Paulo R. H. Moreno

Cell and tissue cultures of Catharanthus roseus: A literature survey

The literature concerning the regulation and the biosynthesis of secondary metabolites in cell and tissue cultures of Catharanthus roseus is reviewed. The aim of this review is to summarise the progress achieved since the previous review of this subject from 1988 to December 1993. Several factors influencing the production of indole alkaloids are discussed. Special attention is given to large-scale cultivation methods. Some economic considerations on the production of ajmalicine are also discussed.

Effects of elicitation on different metabolic pathways in Catharanthus roseus (L.)G.Don cell suspension cultures

Abstract Cell suspension cultures of Catharanthus roseus were elicited with an-autoclaved cell-free filtrate of Pythium aphanidermatum . The regulation of alkaloid, terpenoid, and phenolic biosynthetic pathways was studied by product formation and assay of the enzyme activity of anthranilate synthase (AS), tryptophan decarboxylase (TDC), strictosidine synthase (SSS), strictosidine-β-glucosidase (SG), isopentenyl diphosphate isomerase (IPP-isomerase), geraniol-10-hydroxylase (G10H), chorismate mutase (CM), and phenylalanine ammonia lyase (PAL). After elicitation, AS and TDC activities were induced which resulted in an increase in the amount of tryptamine in the cells. For SSS and SG, no significant induction was observed. Ajmalicine accumulation was not increased compared with that in controls. An increased amount of phenolic compounds was found in the culture medium, although CM activity was not induced and PAL activity was inhibited after elicitor treatment. The activities of the enzymes IPP-isomerase and G10H were inhibited after elicitation. Also, the incorporation of [ 14 C]IPP in terpenoid products such as squalene was inhibited. These findings indicate a limitation in the terpenoid pathway which could promote a shortage of secoiridoid precursors for terpenoid indole alkaloid biosynthesis.

Elicitor-mediated induction of isochorismate synthase and accumulation of 2,3-dihydroxy benzoic acid in Catharanthus roseus cell suspension and shoot cultures.

After elicitation, cell suspension cultures of Catharanthus roseus accumulate phenolic compounds. The major phenolic compound produced was isolated and identified as 2,3-dihydroxybenzoic acid (DHBA). The accumulation of this compound is a rapid response to the addition of elicitor; within 6 h after the addition of elicitor, DHBA concentration reached 6.3 mg/l cell suspension. DHBA was not detected in non-elicited cells. The formation of DHBA in elicited cells was correlated with the induction of the enzyme isochorismate synthase (ICS). Shoot cultures of C. roseus also presented a strong induction of ICS after elicitation. Due to its biological activity, DHBA could play a role in the defence mechanism of C. roseus.

Induction of ajmalicine formation and related enzyme activities in Catharanthus roseus cells: effect of inoculum density

In Catharanthus roseus cell cultures the time courses of four enzyme activities, tryptophan decarboxylase (TDC), strictosidine synthase (SSS), geraniol-10-hydroxylase (G10H) and anthranilate synthase (AS), and alkaloid accumulation were compared under two different culture conditions (low-inoculum density and high-inoculum density on induction medium) and a control on growth medium. In growth medium a transient increase in TDC activity was first observed after which G10H reached its maximum activity; only tryptamine accumulated, no ajmalicine could be detected. Apparently, a concerted induction of enzyme activities is required for ajmalicine formation. Cells inoculated in induction medium showed such a concerted induction of AS, TDC and G10H activities. After 30 days the low-density culture had accumulated six times more ajmalicine (in μmoles/g) than the high-density culture. Thus, increase in biomass concentration (high-density cultures) did not enhance the total alkaloid production. The major differences observed in enzyme levels between high-and low-density cultures were in the AS and TDC activities, which were two to three times higher in the low-density culture, indicating that there is a positive correlation between ajmalicine formation and AS and TDC activities.

Ajmalicine production by cell cultures of Catharanthus roseus: from shake flask to bioreactor

The productivity of a cell culture for the production of a secondary metabolite is defined by three factors: specific growth rate, specific product formation rate, and biomass concentration during production. The effect of scaling-up from shake flask to bioreactor on growth and production and the effect of increasing the biomass concentration were investigated for the production of ajmalicine by Catharanthus roseus cell suspensions. Growth of biomass was not affected by the type of culture vessel. Growth, carbohydrate storage, glucose and oxygen consumption, and the carbon dioxide production could be predicted rather well by a structured model with the internal phosphate and the external glucose concentration as the controlling factors. The production of ajmalicine on production medium in a shake flask was not reproduced in a bioreactor. The production could be restored by creating a gas regime in the bioreactor comparable to that in a shake flask. Increasing the biomass concentration both in a shake flask and in a stirred fermenter decreased the ajmalicine production rate. This effect could be removed partly by controlling the oxygen concentration in the more dense culture at 85% air saturation.

Chapter 3 Biosynthesis of Terpenoid Indole Alkaloids in Catharanthus roseus Cells

Publisher Summary This chapter studies the biosynthesis of the terpenoid indole alkaloids in C. roseus. On the level of the enzymes, certain steps have now been well characterized, but others remain unknown. The conversion of loganin into secologanin is one of the intriguing unresolved problems, although it is not a rate-limiting step. Considering the activities of the enzymes tryptophan decarboxylase (TDC), strictosidine synthase (SSS), and strictosidine glucosidase (SG) in cultured C. roseus cells, it is found that the actual production of alkaloids is considerably lower than that which could have been produced by these enzymes. The chapter deals with alkaloid biosynthesis in C. roseus; the biochemistry of this plant has also been studied for other aspects, such as anthocyanin production, phosphate metabolism, cell growth, and cell division cycle. Most of the studies concerning the primary metabolism are not linked with those of secondary metabolism. However, one may expect that in the future the studies on secondary metabolism, such as chorismate-derived products and terpenoid-derived products such as the alkaloids, will be integrated.

Determination of the activity of the cytochrome p450 enzyme geraniol 10‐hydroxylase in plants by high‐performance liquid chromatography

The cytochrome P450 enzyme geraniol 10-hydroxylase plays an important role in the biosynthesis of pharmaceutically important alkaloids in Catharanthus roseus. An HPLC assay was developed for this enzyme based on the UV detection of the product 10-hydroxygeraniol after its separation from the substrate geraniol on a reversed-phase C-18 column. Furthermore, this system can be used for the UV detection of nerol, which is also a substrate for geraniol 10-hydroxylase, and its product 10-hydroxynerol. The presence of interfering enzymes which epoxidize rather than hydroxylate geraniol and nerol could also be detected. The developed HPLC assay was validated with respect to the incubation time (linear up to 45 min) and the amount of protein added per incubation (linear up to 400u2005µg of protein). The HPLC assay will be a useful tool during the purification of geraniol 10-hydroxylase from cell suspension cultures of Catharanthus roseus. Copyright