Alkaloid Production by Cell Cultures

Alkaloid Production by Cell Cultures 1. Growth Conditions 

Although very low levels of alkaloids are produced by the Cinchona cell cultures, they are capable of producing considerable amounts of anth-raquinones, particuleu-ly after elicitation with fungal elicitors. The anth-raquinones are thought to act as phytoalexins in this plant genus (575). Anthraquinone production could be stimulated by adding polymeric adsorbents like Amberlite XAD-7 to the medium. A production rate of 20 mg/liter/day could be obtained in this way (576). 

Efficient immobilization of C. pubescens cells is possible in a semirigid matrix of polyurethane foam particles (107,162). The influence of immobilization on alkaloid production has not been studied. 

Permeabilization of C. ledgeriana cells to promote release of alkaloids is possible with DMSO. However, for complete release of the alkaloids DMSO levels of 20% are necessary, and at these levels cells are severely damaged, hampering cell recovery after treatment (553). Fineilly, cell cultures might excrete up to 70% of the alkaloids into the medium (561). 

Addition of XAD-7 to the medium did not stimulate total alkaloid production, but most of the alkaloids produced were excreted (80-90%) 42). Hairy root cultures released only about 1% of the alkaloids into the medium 574). 

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Figure 96. Diagram of alkaloid production by cell culture. Abbreviations A - alkaloid synthesis.

CANEL, C., LOPES-CARDOSO, M.I., WH1TMER, S VAN DER FITS, L., PASQUALI, G., VAN DER HEIJDEN, R HOGE, J.H., VERPOORTE, R., Effects of over-expression of strictosidine synthase and tryptophan decarboxylase on alkaloid production by cell cultures of Catharanthus roseus. Planta, 1998,205, 414-419. 

Canel C et al (1998) Effects of over-expression of strictosidine synthase and tryptophan decarboxylase on alkaloid production by cell cultures of Catharanthus roseus. Planta 205 414 19... 

The hybrid is able to produce more alkaloids than the basic callus, which is an undifferentiated mass of cells. Alkaloid production in cell cultures can be more successful with the immobihzation of plant cells and enzymes and by using bioreactor systems . Alkaloid produced in cell cultures can be isolated directly from this culture or from young plants grown from this culture. More than 250 alkaloids are reported to be produced by cell-culture techniques. Only a limited number of species have been researched in this respect. The species studied are known to produce alkaloids with special use in applications. The most researched alkaloids produced by cell cultures are mentioned in Table 25. 

Some plant cells can be encouraged to grow in vitro under specific controlled conditions, which can be used to produce secondary plant metabolites at higher yields than those found in plants, but at a high cost. There are few examples where the technological difficulties and costs involved have led to commercialisation currently, but the valuable pharmaceutical alkaloid berberine, the quinnone shikonin and the terpenoid paclitaxel are all potential candidates for production by cell culture. 

Elicitation is effective in enhancing metabolite synthesis in some cases, such as in production of paclitaxel by Taxus cell suspension cultures [2] and tropane alkaloid production by suspension cultures of Datura stramonium [3]. Increasing the activity of metabolic pathways by elicitation, in conjunction with end-product removal and accumulation in an extractive phase, has proven to be a very successful strategy for increasing metabolite productivity [4]. For example, two-phase operation with elicitation-enhanced alkaloid production in cell suspension cultures of Escherichia californica [5,6]. 

Alkaloid Plant source Price (DM/gram) Production by cell cultures Source Yield ... 

The development of plant cell biotechnological production of berberine by Coptis cells seems so far to be quite successful. Yields have been improved from a meager 2.4 mg of berberine hydrochloride isolated from 3.8 g of callus (0.06% of DW) (512) to 7 g/liter (10% of DW) (59), that is, an increase of more than 150 times the alkaloid levels in the cells. Several aspects can be pointed out that have contributed to this. First, all callus cultures induced do produce the desired product. Further, because of the yellow color of the alkaloid subsequent selection is easy to perform visually. Finally, the alkaloid production of cell cultures proved to be stable after repetitive selection. 

Akad. Wissenschaft DDR. DD-205-184-A. 26-04-1982-DD-239293 (21-12-1983). Cinchona alkaloid production by cell or callus culture giving production free of plant material. 

Anderson, L. A., Phillipson, J. D. and Roberts, M. F. 1987. Alkaloid production by plant cells. In Plant and Animal Cell Cultures, Process, Possibilities (Webb, C. and Mavituna, F. eds.), pp. 172-192. Chichester Ellis Horwood. 

Villegas, M., Leon, R. and Brodelius P. E. 1999. Effects of alginate and immobilization by entrapment in alginate on benzophenanthridine alkaloid production in cell suspension cultures of Eschscholtzia californica. Biotechnology Letters, 21(1) 49-55. 

In vitro tissue and cell cultures of lupin plants are not appropriate systems for the study of biosynthesis of lupin alkaloids, because the production ability by in vitro culture is rather low, i.e., 10 2 to lO times compared with that of differentiated plants. The production of the alkaloids of lupinine- and sparteine-groups by cell culture have been reported by us [59] and by Wink s group [60]. We have also successfully produced matrine in green callus culture and in multiple shoots of Sophora flavescens [61]. The producibility of matrine was positively correlated with the chloroplast formation. This indicates that the formation of carbon skeleton of matrine-type alkaloids also likely takes place in chloroplasts in plant cells as postulated in that of sparteine-type alkaloids [62]. 

The synthesis of indole alkaloids from the common precursors tryptamine and secologanin by cell cultures of C, roseus has been studied in detail (IT). Cells entrapped in agar, agarose, or carrageenan produce ajmalicine isomers at about the same rate as the freely suspended cells, while cells entrapped in alginate showed up to 160 fold increased synthesis. A major reason for this increased synthesis is believed to be the restricted growth of the entrapped cells, as has been discussed above for Capsiaum. The yield of ajmalicine is above 12 times as much product formed from added precursors as by de novo synthesis. In this case too, alginate entrapped cells were much more productive than freely suspended cells (140% increase), and were also used to synthesize the related alkaloid serpentine in a batch procedure (17). 

The production of alkaloids in plant cell cultures is a result of an enormously complex set of interactions between cellular and extracellular compartments. The extracellular compartment should at least offer possibilities for survival of the cellular compartment, but often cell growth and cell differentiation are prerequisites. The cellular compartment, however, is continuously changing the extracellular compartment by uptake of nutri-... 

The internal storage capacity is limited by the number of storage cells. Excreted alkaloids which are dissolved in the medium are exposed to catalytic activities in the medium (e.g., peroxidases) and can thus be degraded as was demonstrated for quinolizidine alkaloids in lupine cell cultures (92). A role for peroxidases in product degradation has been recently demonstrated for indole alkaloids produced by suspension cultures of Tabemaemontana. Reduction of peroxidase activity by removal of Ca ions from the medium resulted in the formation of the alkaloid... 

In the following ptu-agraphs this approach will be applied to production of an alkaloid from plant cells cultured in a bioreactor. Regime analysis can be performed by comparison of characteristic parameters of the mechanisms involved in the process. Here the characteristic time concept will be used. The characteristic time is a measure for the rate of a mechanism. A fast mechanism has a short characteristic time. Other terms used are relaxation time, process time, or time constant. A time constant is formally only defined for first-order linear processes. Not all mechanisms involved in a plant cell production process are first order, therefore the term characteristic time is used. The characteristic time is defined as the ratio of a capacity and a flow for example, the characteristic time for oxygen transfer to the liquid phase in a aerated bioreactor q.l becomes... 

Production of Alkaloids by Cell Cultures of Papaver Species ... 

A study on alkaloid production by nongrowing (owing to the absence of 2,4-D) cell suspension cultures of C. roseus in a continuous flow reactor with cell retention was performed by Pareilleux and Vinas (626). It was shown by the authors that part of the produced alkaloids were excreted into the culture medium. Although the continuous flow system seemed to be suitable for secondary metabolite production, the reported specific production rate was relatively low (0.012 mg ajmalicine/g DW/day). To obtain ajmalicine production 1.2 mM tryptamine had to be added to the infiuent medium. The duration of the continuous flow experiment was only... 

The possibilities of surface immobilization of C. roseus cells was investigated by Archambault et al. (630), Facchini and Dicosmo (631), and Rho et al. (632). Archambault et al. (630) used a fibrous polyester sheet and studied the effect of surface immobilization on alkaloid production. The cells were grown on the polyester matrix in shake flasks, a stirred vessel, or an airlift reactor. From the results presented it is clear that surface immobilization had a negative effect on alkaloid production in these experiments (Table XXXIX). Suspension cultures were found to produce significantly higher amounts of alkaloids than surface immobilized cells. Cultivation in bioreactors further diminished alkaloid production. 

Synthelabo. 04-02-1975 FR-003347 (05-08-1976). Alkaloid production by culturing Vinca minor cells in undifferentiated state in liquid medium. 

Modern biotechnological methods of producing potentially useful alkaloids, particularly plant cell culturing, is an area of fundamental importance and has been receiving a lot of attention. An analysis of the state of the art and where it is headed is discussed here by a well-known group from The Netherlands in Plant Biotechnology for the Production of Alkeiloids Present Status and Prospects. ... 

The cell-free synthesis of strictosidine (79) and cathenamine (82) has been further explored, and the conditions under which these key compounds are formed have been optimized. Strains from C. roseus suspension cultures that were resistant to inhibition of their growth by various tryptophan analogues have been selected. The free tryptophan level in cells of these strains could be 30—40 times higher than in normal cells. Tryptophan at this level did not induce tryptophan decarboxylase, nor the production of alkaloids. It is to be noted, however, that stimulation of alkaloid production by tryptophan and tryptamine ° in cultures of normal cells has been reported. In the case of tryptamine the two most prominent metabolites were A/ -acetyltryptamine and JVN-dimethyltrypt-amine. 

Cell mass production and metabolite production by cells developed over the last 50 years. Muir et al. reported the first suspension culture of plant cells and the first cultivation of cells in bioreactors in 1955 by Melchers and Engelmann. The development of bioreactors since this time was extremely fast, from small flask type to multicubic meters. One of the largest bioreactors in the production of alkaloids by transformed hairy roots is reported to be of 500 m in size and has been used in a pilot scale in 1990 by the research group of Wilson. In recent studies, the different bioreactors have been compared to produce galanthamine from shoots of... 

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