Tropane Alkaloid Biosynthesis Research Articles

Induction of Tropane Alkaloid Formation in Transformed Root Cultures of Brugmansia suaveolens (Solanaceae)

Hairy root cultures of Brugmansia suaveolens were set up by infection of root tips with Agrobacterium rhizogenes. The successful transformation was confirmed by analysing rolC and virC genes using polymerase chain reaction (PCR). Hairy root cultures were employed to study the formation of tropane alkaloids, such as hyoscyamine. The transformed cultures were incubated with potential elicitors, such as methyljasmonate, quercetin and salicylic acid in order to stimulate the biosynthesis of tropane alkaloids. Profile and amounts of tropane alkaloids were analysed using capillary GLC-MS. At least 18 different tropane alkaloids could be identified. Treatment of the cultures with 200 microM methyljasmonate increased the alkaloid accumulation 25-fold up to a level of 1 mg/g fresh weight as compared to untreated controls. Quercetin enhanced the alkaloid production 10 fold (0.4 mg/g fresh weight) within 24 h. In contrast 100 microM salicylic acid decreased alkaloids to a level of 1 microg/g fresh weight.

Effects of methyl jasmonate and salicylic acid on the production of tropane alkaloids and the expression of PMT and H6H in adventitious root cultures of Scopolia parviflora

The effects of methyl jasmonate (MJ) and salicylic acid (SA) on the production of tropane alkaloids (TA) and the expression of putrescine N-methyltransferase (PMT) and hyoscyamine 6β-hydroxylase (H6H) were studied in adventitious root cultures of Scopolia parviflora. MJ treatments increased the amounts of both scopolamine and hyoscyamine, with growth inhibition of the roots, while SA increased the amount of scopolamine without negative effects on growth. Regarding both MJ and SA, their concentrations and exposure times were factors that strongly affected the production of TA. Western blot analysis showed that treatments with MJ or SA regulated the expression of the key enzymes PMT and H6H and the biosynthesis of TA. The results from this study suggest that MJ and SA regulated directly or indirectly the expression of PMT or the H6H gene and, finally, increased the production of TA and stimulated the excretion of TA into the culture medium.

Enhanced production of scopolamine by bacterial elicitors in adventitious hairy root cultures of Scopolia parviflora

In an attempt to increase productivity, the effect of elicitation on tropane alkaloids (TA) biosynthesis was studied in adventitious hairy root cultures of Scopolia parviflora. Two Gram-positive strains and one Gram-negative strain of bacteria were used as biotic elicitors. The raw bacterial elicitors affected the tropane alkaloid profile by increasing the scopolamine concentration, while the autoclaved bacterial elicitors produced similar effects on the control. The conversion ratio of hyoscyamine to scopolamine was increased following elicitation using raw bacterial elicitors. The bacterial elicitor inhibited the expression of H6H (hyoscyamine 6β-hydoxylase) whereas the expression of PMT (putrescine N-methyltransferase) was raised by elicitation. These results have important implications for the large-scale production of tropane alkaloids.

Tropane alkaloid의 생합성과 분자육종

The tropane alkaloids hyoscyamine (its racemic form being atropine) and scopolamine are used medicinally as anticholinergic agents that act on the parasympathetic nerve system. Because they differ in their actions on the central nervous system, currently there is a 10-fold higher commercial demand for scopolamine, in the N-butylbromide form, than there is for hyoscyamine and atropine combined. Several solanaceous species have been used as the commercial sources of these alkaloids, but the scopolamine contents in these plants often are much lower than those of hyoscyamine. For this reason there has been long-standing interest in increasing the scopolamine contents of cultivated medicinal plants. Naturally occurring and artificial interspecific hybrids of Duboisia have high scopolamine contents and are cultivated as a commercial source of scopolamine in Australia and other countries. Anther culture combined with conventional interspecific hybridization also has been used to breed high scopolamine-containing plants in the genera Datura and Hyoscyamus, but without much success. The use of recombinant DNA technology for the manipulation of metabolic processes in cells promises to provide important contributions to basic science, agriculture, and medicine. In this review, I introduce on the enzymes and genes involved in tropane alkaloid biosynthesis and current progress in metabolic engineering approaches for tropane alkaloid, especially scopolamine, production.

Kinetic study of littorine rearrangement in Datura innoxia hairy roots by (13)C NMR spectroscopy.

The kinetics of tropane alkaloid biosynthesis, particularly the isomerization of littorine into hyoscyamine, were studied by analyzing the kinetics of carbon-13 ((13)C) in metabolites of Datura innoxia hairy root cultures fed with labeled tropoyl moiety precursors. Both littorine and hyoscyamine were the major alkaloids accumulated, while scopolamine was never detected. Feeding root cultures with (RS)-phenyl[1,3-(13)C(2)]lactic acid led to (13)C spin-spin coupling detected on C-1' and C-2' of the hyoscyamine skeleton, which validated the intramolecular rearrangement of littorine into hyoscyamine. Label from phenyl[1-(13)C]alanine or (RS)-phenyl[1,3-(13)C(2)]lactic acid was incorporated at higher levels in littorine than in hyoscyamine. Initially, the apparent hyoscyamine biosynthesized rate (v(app)()hyo = 0.9 micromol (13)C.flask(-1).d(-1)) was lower than littorine formation (v(app)()litto = 1.8 micromol (13)C.flask(-1).d(-1)), suggesting that the isomerization reaction could be rate limiting. The results obtained for the kinetics of littorine biosynthesis were in agreement with the role of this compound as a direct precursor of hyoscyamine biosynthesis.

Secondary metabolism in root and callus cultures of Hyoscyamus muticus L.: the relationship between morphological organisation and response to methyl jasmonate

Production of acid-soluble conjugated di- and polyamines, like that of other secondary metabolites, is enhanced by exposure to methyl jasmonate (MJ). We investigated this metabolic response, and activities of enzymes involved in putrescine (Put) and tropane alkaloid biosynthesis, in root cultures of Hyoscyamus muticus and compared it with that of callus cultures. In root cultures, free Put and N-methylputrescine (mPut) increased upon treatment with MJ, whereas in callus cultures mPut levels were not affected. Differently from roots, conjugated amines were scarce or absent in callus cultures, and accumulated only transiently upon treatment with MJ. Arginine decarboxylase, ornithine decarboxylase and diamine oxidase activities in root cultures were strongly stimulated by treatment with MJ, but were inhibited in callus cultures. Exposure to MJ also enhanced putrescine N-methyltransferase activity in root cultures more than in callus cultures. These results are discussed in relation to the different capacity for tropane alkaloid production in the two culture systems.

Tropane alkaloid biosynthesis. A century old problem unresolved.

Tropane alkaloid biosynthesis. A century old problem unresolved.

Effects of the rol C gene on hairy root: induction development and tropane alkaloid production by Atropa belladonna.

Two series of Atropa belladonna hairy root lines were obtained, the first one transformed via Agrobacterium tumefaciens harboring rol C and npt II genes, and the other transformed with rol ABC and npt II genes. Thirteen hairy root lines were obtained and selected on hormone-free medium. The transformation was confirmed by PCR analysis, and these root lines were first examinated for their growth rate. Then hyoscyamine and scopolamine production was measured after 3 and 4 weeks of culture to evaluate the possible role of rol C gene in tropane alkaloid formation. The rol C gene alone played a significant role in the hairy root growth rate (17-fold increase). However this effect was much lower than that induced by the rol ABC genes together (75-fold increase). In contrast, the rol C gene alone was as efficient as the rol ABC genes together (mean value of total alkaloids: 0.36% dry weight, i.e., 12-fold times more than in untransformed roots) to stimulate the biosynthesis of tropane alkaloids in A. belladonna hairy root cultures.

Structures and expression patterns of two tropinone reductase genes from Hyoscyamus niger.

In the biosynthesis of tropane alkaloids, two tropinone reductases (TRs) catalyze reduction of tropinone to different stereoisomers, tropine and pseudotropine. Two TRs from Hyoscyamus niger have 64% of identical amino acids and hence a common evolutionary origin. In this study, genomic clones of TRs were isolated from H. niger. Their sequence comparison showed that although they have the same exon/intron organization, sequence similarity was restricted to the coding regions. In H. niger transgenic hairy roots, both TR promoters activated transcription of the reporter genes in endodermis and pericycle of the roots. A quantitative reporter assay and a nuclear run-on experiment indicated that the two genes are transcribed at a similar rate. The results indicate that although different activity levels have been observed for the TR enzymes in the H. niger root, the TR genes per se conserve similar tissue-specific expression pattern and transcriptional rate.

Two tropinone reductases, that catalyze opposite stereospecific reductions in tropane alkaloid biosynthesis, are localized in plant root with different cell-specific patterns.

In the plant species that produce tropane alkaloids, two tropinone reductases (TRs) catalyze the stereospecific reductions of the 3-carbonyl group of tropinone. This reduction is a key branch point that determines the metabolite flow into the separate alkaloid groups, each with different stereospecific configurations. In this study, a specific antibody was prepared for each of the TRs by immunizing mice with recombinant TR protein and subsequent immuno-affinity purification of the antiserum. Immunoblot analyses revealed that accumulation of both TRs was highest in the lateral roots of Hyoscyamus niger throughout its development. In cultured roots, TR proteins were accumulated in a basal region but not in root apex. These patterns were similar to that of hyoscyamine 6 beta-hydroxylase (H6H), an enzyme that catalyzes a downstream step in the same biosynthetic pathway. However, an immunohistochemical analysis revealed that the two TRs and H6H were accumulated with different cell-specific patterns in the cultured root, suggesting transportation of the alkaloid intermediate(s) across the different cell layers.

Expression of Atropa belladonna putrescine N-methyltransferase gene in root pericycle.

The cDNAs encoding putrescine N-methyltransferase (PMT), which catalyzes the S-adenosylmethionine-dependent N-methylation of putrescine at the first committed step in the biosynthetic pathways of tropane alkaloids, were isolated from Atropa belladonna and Hyoscyamus niger. These PMTs, however, lacked the N-terminal tandem repeat arrays previously found in Nicotiana PMTs. AbPMT1 RNA was much more abundant in the root of A. belladonna than was AbPMT2 RNA. The 5'-flanking region of the AbPMT1 gene was fused to the beta-glucuronidase (GUS) reporter gene and transferred to A. belladonna. Histochemical analysis showed that GUS is expressed specifically in root pericycle cells and that the 0.3-kb 5'-upstream region was sufficient for pericycle-specific expression. Treatment of A. belladonna roots with methyl jasmonate did not up-regulate the expression of GUS or endogenous AbPMT genes. The regulation of tropane alkaloid biosynthesis is discussed and compared with that of nicotine biosynthesis.

The role and source of 5′-deoxyadenosyl radical in a carbon skeleton rearrangement catalyzed by a plant enzyme

The last step in the biosynthesis of tropane alkaloids is the carbon skeleton rearrangement of littorine to hyoscyamine. The reaction is catalyzed by a cell-free extract prepared from cultured hairy roots of Datura stramonium. Adenosylmethionine stimulated the rearrangement 10–20-fold and showed saturation kinetics with an apparent K m of 25 μM. It is proposed that S-adenosylmethionine is the source of a 5′-deoxyadenosyl radical which initiates the rearrangement in a similar manner as it does in analogous rearrangements catalyzed by coenzyme B 12-dependent enzymes. Possible roles of S-adenosylmethionine as a radical source in higher plants are discussed.

The Biosynthesis of Tropane Alkaloids in Datura stramonium: The Identity of the Intermediates between N-Methylpyrrolinium Salt and Tropinone

The biosynthesis of the tropane alkaloids in transformed root cultures of Datura stramonium has been studied using sodium [1,2-13C2]acetate, (R,S)-[2,3-13C2]-1-(1-methyl-2-pyrrolidinyl)propan-2-one {(R,S)-[2‘,3‘-13C2]hygrine}, ethyl (R,S)-[1,2-13C2,2-14C]-2-(1-methyl-2-pyrrolidinyl)acetate, and ethyl (R,S)-[2,3-13C2,3-14C]-4-(1-methyl-2-pyrrolidinyl)-3-oxobutanoate. The incorporation of (R,S)-[2‘,3‘-13C2]hygrine into cuscohygrine and several other condensation products was high (15−40% specific incorporation), but label was not recovered in either tropine or tropine esters (hyoscyamine; 0.0 ± 0.5% specific incorporation). None of the recovered alkaloids was labeled when ethyl (R,S)-[1,2-13C2,2-14C]-2-(1-methyl-2-pyrrolidinyl)acetate was fed to the cultures. In contrast, sodium [1,2-13C2]acetate and ethyl (R,S)-[2,3-13C2,3-14C]-4-(1-methyl-2-pyrrolidinyl)-3-oxobutanoate were incorporated into hyoscyamine (9 and 2% specific incorporation, respectively) and a number of other tropane alkaloids (up to 12% spec...

Tigloyl-CoA:pseudotropine acyl transferase—An enzyme of tropane alkaloid biosynthesis

Tigloyl-CoA:pseudotropine acyl transferase esterifies the 3β-hydroxyl group of pseudotropine (3β-hydroxytropane) with the tiglic acid moiety of tigloyl-Coenzyme A. This enzyme was purified to near electrophoretic homogeneity—about 330-fold—from transformed root cultures of Datura stramonium. The protocol involved the sequential use of ammonium sulphate precipitation, hydrophobic interaction, anion-exchange, chromatofocusing and gel filtration chromatography. The enzyme has a M r of 65 000, as determined by gel filtration chromatography on Sepharose 6 and SDS-PAGE electrophoresis, indicating it to be active as a monomer. Maximal activity occurs at pH 9.0 but the enzyme is still about 30% active at pH 7.0. The purified protein shows simple Michaelis-Menten kinetic behaviour, with K m values of 0.36 and 1.31 mM for pseudotropine and tigloyl-CoA, respectively. The enzyme is specific for the acyl group receptor. Of a range of potential acceptors tested, only pseudotropine and 4-hydroxy-1-methylpiperidine (14%) were used: tropine (3α-hydroxytropane) and norpseudotropine were not acylated. In contrast, the enzyme possesses the ability to transfer the acyl moiety to pseudotropine from a wide range of aliphatic acyl-CoA thioesters. Tigloyl-CoA and acetyl-CoA act recuprocally as competitive inhibitors, suggesting that they compete for the same active site on the enzyme. With acetyl-CoA the K m is 0.33 mM, indicating that the enzyme has a higher affinity for this acyl donor than for tigloyl-CoA. Neither CoA nor any of the alkamine acceptors tested were able to inhibit the acylation of pseudotropine.

Establishment and characterization of photosynthetic hairy root cultures of Datura stramonium

Twelve different lines of Datura stramonium (normal and hairy) root cultures were subjected to conditions which induce photoautotrophy. Two of the hairy root lines responded to induction, showing clearly a diminished growth rate when compared to heterotrophic cultures, an increase in chlorophyll, a net O2 evolution, CO2 fixation, and de novo synthesis of the ribulose 1,5 biphosphate carboxylase enzyme. A time course of growth and tropane alkaloid levels in the tissue and medium, revealed a correlation between the development of the photosynthetic apparatus and the increase in scopolamine. Although normal cultures did not grow photosynthetically, they showed some greening response under the first step of the induction. The correlation between development of photosynthesis and increase in scopolamine synthesis were corroborated with normal root cultures. This experimental model is used for the basic study of the regulatory enzymes involved in the biosynthesis of tropane alkaloids, as well as for the study of their mechanisms of transport.

Esterification reactions in the biosynthesis of tropane alkaloids in transformed root cultures

Esterification reactions in the biosynthesis of tropane alkaloids in transformed root cultures

Phenyllactic acid but not tropic acid is an intermediate in the biosynthesis of tropane alkaloids in Datura and Brugmansia transformed root cultures

(S)-(-)-Tropic acid is the acidic moiety of the tropane ester alkaloids, hyoscyamine and scopolamine (hyoscine). When tropic acid is fed to transformed root cultures of Datura stramonium L. or a Brugmansia (Datura) Candida x B. aurea hybrid, the formation of these alkaloids is inhibited. Phenyllactic acid, from which the tropoyl moiety is derived, is considerably less inhibitory. Label from (RS)-phenyl[1,3-13C2]lactic acid is incorporated at high levels into apoatropine, littorine, aposcopolamine, hyoscyamine, 7β-hydroxyapoatropine, scopolamine and 7β-hydroxyhyoscyamine when fed to these cultures. The presence of an excess concentration of unlabelled tropic acid has little influence on the specific incorporation into these products. It is concluded that free tropic acid is not an intermediate in hyoscyamine biosynthesis but rather that the rearrangement of phenyllactic acid occurs subsequent to its esterification.

CDNA encoding tropinone reductase-II from Hyoscyamus niger.

Two stereospecific NADPH-dependent reductases, TR-I and TR-11, constitute a branching point in the biosynthesis of tropane alkaloids. TR-I catalyzes the stereospecific reduction of tropinone to tropine (Koelen and Gross, 1982), whereas TR-I1 reduces tropinone to pseudotropine (Drager et al., 1988). We previously characterized TRs that had been purified from cultured roots of Hyoscyamus niger (Hashimoto et al., 1992) and showed that the two TRs had both common and different biochemical and kinetic properties. To obtain a better understanding of the structure and evolutionary relationship of these reductases, we isolated cDNA clones coding for TR-I1 from H. niger (Table I). An intemal amino acid sequence was found in both TR-I and TR-I1 that had been purified from the cultured roots of Datura stramonium and H. niger, respectively (Nakajima et al., 1993). An oligonucleotide probe corresponding to this sequence was synthesized and used to screen the cDNA library from cultured roots of H. niger. DNA sequencing analysis revealed that a11 four of the isolated cDNA clones encoded the TR-I1 polypeptide, probably due to the low concentration of the TR-I transcript in this genus. None of the cDNA clones contained a full ORF; some lacked the amino-terminal part and others the carboxy-terminal part and the 3‘ nontranslated region. Because the nucleotide sequence of the overlapping part (0.4-0.8 kb) matched perfectly, we concluded that these clones were derived from a single gene. The combined nucleotide sequence (1049 bp) contained a 783-bp ORF coding for a polypeptide composed of 260 amino acids, and the calculated mo1 wt of 28,436 agreed well with the molecular mass for the TR-I1 subunit (29 kD) that had been purified from H. niger (Hashimoto et al., 1992). The isolated cDNA was expressed in Escherichia coli as a fusion protein to /3-galactosidase under the control of the Zac promoter. The fusion protein was induced by isopropyl-P-Dthiogalactopyranoside, and the bacterial lysate was assayed for TR activities as described elsewhere (Nakajima et al., 1993). The fusion protein catalyzed the same highly stereospecific reduction of tropinone as the TR-I1 from H. niger; pseudotropine was the sole reaction product detected. The deduced amino acid sequence of TR-I1 from H. niger is highly homologous to that from D. stramonium (Nakajima

Putrescine N-Methyltransferase in Cultured Roots of Hyoscyamus albus

Biosynthesis of tropane alkaloids is thought to proceed by way of the diamine putrescine, followed by its methylation by putrescine N-methyltransferase (PMT; EC 2.1.1.53). High PMT activities were found in branch roots and/or cultured roots of several solanaceous plants. PMT was partially purified and characterized from cultured roots of Hyoscyamus albus that contain hyoscyamine as the main alkaloid. Initial velocity studies and product inhibition patterns of PMT are consistent with an ordered bi-bi mechanism, in which the K(m) values for putrescine and S-adenosyl-l-methionine are 277 and 203 mum, respectively, and the K(i) value for S-adenosyl-l-homocysteine is 110 mum. PMT efficiently N-methylated amines that have at least two amino groups separated by three or four methylene groups. Monoamines were good competitive inhibitors of PMT, among which n-butylamine, cyclohexylamine, and exo-2-aminonorbornane were most inhibitory, with respective K(i) values of 11.0, 9.1, and 10.0 mum. When n-butylamine was fed to root cultures of H. albus, the alkamine intermediates (tropinone, tropine, and pseudotropine) drastically decreased at 1 mm of the exogenous monoamine, and the hyoscyamine content decreased by 52% at 6 mm, whereas the contents of 6beta-hydroxyhyoscyamine and scopolamine did not change. Free and conjugated forms of polyamines were also measured. The n-butylamine treatment caused a large increase in the putrescine content (especially in the conjugated pool), and the spermine content also increased slightly, whereas the spermidine content decreased slightly. The increase in the putrescine pool size (approximately 40 nmol/mg dry weight) was large enough to account for the decrease in the total alkaloid pool size. Similar results were also obtained in root cultures of Datura stramonium. These studies further support the role of PMT as the first committed enzyme specific to alkaloid biosynthesis.

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