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Influence of Precursor Availability on Alkaloid Accumulation by
Transgenic Cell Line of Catharanthus roseus 总被引:7,自引:0,他引:7 下载免费PDF全文
Serap Whitmer Camilo Canel Didier Hallard Cecilia Gon?alves Robert Verpoorte 《Plant physiology》1998,116(2):853-857
We have used a transgenic cell line of Catharanthus roseus (L.) G. Don to study the relative importance of the supply of biosynthetic precursors for the synthesis of terpenoid indole alkaloids. Line S10 carries a recombinant, constitutively overexpressed version of the endogenous strictosidine synthase (Str) gene. Various concentrations and combinations of the substrate tryptamine and of loganin, the immediate precursor of secologanin, were added to suspension cultures of S10. Our results indicate that high rates of tryptamine synthesis can take place under conditions of low tryptophan decarboxylase activity, and that high rates of strictosidine synthesis are possible in the presence of a small tryptamine pool. It appears that the utilization of tryptamine for alkaloid biosynthesis enhances metabolic flux through the indole pathway. However, a deficiency in the supply of either the iridoid or the indole precursor can limit flux through the step catalyzed by strictosidine synthase. Precursor utilization for the synthesis of strictosidine depends on the availability of the cosubstrate; the relative abundance of these precursors is a cell-line-specific trait that reflects the metabolic status of the cultures. 相似文献
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A. Geerlings D. Hallard A. Martinez Caballero I. Lopes Cardoso R. van der Heijden R. Verpoorte 《Plant cell reports》1999,19(2):191-196
Cinchona officinalis 'Ledgeriana', former called Cinchona ledgeriana, hairy roots were initiated containing constitutive-expression constructs of cDNAs encoding the enzymes tryptophan decarboxylase
(TDC) and strictosidine synthase (STR) from Catharanthus roseus, two key enzymes in terpenoid indole and quinoline alkaloid biosynthesis. The successful integration of these genes and the
reporter gene gus-int was demonstrated using Southern blotting and the polymerase chain reaction. The products of TDC and STR, tryptamine and
strictosidine, were found in high amounts, 1200 and 1950 μg g–1 dry weight, respectively. Quinine and quinidine levels were found to rise up to 500 and 1000 μg g–1 dry weight, respectively. The results show that genetic engineering with multiple genes is well possible in hairy roots of
C. officinalis. However, 1 year after analyzing the hairy roots for the first time, they had completely lost their capacity to accumulate
alkaloids.
Received: 15 October 1997 / Accepted after revision: 21 March 1999 相似文献
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E. Lucumi M. Luczkiewicz A. Vera D. Hallard R. van der Heijden R. Verpoorte 《Biotechnology letters》2001,23(20):1691-1696
A cell suspension culture of Tabernaemontana divaricata, that had lost alkaloid production, was still capable of producing a similar pattern of alkaloids as directly after its initiation. When fed with early precursors, such as tryptamine and loganin, 57% of the precursors was converted into indole alkaloids such as strictosidine, vallesamine, O-acetylvallesamine and voaphylline. Apparently most of the cell factory has remained stable during the many years of subculturing. Only an early step of the biosynthesis the flux seems to be diverted to other pathways. 相似文献
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Lucumi E. Vera A. Hallard D. van der Heijden R. Verpoorte R. 《Plant Cell, Tissue and Organ Culture》2002,68(3):293-299
A cell suspension culture of Tabernaemontana elegans lost its ability to produce alkaloids after a prolonged period of subculture. To determine whether it was still capable of performing the later steps of the alkaloid biosynthetic pathway, the culture was fed with tryptamine and loganin. The precursors and alkaloids were determined in the biomass and in the medium during a growth cycle. In this culture, an increase in the amount of serotonin was found in the biomass after feeding of tryptamine and loganin. Secologanin was detected in small amounts but strictosidine was not. Therefore, a limitation in alkaloid formation in this T. elegans cell line occured in the formation of secologanin from loganin. After feeding of secologanin alone, strictosidine, 10-hydroxy strictosidine, strictosidinic acid and two other indole alkaloids, as yet unidentified, were formed. However, the alkaloids originally produced by this cell line were not found. As the biosynthesis is impaired at several steps, it seems that the loss of productivity is more likely to be to a change on the level of the regulation of the pathway, than due to the loss of the capacity to express an individual biosynthetic gene of the pathway. 相似文献
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D. Hallard R. van der Heijden R. Verpoorte M. I. Lopes Cardoso G. Pasquali J. Memelink J. H. C. Hoge 《Plant cell reports》1997,17(1):50-54
A transgenic cell suspension culture of Nicotiana tabacum L. `Petit Havana' SR1 was established expressing tryptophan decarboxylase and strictosidine synthase cDNA clones from Catharanthus roseus (L.) G. Don under the direction of cauliflower mosaic virus 35S promoter and nopaline synthase terminator sequences. During
a growth cycle, the transgenic tobacco cells showed relatively constant tryptophan decarboxylase activity and an about two-
to sixfold higher strictosidine synthase activity, enzyme activities not detectable in untransformed tobacco cells. The transgenic
culture accumulated tryptamine and produced strictosidine upon feeding of secologanin, demonstrating the in vivo functionality
of the two transgene-encoded enzymes. The accumulation of strictosidine, which occurred predominantly in the medium, could
be enhanced by feeding both secologanin and tryptamine. No strictosidine synthase activity was detected in the medium, indicating
the involvement of secologanin uptake and strictosidine release by the cells.
Received: 25 February 1996 / Revision received: 16 August 1996 / Accepted: 30 September 1996 相似文献
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Frédérique Garnier Philippe Label Didier Hallard Jean-Claude Chénieux Marc Rideau Saïd Hamdi 《Plant Cell, Tissue and Organ Culture》1996,45(3):223-230
Cytokinins play a critical role in several aspects of plant growth, metabolism and development. We previously reported that adding cytokinins to the culture medium of a suspension-cultured cell line of periwinkle increased the accumulation of indole alkaloids, and our aim was to compare the effect of exogenously-applied cytokinins with that of elevated levels of endogenous cytokinins on indole alkaloid production. We used an Agrobacterium tumefaciens strain yielding a plasmid with the isopentenyl transferase gene under control of its own promoter. Co-culture of suspension cells with the bacteria caused a severe stress response leading to cell necrosis. Therefore, we failed to transform this material but we succeeded in transforming periwinkle cotyledons. We verified that callus cultures generated from the isopentenyl transferase-transgenic cotyledons accumulated high cytokinin concentrations. Treating normal callus cultures (generated from untransformed cotyledons) with cytokinins enhanced their alkaloid production. By contrast, the enhanced concentration of endogenous cytokinins in transgenic calli did not increase indole alkaloid production, and thus did not mimic the effect of exogenously-applied cytokinins. Hypothesis to explain this discrepancy are discussed.Abbreviations 2,4-d
2,4-dichlorophenoxyacetic acid
- DW
dry cell mass
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ipt
isopentenyl transferase gene 相似文献
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