Yukihito Yukimune

Tropane alkaloid production in Hyoscyamus root cultures

Abstract Tropane alkaloid production was studied in callus and root cultures of seven Hyoscyamus species. Cultured roots grew well (2- to 6-fold in 8 days) without addition of growth regulators. Hyoscyamine content in these roots varied from 0.04 % DW to more than 1.1 % DW, and scopolamine content from 0.06 % DW to 0.3 % DW, depending on the plant species. Calli grew slowly (2- to 7-fold in 4 weeks) and had much lower alkaloid content than their respective root cultures. Hyoscyamine was the main alkaloid in all callus cultures. Alkaloid production in root cultures of H. niger and H. albus was studied under various culture conditions. When roots were cultured in medium without growth regulators, the main alkaloids produced were scopolamine (0.2% DW) in H. niger and hyoscyamine (0.5 to 1.2% DW) in H. albus . In H. albus root cultures, an inverse relationship between growth and hyoscyamine content was found. Auxin, especially IBA at 10 −6 M to 10 −4 M, stimulated growth of the root cultures, which was accompanied by increased lateral root induction. Alkaloid biosynthesis, however, was inhibited as auxin concentration increased. In H. niger root culture, scopolamine formation was inhibited by auxin more strongly than hyoscyamine formation. These effects of auxin on growth and alkaloid biosynthesis were confirmed in Datura , Atropa and Duboisia root cultures. Effects of other growth regulators and several basal media were also examined.

Putrescine and putrescine N-methyltransferase in the biosynthesis of tropane alkaloids in cultured roots of Hyoscyamus albus : I. Biochemical studies.

The activity of arginine decarboxylase (EC 4.1.1.19) in cultured roots of Hyoscyamus albus L., which produce considerable amounts of tropane alkaloids, was twice that of ornithine decarboxylase (EC 4.1.1.17), both activities being highest during active root growth, whereas arginase (EC 3.5.3.1) activity was negligible. Actively growing roots had putrescine conjugates as their major polyamines, and spermidine was the most abundant free polyamine. Putrescine N-methyltransferase (PMT; EC 2.1.1.53) activity was high, the peak occurring on the sixth day of culture when root growth became slower. Thereafter, the free N-methylputrescine content of the roots increased and was followed by an increase in alkaloid content (mostly hyoscyamine). The amounts of arginine and, especially, of ornithine were low. No N-methylornithine was detected. The PMT activity was present only in root, shoot and cell-suspension cultures of plants that synthesized tropane alkaloids or nicotine; no enzyme activities that methylate ornithine at the δ-amino group or that decarboxylate δ-N-methylornithine were detected in any of the cultures tested. Our data indicate that tropane alkaloids in H. albus roots are synthesized by way of the symmetrical putrescine, i.e. a pathway different from that proposed by E. Leete (1962, J. Am. Chem. Soc. 84, 55) according to which these alkaloids are synthesized by way of asymmetrical δ-N-methylornithine.

Putrescine and putrescine N-methyltransferase in the biosynthesis of tropane alkaloids in cultured roots of Hyoscyamus albus : II. Incorporation of labeled precursors.

Abstract[1,4-14C]Putrescine, administered to cultured roots of Hyoscyamus albus L., was converted to spermidine and spermine as well as to conjugated forms. A substantial fraction of its radioactivity (approx. 6%) was, however, incorporated into N-methylputrescine within 4 h, followed by an increase in radioactive tropane alkaloids (mostly hyoscyamine). Incubation of the roots with dl-[5-14C]ornithine and l-[2,3-3H]arginine separately resulted in rapid incorporation of each label into free putrescine and N-methylputrescine, followed by incorporation into hyoscyamine. During active alkaloid biosynthesis in H. albus roots, scarcely any radioactivity was recovered in δ-N-methylornithine. When the roots had been treated with 5 mM dl-α-difluoromethylornithine for 1 d prior to the administration of labeled ornithine, the total uptake of radioactivity into the root cells was not reduced, even though ornithine decarboxylase was specifically inactivated and the incorporation of the label to putrescine, N-methylputrescine, hyoscyamine and scopolamine markedly reduced. These and other results (Hashimoto et al., 1989, Planta 178, 123–130) are strong indications that tropane alkaloids in H. albus root cultures are synthesized from both ornithine and arginine by way of putrescine.

The configuration of methyl jasmonate affects paclitaxel and baccatin III production in Taxus cells.

All stereoisomers of methyl jasmonate (MJA) were prepared, and their effects on cell yield and promotion of paclitaxel (Taxol) and baccatin III production investigated in cell suspension cultures of Taxus media. (3R,7S)-MJA showed the strongest cell growth inhibition, followed by (3R,7R)-MJA. In contrast, (3S,7R)- and (3S,7S)-MJA had very low inhibitory effects, indicating that this inhibition depends largely on the (3R)-configuration. In terms of the promotion of paclitaxel and baccatin III production, (3R,7R)-MJA had the highest activity. Although it showed considerable activity at low concentration, at higher concentrations the activity was decreased due to strong inhibition of cell growth. Interestingly, paclitaxel and baccatin III contents increased even at a high (3S,7R)-MJA concentration, whereas the other isomers had the opposite effects. These findings are interpreted to suggest that the optimum configuration is (3R,7R), the (3R)-configuration not being indispensable, and that the (7R)-configuration is suitable for the promotion of paclitaxel and baccatin III production.