enzyme level of at least two tetrahydrobenzylisoquinoline alkaloids, the benzophenanthridine alkaloid sanguinarine in the California poppy, Eschscholtzia californica, and the bisbenzylisoquinoline alkaloid berbamunine in barberry, Berberis stolonifera, has been elucidated in detail starting from the aromatic amino acid (aa) l-tyrosine. In an initial attempt to develop alternate systems for the production of medicinally important alkaloids, one enzyme from each pathway (BBE, a covalently flavinylated enzyme of benzophenanthridine alkaloid biosynthesis and CYP80, a phenol coupling cytochrome P-450-dependent oxidase of bisbenzylisoquinoline alkaloid biosynthesis) has been purified to homogeneity, a partial aa sequence determined, and the corresponding cDNAs isolated with aid of synthetic oligos based on the aa sequences. The recombinant enzymes were actively expressed in Spodoptera frugiperda Sf9 cells using a baculovirus vector, purified and then characterized. Insect cell culture has proven to be a powerful system for the overexpression of alkaloid biosynthetic genes.

Involvement of protein kinase and G proteins in the signal transduction of benzophenanthridine alkaloid biosynthesis

Benzophenanthridine alkaloid biosynthesis and biosynthetic enzyme activity were induced in suspension-cells of Sanguinaria canadensis L. by compounds that stimulate the activities of protein kinase and GTP-binding proteins. The results indicate that one or more protein kinases, and putative G proteins are involved in the signal transduction pathway that mediates ABA and fungal-induced benzophenanthridine alkaloid biosynthesis. We have previously reported that elicitor-induced benzophenanthridine alkaloid biosynthesis in suspension-cell cultures of Sanguinaria canadensis L. (SCP-GM) is mediated by a signal transduction system that involves calcium and possibly protein kinase(s). In this work, a number of exogenous agents were employed to further investigate the components of the signal transduction pathway involved in the induction of alkaloid biosynthesis by a fungal elicitor and abscisic acid (ABA). SCP-GM suspension-cells were treated with compounds that modify protein kinase activity, including phorbol esters, and 1-oleoyl-2-acetyl-rac-glycerol (OAG), a synthetic diacylglycerol analogue. Phorbol-12-myristate-13-acetate induced alkaloid accumulation by as much as 65-fold over control values, while the negative control, phorbol-13-monoacetate, had no effect. OAG also increased alkaloid production by approximately 25-fold as compared to controls. Likewise, pretreatment of the suspension-cell cultures with H-7 or staurosporine, significantly suppressed ABA- or fungal-induction of benzophenanthridine alkaloid biosynthesis. Modulators of GTP-binding protein activity were also active in this system. Treatment of the suspension-cells with cholera toxin (CHX) induced alkaloid accumulation by 25-fold, which increased to 34-fold when CHX was combined with a fungal elicitor derived from Penicillium expansum (PE), and 32-fold when CHX was combined with ABA. Treatment of SCP-GM cells with CHX also enhanced the activities of two N-methyltransferases in the benzophenanthridine biosynthetic pathway namely, tetrahydroberberine- N-methyltransferase and tetrahydrocoptisine- N-methyltransferase, by six and seven fold, respectively. Furthermore, benzophenanthridine alkaloid biosynthesis was induced by treating the suspension-cells with the G-protein activators, mastoparan, mas-7 or melittin, while the inactive homologue, mas-17, did not. Suppression of alkaloid accumulation occurred when the susoension-cells were treated with GDPßS or pertussis toxin prior to treatment of the SCP-GM cells with either PE or ABA. The results support the hypothesis that one or more protein kinases, and putative G proteins are involved in the signal transduction pathway that mediates ABA and fungal-induced benzophenanthridine alkaloid biosynthesis.

Isolation and analysis of a gene bbe1 encoding the berberine bridge enzyme from the California poppy Eschscholzia californica.

A genomic clone, bbe1 was isolated that encodes the methyl jasmonate-inducible berberine bridge enzyme of antimicrobial benzophenanthridine alkaloid biosynthesis in the California poppy Eschscholzia californica. Genomic DNA gel blot analysis indicates that two genes are present in the E. californica genome that code for the berberine bridge enzyme reading frame. Each coding region is apparently preceeded by a unique promoter sequence. The bbe1 gene contains no introns and one transcriptional start site. A 41 nucleotide region between -496 and -455 of the 5'-flanking region appears to be essential for promoter activity in E. californica. The promoter displayed an unexpectedly high species specificity, being active in only E. californica and Thalictrum bulgaricum, out of 28 cell suspension cultures tested.

Induction of Benzo[c]phenanthridine Alkaloid Biosynthesis in Suspension Cell Cultures ofSanguinaria canadensisby Retinoic Acid Derivatives

Abstract Treatment of suspension-cell cultures of Sanguinaria canadensis L. (SCP-GM) with a series of natural and synthetic retinoic acid derivatives induced benzophenanthridine alkaloid biosynthesis in a dose-dependent manner. Suspension cells were treated with one of the following: retinoic acid (RA), retinol (ROH), retinal (RHO). 13- cis-retinoic acid (cis-RA), or retinyl acetate (ROAc) in concentrations ranging from 1 to 50 μM (24 h). Each of the RA derivatives tested increased the cellular alkaloid concentrations of sanguinarine and chelerythrine by 97 to 470% (approximately 0.045–0.23% dry wt) depending on the reinoid and the dosage employed. Control SCP-GM suspension-cells accumulated only trace amounts of sanguinarine and chelerythrine, approximately 0.005% dry wt and 0.004% dry wt, respectively. The activity of S-adenosyl methionine: tetrahydroberberine-.V-methyltransferase, a key branch point enzyme in the benzophenanthridine biosynthetic pathway, was also induced over 24 h after treatment of th...

Heterologous expression of alkaloid biosynthetic genes — a review

Tetrahydrobenzylisoquinoline alkaloids comprise a diverse class of secondary metabolites with many pharmacologically active members. The biosynthesis at the enzyme level of at least two tetrahydrobenzylisoquinoline alkaloids, the benzophenanthridine alkaloid sanguinarine in the California poppy, Eschscholtzia californica, and the bisbenzylisoquinoline alkaloid berbamunine in barberry, Berberis stolonifera, has been elucidated in detail starting from the aromatic amino acid (aa) l-tyrosine. In an initial attempt to develop alternate systems for the production of medicinally important alkaloids, one enzyme from each pathway (BBE, a covalently flavinylated enzyme of benzophenanthridine alkaloid biosynthesis and CYP80, a phenol coupling cytochrome P-450-dependent oxidase of bisbenzylisoquinoline alkaloid biosynthesis) has been purified to homogeneity, a partial aa sequence determined, and the corresponding cDNAs isolated with aid of synthetic oligos based on the aa sequences. The recombinant enzymes were actively expressed in Spodoptera frugiperda Sf9 cells using a baculovirus vector, purified and then characterized. Insect cell culture has proven to be a powerful system for the overexpression of alkaloid biosynthetic genes.

Characterization and Mechanism of the Berberine Bridge Enzyme, a Covalently Flavinylated Oxidase of Benzophenanthridine Alkaloid Biosynthesis in Plants

The berberine bridge enzyme ((S)-reticuline:oxygen oxidoreductase (methylene-bridge-forming), EC 1.5.3.9) catalyzes the oxidative cyclization of the N-methyl moiety of (S)-reticuline into the berberine bridge carbon, C-8, of (S)-scoulerine. This is a reaction that has neither an equivalent in organic chemistry nor a parallel in nature. The uniqueness of this catalytic reaction prompted an in depth study that began with the isolation of the cDNA encoding the berberine bridge enzyme followed by the overexpression of this cDNA in insect cell culture. The heterologously expressed enzyme has herein been shown to contain covalently attached FAD in a molar ratio of cofactor to protein of 1:1.03. Site-directed mutagenesis and laser desorption time-of-flight mass spectrometry suggest that the site of covalent attachment is at His-104. The holoenzyme exhibited absorbance maxima at 380 and 442 nm and a fluorescence emission maximum at 628 nm (310 nm excitation). Enzymic transformation of a series of (S)-reticuline derivatives modified with respect to the stereochemistry at C-1 or in the aromatic ring substitution suggests that ring closure proceeds in two steps: formation of the methylene iminium ion and subsequent ring closure via an ionic mechanism.

Quercetin-induced benzophenanthridine alkaloid production in suspension cell cultures of Sanguinaria canadensis.

Addition of micromolar concentrations of quercetin or rutin to suspension cell cultures of Sanguinaria canadensis L. (bloodroot) induced the biosynthesis of sanguinarine and chelerythrine in a dose-dependent manner. In contrast, related compounds: baicalein, naringin, naringenin, catechin, caffeic acid and benzoic acid displayed very weak inductive activity. Of the two active flavonoids, quercetin was the most effective for inducing benzophenanthridine alkaloid biosynthesis, with doses of 100 microM increasing alkaloid production over 375% as compared to negative controls. Quercetin's inductive effects were similar to that of an elicitor derived from fungus Penicillium expansum (PE-elicitor). Suppression of quercetin and PE-induced alkaloid biosynthesis by low doses of actinomycin D (5 micrograms/ml, alpha-amanitin (20 micrograms/ml), or cycloheximide (1 microgram/ml) demonstrate a requirement for both RNA and de novo cytoplasmic protein synthesis and suggest that alterations in gene expression are involved in the inductive mechanism. Furthermore, quercetin-induced alkaloid biosynthesis was significantly reduced by pretreatment of the cells with the calcium chelator, EGTA (3 mM), or the calcium channel inhibitor, verapamil (100 microM), suggesting that this process was calcium dependent.

Elicitor-stimulated benzophenanthridine alkaloid biosynthesis in bloodroot suspension cultures is mediated by calcium

Addition of an elicitor derived from the fungus Penicillium expansum Link (PE-elicitor) or the calcium ionophore A23187, to a suspension-cell culture of Sanguinaria canadensis induced the production of the benzophenanthridine alkaloids, sanguinarine and chelerythrine, in a dose-dependent manner. Pretreatment of the cells with the specific calcium chelatant EGTA (3 mM) or the calcium channel inhibitor verapamil (100 μM), for 1 hr prior to the addition of the PE-elicitor, decreased the accumulation of both sanguinarine and chelerythrine. Moreover, A23187-stimulated alkaloid accumulation was almost completely inhibited by pretreating the suspension cells with EGTA (3 mM) for 1 hr and this suppression was reversed by the readdition of calcium ions to the medium. Furthermore, addition of trifluoperazine (100 μM) to the suspension-cell cultures 1 hr before the PE-elicitor (35 μg Glc equ ml −1) treatment suppressed the accumulation of benzophenanthridine alkaloids by 51% as compared to suspension cells treated with PE-elicitor alone. These results demonstrate that an external source of calcium ions is required for elicitor-induced benzophenanthridine alkaloid accumulation and suggest that calcium and possibly calmodulin and/or protein kinase C may participate in a signal transduction system that mediates this process.

Stereochemical Studies on the Biosynthesis of Protoberberine, Protopine, and Benzophenanthridine Alkaloids Using Papaveraceae Plant Cell Cultures

ADVERTISEMENT RETURN TO ISSUEPREVArticleNEXTStereochemical Studies on the Biosynthesis of Protoberberine, Protopine, and Benzophenanthridine Alkaloids Using Papaveraceae Plant Cell CulturesKinuko Iwasa, Miyoko Kamigauchi, Narao Takao, and Mark CushmanCite this: J. Nat. Prod. 1993, 56, 12, 2053–2067Publication Date (Print):December 1, 1993Publication History Published online1 July 2004Published inissue 1 December 1993https://doi.org/10.1021/np50102a005RIGHTS & PERMISSIONSArticle Views267Altmetric-Citations7LEARN ABOUT THESE METRICSArticle Views are the COUNTER-compliant sum of full text article downloads since November 2008 (both PDF and HTML) across all institutions and individuals. These metrics are regularly updated to reflect usage leading up to the last few days.Citations are the number of other articles citing this article, calculated by Crossref and updated daily. Find more information about Crossref citation counts.The Altmetric Attention Score is a quantitative measure of the attention that a research article has received online. Clicking on the donut icon will load a page at altmetric.com with additional details about the score and the social media presence for the given article. Find more information on the Altmetric Attention Score and how the score is calculated. Share Add toView InAdd Full Text with ReferenceAdd Description ExportRISCitationCitation and abstractCitation and referencesMore Options Share onFacebookTwitterWechatLinked InReddit PDF (811 KB) Get e-Alerts Get e-Alerts

Enzymic 10-hydroxylation and 10- O-methylation of dihydrosanguinarine in dihydrochelirubine formation by Eschscholtzia

Two new enzymes involved in benzophenanthridine alkaloid biosynthesis, dihydrosanguinarine-10-hydroxylase and 10-hydroxydihydrosanguinarine-10- O-methyltransferase, have been found in cell-free extracts derived from Eschscholtzia californica cell suspension cultures. The hydroxylase is a microsome-associated, cytochrome P-450-dependent monooxygenase which acts specifically at C-10 of dihydrosanguinarine. The methyltransferase, enriched 60-fold and partially characterized, methylates only the hydroxyl moiety at C-10 to form dihydrochelirubine. Both enzymes were found to be highly substrate specific.