Putrescine and putrescine N-methyltransferase in the biosynthesis of tropane alkaloids in cultured roots of Hyoscyamus albus

Hashimoto, Takashi; Yukimune, Yukihito; Yamada, Yasuyuki

doi:10.1007/bf00392535

Cited by 75 publications

(26 citation statements)

References 33 publications

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“…On the other hand, the levels of ODC and ADC are each some fivefold lower than that of PMT and only represent, in combination, about a fourfold excess over the production of hygrine-derived alkaloids. As also found for H. albus (Hashimoto et al 1989a), ADC is present at a slightly greater level than ODC. The relative importance of these two activities in alkaloid biosynthesis is yet to be established but, even when these activities were determined under near-optimal conditions, it is apparent that their low levels are likely to make a significant contribution to the limitation of alkaloid formation by restricting the supply of putrescine for N-methylation by PMT.…”

Section: Discussionmentioning

confidence: 49%

“…What is apparent, however, is that this is the principal enzyme in the formation of N-methyl putrescine and is present at a level amply sufficient to account for the determined flux. Evidence has recently been presented (Hashimoto et al 1989 a) that in H. albus the alternative pathway via 6-Nmethylornithine (Leete 1979) is unlikely to contribute significantly to the supply of this key intermediate, the first metabolite committed to the alkaloidal pathway. On the other hand, the levels of ODC and ADC are each some fivefold lower than that of PMT and only represent, in combination, about a fourfold excess over the production of hygrine-derived alkaloids.…”

Section: Discussionmentioning

confidence: 99%

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Factors regulating tropane-alkaloid production in a transformed root culture of a Datura candida � D. aurea hybrid

Robins

Parr

Payne

et al. 1990

Planta

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Using in combination an analysis of (i) the levels of enzyme activities present, (ii) the pool sizes of metabolic intermediates and end products and (iii) the effects of feeding metabolic intermediates, the limitations ℴ flux into tropane alkaloids in a Datura root culture have been examined. This culture, produced by transforming a Datura candida × D. aurea hybrid with Agrobacterium rhizogenes, is found to be highly competent in the biosynthesis of both hyoscyamine and scopolamine as well as a wide range of other hygrine-derived alkaloids. It has been found that, of six enzymes which are involved in this pathway, the two initial activities, ornithine decarboxylase (EC 4.1.1.17) and arginine decarboxylase (EC 4.1.1.19), are present at potentially flux-limiting levels, in contrast to those other enzymes assayed which act further down the pathway. An additional limitation to flux, involving the supply of activated acids for condensation with tropine to form the identified tropoyl and tigloyl derivatives, is also indicated from the observed effect of feeding free acids. The relative contribution to flux limitation caused by these two interacting phenomena is inferred from an analysis of the changing relative levels of metabolic intermediates and end products as cultures mature.

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Section: Discussionmentioning

confidence: 49%

Section: Discussionmentioning

confidence: 99%

Factors regulating tropane-alkaloid production in a transformed root culture of a Datura candida � D. aurea hybrid

Robins

Parr

Payne

et al. 1990

Planta

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“…Mass spectra were obtained with an API165 (Perkin-Elmer) after reverse-phase HPLC separation [mobile phase, linear gradient of 10-70% acetonitrile͞H 2 O containing 1% acetic acid for 15 min; column, TSK-gel ODS-80 (Toyo-Soda; 3.9 ϫ 280 mm); flow rate, 1.0 ml͞min], with enzymatically prepared columbamine Biosynthetic pathways of tropane alkaloids and nicotine. Tropane alkaloids and nicotine are derived from diamine putrescine produced from ornithine by ornithine decarboxylase (ODC), arginine, or both (28,29). Putrescine is N-methylated by PMT then oxidatively deaminated by diamine oxidase to the 1-methyl-⌬ 1 -pyrrolinium cation (30).…”

Section: H-labeled Methyl Group Of S-adenosyl-l-[methyl-mentioning

confidence: 99%

Metabolic engineering of plant alkaloid biosynthesis

Sato

Hashimoto

Hachiya

et al. 2000

Proc. Natl. Acad. Sci. U.S.A.

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241

114

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Plant alkaloids, one of the largest groups of natural products, provide many pharmacologically active compounds. Several genes in the biosynthetic pathways for scopolamine, nicotine, and berberine have been cloned, making the metabolic engineering of these alkaloids possible. Expression of two branching-point enzymes was engineered: putrescine N-methyltransferase (PMT) in transgenic plants of Atropa belladonna and Nicotiana sylvestris and (S)-scoulerine 9-Omethyltransferase (SMT) in cultured cells of Coptis japonica and Eschscholzia californica. Overexpression of PMT increased the nicotine content in N. sylvestris, whereas suppression of endogenous PMT activity severely decreased the nicotine content and induced abnormal morphologies. Ectopic expression of SMT caused the accumulation of benzylisoquinoline alkaloids in E. californica. The prospects and limitations of engineering plant alkaloid metabolism are discussed.berberine ͉ nicotine ͉ polyamine ͉ sanguinarine ͉ scopolamine H igher plants constitute one of our most important natural resources. They provide not only foodstuffs, fibers, and woods, but many chemicals, such as oils, flavorings, dyes, and pharmaceuticals. Although plants are renewable resources, some species are becoming more difficult to obtain in sufficient amounts to meet increasing demands. Destruction of natural habitats and technical difficulties in cultivation also are driving the drastic reductions in plant availability. For example, it is claimed that the demand for paclitaxel, a potent anticancer compound, could endanger forests of Taxus brevifolia (Pacific yew) because of the low paclitaxel content (40-100 mg͞kg of bark) in and slow growth of the trees (1).For many natural chemicals it is possible to synthesize alternatives from petroleum, coal, or both. The economic limitations of chemical synthesis and the pollution that accompanies this type of chemical synthesis, however, have led to the development of cell culture and molecular engineering of plants for the production of important and commodity chemicals. Plant cell and organ culture offer promising alternatives for the production of chemicals because totipotency enables plant cells and organs to produce useful secondary metabolites in vitro (2). Cell culture is also advantageous in that useful metabolites are obtained under a controlled environment, independent of climatic changes and soil conditions. In addition, the products are free of microbe and insect contamination. Fermentation technology also can be used to produce desired metabolites and can be optimized to maintain high and stable yields of known quality by cellular and molecular breeding techniques to further improve productivity and quality. After extensive empirical trials, some metabolites are now being produced by large-scale cell culture (e.g., shikonin and berberine; ref. 2), but the numbers of compounds that are producible commercially by cell culture technology are still very few. The main limitations are low productivity and the necessity of the down-stream pr...

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“…1). Putrescine (PUT) is a common precursor of both polyamines such as spermidine (SD) and spermine (SM) and tropane/pyridine alkaloids (Guggisberg and Hesse 1983;Hashimoto et al 1989;Hibi et al 1994). Putrescine N-methyltransferase (PMT; EC.2.1.1.53) is the enzyme involved in the removal of PUT from the polyamine pool, since it catalyzes the N-methylation of this diamine to form N-methylputrescine (MPUT).…”

Section: Introductionmentioning

confidence: 99%

Tropane alkaloids production in transgenic Hyoscyamus niger hairy root cultures over-expressing Putrescine N-methyltransferase is methyl jasmonate-dependent

Zhang

Yang

Lü

et al. 2006

Planta

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The cDNA from Nicotiana tabacum encoding Putrescine N-methyltransferase (PMT), which catalyzes the first committed step in the biosynthesis of tropane alkaloids, has been introduced into the genome of a scopolamine-producing Hyoscyamus niger mediated by the disarmed Agrobacterium tumefaciens strain C58C1, which also carries Agrobacterium rhizogenes Ri plasmid pRiA4, and expressed under the control of the CaMV 35S promoter. Hairy root lines transformed with pmt presented fivefold higher PMT activity than the control, and the methylputrescine (MPUT) levels of the resulting engineered hairy roots increased four to fivefold compared to the control and wild-type roots, but there was no significant increase in tropane alkaloids. However, after methyl jasmonate (MeJA) treatment, a considerable increase of PMTase and endogenous H6Hase as well as an increase in scopolamine content was found either in the transgenic hairy roots or the control. The results indicate that hairy root lines over-expressing pmt have a high capacity to synthesize MPUT, whereas their ability to convert hyoscyamine into scopolamine is very limited. Exposure to MeJA strongly stimulated both polyamine and tropane biosynthesis pathways and elicitation led to more or less enhanced production simultaneously.

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Putrescine and putrescine N-methyltransferase in the biosynthesis of tropane alkaloids in cultured roots of Hyoscyamus albus

Cited by 75 publications

References 33 publications

Factors regulating tropane-alkaloid production in a transformed root culture of a Datura candida � D. aurea hybrid

Factors regulating tropane-alkaloid production in a transformed root culture of a Datura candida � D. aurea hybrid

Metabolic engineering of plant alkaloid biosynthesis

Tropane alkaloids production in transgenic Hyoscyamus niger hairy root cultures over-expressing Putrescine N-methyltransferase is methyl jasmonate-dependent

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