Abstract
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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Abbreviations
- ADC:
-
arginine decarboxylase
- GC-MS:
-
gas chromatography-mass spectrometry
- HPLC:
-
high-performance liquid chromatography
- H6H:
-
hyoscyamine 6-β-hydroxylase
- MPO:
-
N-methyl putrescine oxidase
- NMR:
-
nuclear magnetic resonance
- ODC:
-
ornithine decarboxylase
- PMT:
-
putrescine N-methyl transferase
- TLC:
-
thin-layer chromatography
- TDH:
-
tropinone dehydrogenase
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We are grateful to a number of people for assistance with parts of the work presented here. Judy Furze, Liz Bent, Martin Hilton and Richard Hulme helped with some of the experimentation. Dr. I. Colquhoun ran the NMR spectra and John Eagles the GC-MS analyses. We are particularly grateful to Dr. J. Woolley (School of Health and Life Sciences, Leicester Polytechnic, Leicester, UK) for discussions on the mechanism of tropic-acid formation and for kindly supplying an authentic sample of littorine. We are especially grateful to Dr. W.C. Evans (Buddlehayes, Southleigh, Devon, UK) for supplying us with his Datura hybrids and a number of authentic alkaloids. His comments on the manuscript were much appreciated.
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Robins, R.J., Parr, A.J., Payne, J. et al. Factors regulating tropane-alkaloid production in a transformed root culture of a Datura candida × D. aurea hybrid. Planta 181, 414–422 (1990). https://doi.org/10.1007/BF00195896
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DOI: https://doi.org/10.1007/BF00195896