Phytochemistry Reviews 1: 1325, 2002. 2002 Kluwer Academic Publishers. Printed in the Netherlands.

13

Biotechnology for the production of plant secondary metabolites

R. Verpoorte1, , A. Contin1 & J. Memelink2
of Pharmacognosy, Leiden/Amsterdam Center for Drug Research, Leiden University, Leiden, The Netherlands; 2 Institute of Plant Molecular Sciences, Leiden University, Leiden, The Netherlands; Author for correspondence (E-mail: Verpoort@LACDR.LeidenUniv.NL)
1 Division

Key words: biosynthetic pathway mapping, economic feasibility, large-scale plant cell cultures, metabolic engineering, optimization productivity, regulation biosynthesis, terpenoid indole alkaloids

Abstract The production of plant secondary metabolites by means of large-scale culture of plant cells in bioreactors is technically feasible. The economy of such a production is the major bottleneck. For some costly products it is feasible, but unfortunately some of the most interesting products are only in very small amounts or not all produced in plant cell cultures. Screening, selection and medium optimization may lead to 20- to 30-fold increase in case one has producing cultures. In case of phytoalexins, elicitation will lead to high production. But for many of the compounds of interest the production is not inducible by elicitors. The culture of differentiated cells, such as (hairy) root or shoot cultures, is an alternative, but is hampered by problems in scaling up of such cultures. Metabolic engineering offers new perspectives for improving the production of compounds of interest. This approach can be used to improve production in the cell culture, in the plant itself or even production in other plant species or organisms. Studies on the production of terpenoid indole alkaloids have shown that the overexpression of single genes of the pathway may lead for some enzymes to an increased production of the direct product, but not necessarily to an increased alkaloid production. On the other hand feeding of such transgenic cultures with early precursors showed an enormous capacity for producing alkaloids, which is not utilized without feeding precursors. Overexpression of regulatory genes results in the upregulation of a series of enzymes in the alkaloid pathway, but not to an improved ux through the pathway, but feeding loganin does result in increased alkaloid production if compared with wild-type cells. Indole alkaloids could be produced in hairy root cultures of Weigelia by overexpression of tryptophan decarboxylase and strictosidine synthase. Alkaloids could be produced in transgenic yeast overexpressing strictosidine synthase and strictosidine glucosidase growing on medium made out the juice of Symphoricarpus albus berries to which tryptamine is added. Metabolic engineering thus seems a promising approach to improve the production of a cell factory. Abbreviations: CaMV cauliower mosaic virus; DW dry weight; FW fresh weight; SGD strictosidine glucosidase; STR strictosidine synthase; TDC tryptophan decarboxylase Introduction Secondary metabolism in a plant plays a major role in the survival of the plant in its environment. Attraction of pollinators, defence against predators and diseases, etc., are examples of the roles of secondary metabolites (Harborne, 1978; Wink, 1988). The secondary metabolites formed also are an important trait for our food plants (taste, colour, scent, etc.) and ornamental plants. Moreover, numerous plant secondary metabolites such as alkaloids, anthocyanins, avonoids, quinones, lignans, steroids, and terpenoids have found commercial application as drug, dye, avour, fragrance, insecticide, etc. Such ne chemicals are extracted and puried from plant materials. Some are used in large amounts like for example

14 quinine and hop bitter acids with a world market of respectively ca. 500 and 7,500 tonnes a year. Others such as vincristine and paclitaxel have a world market of just a few kilograms. Obviously prices will be linked with the total market volume, the latter compounds have prices in the range of US $ 5,000 per gram and the former of US $ 100 per kilogram. Production by plants is not always satisfactory. It is often restricted to a species or genus and might be activated only during a particular growth or developmental stage, or under specic seasonal, stress or nutrient availability conditions. For example some plants are difcult to cultivate, necessitating collection in the wild and thus the risk of the plant getting extinct. Or plants may grow very slowly, like Cinchona trees that need about 10 years before ready for harvesting, thus requiring long term planning of possible market demands. For pharmaceutical products there is also the need for a production that falls under GMP-rules. For these reasons in the past years a lot of effort has been put into plant cell cultures as a possible production method for plant secondary metabolites of commercial interest (Berlin, 1986; Verpoorte et al., 1993, 1998; Buitelaar and Tramper,1992; Lipsky, 1992; Su, 1995; Alfermann and Petersen, 1995). In some cases one has been able to come up with industrially feasible processes, like for pure compounds such as shikonin, taxol and berberine, or for biomass as in case of ginseng roots. However, for many of the pharmaceuticals of interest the production is too low or even zero in the cultured cells, despite extensive studies on the optimisation of growth and production media and cell line selection for high producing strains. This is usually due to the fact that production is controlled in a tissue-specic manner, dedifferentiation resulting thus in loss of production capacity. Therefore other approaches such as the growth of differentiated cells (root and shoot cultures) and induction of pathways with various elicitors has been used (for a review see Verpoorte et al., 1991, 1999). Both with limited success so far. The differentiated cells do produce the same products as the plant itself, but the large-scale production remains a bottleneck for the economy of such a production. Elicitation has as major constraint in application, it only activates certain plant speciesspecic pathways, which do not always lead to the product of interest. Therefore in recent years research has focused on the regulation of the biosynthesis, aiming at the possibilities of metabolic engineering. This may be used to improve production of a desired compound in a cell culture, but also in the plant itself, or to achieve production in a related plant species or even in microorganisms.

Production by plant cell cultures The rst questions to be answered for commercial application of plant cell cultures for the production of ne chemicals concern the technological and economical feasibility. In principle plant cell suspension cultures can be obtained from any plant, though some plants are much easier to render cell cultures than others. The major constraint, however, was thought to be the shear sensitivity of plant cells. If compared to microorganisms, plant cells are much bigger, mainly due to the presence of large vacuoles. Plant cells being like a bag of water with a thin cell wall, it was thought that stirring in large fermentors would cause the cells to collapse due to the shear forces produced in stirring the viscous mass of cells (hydrodynamic stress). A study by Wagner and Vogel (1977) in which it was noted that anthraquinone production of a Morinda citrifolia cell line was lower in a stirred fermentor than in a airlift fermentor has been many times cited as the example of the effect of shear forces on cells. However, in this case the effect on cell growth was small. Studies on the effect of shear forces by Meijer et al. (1987) and Leckie et al. (1990) in fact showed that shear sensitivity of plant cells is not such a problem as was thought. In fact plant cells can be grown in stirred tank type of bioreactors, low shear stirrers can be advantageous for more sensitive cell lines. This is also conrmed by the practice, several large-scale experiments have been reported in literature, and commercial production of plant products using stirred tank type of bioreactors has been achieved (Hibino and Ushiyama, 1999; Westphal, 1990). The technology being feasible, the economy of a plant cell biotechnological production is the other important question. Ten Hoopen and co-workers (van Gulik et al. 1988; Verpoorte et al. 1991, 1999) made costs calculations for different types of processes, based on a design for a facility for a plant cell biotechnological production of secondary metabolites. A Catharanthus roseus cell suspension was used as a model, assuming a productivity of 0.3 g/l ajmalicine per 2 weeks, which is about the maximum which has been reported for cell cultures of this plant. A biomass density of 40 g/l (DW) was assumed to be reached in the rst step of the process. After this growth phase of 1 week, the medium was changed into a produc-

15 tion medium containing 8% of sucrose (Knobloch and Berlin, 1980). The cells were harvested after 1 week in the production medium. To produce 3,000 kg of the indole alkaloid ajmalicine 6 bioreactors of 145 m3 are needed. Calculating the costs based on depreciation of the bioreactors, and the costs of media and energy, a price of US $ 1500/kg was concluded. Of this about 65% goes to the depreciation. If the productivity would be increased 10-fold to 3 g/l, a price of US $ 430/kg was calculated. Also the possibilities of a continuous production system were considered. In such a system the cells are kept alive, and the product is collected from the medium. This, however, requires that the product is excreted to free medium outside the cells. In the fed-batch system described above, at a biomass density of 40 g/l there is hardly any free medium. Thus for such a continuous system the biomass density has to be decreased. That requires a larger size of the bioreactors (250 m3 ) and an increase of the depreciation, media and energy costs. In fact this results in a price double of that in the fedbatch culture system. In case that the product is not excreted to the medium, which is most common, an extra step is needed to permeabilize the cells, which even further increases the costs. The conclusion of these calculations was thus that a batch or fed-batch type of cell suspension culture is the most economic. The goal should thus be to improve the productivity of the cells to come to a commercial process. This was for example the case in efforts to select high producing C. roseus cell cultures (Deus-Neumann and Zenk, 1984). Considering all these studies, one may conclude that by this elaborate approach, one may improve productivity with a factor 20-30. But in case that no product is found in the initial cell cultures, this approach will not be successful. For example in case of morphine, vinblastine and vincristine, the production is virtually zero in the cell cultures, all efforts to improve this were not successful. Apparently the biosynthetic pathway is not expressed at all under the culture conditions. Culture of differentiated cells Secondary metabolites are by denition a product of differentiation, in the cell suspension cultures this sort of differentiation does not always occur. For that reason quite some research has been done on the in vitro culture of differentiated cells. Roots, shoots, and embryos have thus been cultured in vitro. As expected, such organ cultures do produce similar patterns of secondary metabolites as the plant. For example the tropane alkaloids hyoscyamine and scopolamine are produced quite well in root cultures (for reviews see, e.g., Oksman-Caldentey et al., 2000; Verpoorte et al., 1991), and essential oils have been produced in shoot cultures (Spencer et al., 1993). By transformation with Agrobacterium rhizogenes so called hairy roots can be obtained, that can grow without plant growth hormones, and have a growth rate, which is similar to cell suspension cultures (Doran, 1997; Giri and Narasu, 2000). Also these hairy roots are good producers of the typical root secondary metabolites, like for example for Hyoscyamus muticus hairy root cultures that are good producers of hyoscyamine and scopolamine (Oksman-Caldentey et al., 2000). Major problem of the organ cultures is the large scale culture. Although all kind of ingenious bioreactors have been described for the culture of roots and/or shoots (Carvalho et al., 1997; Weathers et al., 1997; Giri and Narasu, 2000), for commercial large-scale production these systems are quite expensive. As they have to compete with the cultivation of the whole plant, in most cases such a process is not economically viable. The only commercial example so far, is the growth of ginseng roots (Hibino and Ushiyama, 1999). Immobilised cells Immobilisation of plant cells is another approach, in which it is thought that the improved cell to cell inter-

Improving productivity Screening and selection, medium optimisation The most common approach is the screening and selection for high producing cell lines and the optimisation of the growth and production media. This sort of approaches has extensively been used for the optimisation of microbial production systems. Also for plant cells this approach has been successful as has been shown for berberine production in Coptis japonica cell cultures, where a productivity of 7 g/l has been achieved (Sato et al., 1982; Sato and Yamada, 1984) and for shikonin for which a production of 3 g/l has been reported (Fujita, 1988). On the other hand for other cell cultures one has been less successful. Even though improved productivities were achieved, the high levels were often not stable, and after several subcultures the initial, much lower productivity was observed (for a review see Ohta and Verpoorte, 1992).

16 actions, may result in improved productivity. Indeed in some examples an improved productivity have been reported (Jardin et al., 1991). However, similar as for the organ cultures, also in this case the costs of scaling up to an industrial application is the major constraint. Elicitation As a response to stress factors, such as osmotic shock, addition of heavy metal ions, inorganic salts, microbial homogenates or UV radiation, accumulation of some secondary metabolites can be enhanced in plants or plant cell cultures. In plants certain secondary metabolite pathways are induced by infection with microorganisms. The compounds formed are phytoalexins, low molecular weight compounds with antimicrobial activity (Smith 1996). The induction is due to certain molecules that are formed from degradation of the plant cell wall or the microbial cell wall. These so called biotic elicitors can also be used to trigger the production of phytoalexins in plant cell cultures. This offers an excellent model to study the plant defence system. A number of phenylpropanoid derivatives act as defensive agents against biotic or abiotic stress (Dixon and Paiva, 1995), therefore most progress have been obtained on the elucidation of these pathways and their regulation. Elicitation can also be applied on large scale to induce production of certain compounds at a pre-set time in the process; moreover, the product is often excreted to the medium, thus the cells can be recycled and re-elicited. Kurz et al. (1987) showed that for the production of sanguinarine in poppy cell cultures, a continuous process is feasible in which a sequence of elicitation and medium change can be used to produce this alkaloid. Also in case of the production of paclitaxel elicitation has been shown to cause a clear increase in productivity (Laskaris et al., 1999). Unfortunately, most of the plant secondary metabolites of interest are not phytoalexins. Consequently their production is not induced or increased by elicitation. On the other hand, the elicitation of plant cell cultures can lead to the production of large amounts of for each plant specic secondary metabolites, which offers an interesting source of chemodiversity for screening for new leads for drug development (McAlpine et al., 1999). It is thus concluded that for a commercial production a highly producing plant cell suspension culture is the most promising. Research should thus focus on methods to improve productivity, by increasing growth rate and by increasing secondary metabolite accumulation. For this, metabolic engineering offers interesting perspective to improve the productivity of the plant cell factory. Consequently in the past years one has gone into studies of the biosynthetic pathways, with the aim to map the complete pathways, identify possible rate limiting steps, and learn more about the regulation (Dixon, 1999; Facchini, 2001; Hashimoto and Yamada, 1994; Kutchan, 1995; Verpoorte et al., 1998, 1999; Zenk 1991, 1995). This information should eventually lead to new approaches to improve secondary metabolite production in plants or plant cell cultures. Metabolic engineering obviously requires a thorough knowledge of all the steps in a pathway, and the genes encoding these steps. The biosynthetic pathway mapping is thus the rst step in this approach.

Biosynthetic pathway mapping The knowledge of secondary metabolite pathways in fact is limited. Much of our knowledge is based on work from the 1960s, 1970s, when a lot of work was done with feeding various radioactive labelled intermediates. Based on incorporation and chemical arguments subsequently pathways were predicted for most classes of secondary metabolites, and in particular pharmaceutically important compounds. However, many individual steps remained unclear, as no intermediates were available for testing these steps. Although much good work was done along these lines, looking at incorporation of radioactivity also carries the risk that the radioactivity goes into the nal product along a different way than expected, e.g., after breakdown of the added precursor and subsequent channelling into the nal product along a different pathway. The best example of this probably is the terpenoid biosynthesis. Many studies have been made on the incorporation of mevalonate in a broad range of terpenoids, including also terpenoid indole alkaloids (Banthorpe et al., 1972; Cordell, 1974). But in the past years it has been shown that there is another terpenoid biosynthetic pathway that does not go via mevalonate, but involves deoxyxylose as an important intermediate (Rohmer, 1999; Lichtenthaler, 1999). As it seems this plastidial pathway is leading to carotenoids, monoand diterpenes. Also the terpenoid indole alkaloids were recently shown to be derived from this pathway (Contin et al., 1998; Eichinger et al., 1999). The involvement of this pathway in the alkaloid biosynthesis was done via the retrobiosynthetic approach, in

17 which very early precursors such as sugars or pyruvate, specically labelled on one position with a 13 C label, are fed to the plant, plant organ or plant cells. By means of NMR-analysis of the products, the biosynthetic pathway involved can be deduced (Eisenreich and Bacher, 2000). Still this only shows the route followed, but the individual steps still need to be established. It can only be done by feeding with the proposed labelled intermediates, or by in vitro incubation of a protein extract of the producing plant with the intermediate and if necessary, the co-factors that might be needed for the expected reaction. Both these approaches are hampered by the fact that the intermediate might not be available, and could be difcult to obtain by isolation from plant material or synthesis. In case of in vivo tests there is the possibility that the intermediate does not reach the proper site for its conversion, and instead is converted into other products. In case of in vitro assays, the problem might be that the enzyme cannot be isolated in a stable active form. It might also be that aggregates of enzymes are responsible for a series of subsequent steps, which cannot be separated into individual reactions. For example in avonoid biosynthesis in Arabidopsis several enzymes seems to be aggregated (Burbulis and Winkel-Shirley, 1999). In microorganisms one has used mutants for identication of enzymes and genes involved in certain steps. This approach can be applied when a large number of mutants can be easily obtained and grown. It requires the analysis of all mutants for the accumulation of an intermediate. In case of coloured compounds this can be easily done visually. This is among others the reason that one of the best studied biosynthetic pathways in plants is the one leading to anthocyanins (Davies, 2000; Holton and Cornish, 1995; Mulder-Krieger and Verpoorte, 1994). Mutants in ower colour can easily be distinguished by eye. The availability of the various intermediates for the conrmation of an enzyme activity is also for this approach eventually important. Molecular biology offers several new approaches to cloning genes. In case of secondary metabolism, they have limited applicability. For example, transposon tagging results in plants that might be blocked in a certain step, but just like with mutants, this requires that one is able to identify the block. It means the analysis of large numbers of plants. The more promising approach is the combination of the analysis of the transcriptome, the proteome and the metabolome, and comparing plants or plant cells that produce or do not produce the compounds of interest. This approach is now being developed at many places (Jacobs et al., 2000; Fiehn et al., 2000; Trethewey, 2001). The major problem being that there is no immediate logical link between the metabolome and the proteome, as it is the case for the proteome and the genes. Moreover, the three levels do have different kinetics, so they need to be linked through time by means of statistical methods. A problem with the proteome is that it will be difcult to map all proteins (Jacobs et al., accepted for publication). Moreover, for secondary metabolism there is the problem of very low levels of the proteins involved if compared with those of primary metabolism. In case that one can easily isolate a certain producing tissue, like for example glandular hairs or latex (Fairnbairn and Steele, 1981; Paniego et al., 1999) a more specic set of enzymes can be obtained. In the coming years new methods need to be developed to overcome the mentioned problems. In conclusion for the time being the classical approach of identifying each individual step in a biosynthetic step, and subsequently isolation of the enzymes and cloning of the encoding gene seems the most certain way to reach the goal of mapping a biosynthetic pathway. In this way the tropane, isoquinoline and indole alkaloid biosynthetic pathways are being dissected, and the information obtained from these studies is being applied for metabolic engineering (Facchini, 2001; Hashimoto et al. 1993; Kutchan, 1995; Sato et al., 2001; Verpoorte et al., 2000; Verpoorte and Alfermann, 2000; Yun et al., 1992).

Regulation of the production Metabolic engineering The knowledge on the various biosynthetic pathways has been used already for altering biosynthetic pathways (Verpoorte and Alfermann, 2000). The goals one can envisage for metabolic engineering are many. Some important goals are: improving the production of a desired compound/enzyme for subsequent extraction and isolation improving resistance against pests and diseases lowering level of undesired compounds in food plants increasing the level of a desired compound in food (e.g., vitamins) giving a new trait (colour, taste, smell) to food, owers or ornamental plants.

18 Examples of all these have been reported. Here we will focus on the rst eld of applications, illustrating some of the possibilities as well as the problems with some examples from our own research. Improving the production of a secondary metabolite can be achieved by engineering the plant or plant cell culture already producing that particular compound. But also one can look at the possibility of introducing the production of this compound into other plant species or even microorganisms. Improving production secondary metabolites in plants and plant cells We will illustrate this approach with the example of the production of terpenoid indole alkaloids in Catharanthus roseus plants and plant cells. This plant is an important source of pharmaceuticals (van der Heijden and Verpoorte, 1989; Moreno et al., 1995; Verpoorte et al., 1997) (Figure 1). Ajmalicine from the roots of this plant is used to improve cerebral blood circulation, vinblastine and vincristine are dimeric alkaloids from the leaves that are used as antitumor drugs. The latter two compounds are particularly expensive because of their low levels in the plant. Therefore quite some effort has been put in trying to produce these alkaloids in plant cell cultures. However, these efforts were not successful. The biosynthesis of vindoline, one of the units forming the dimers requires certain steps that only occur in functional chloroplasts (De Luca and Cutler, 1987; St-Pierre et al., 1999; for a review see Facchini, 2001; Verpoorte et al., 1997). There are, however, some claims of cell suspension production of vindoline, while no further accumulation of dimers was observed (Scott et al., 1980; OKeefe et al., 1997). Moreover, the biosynthesis seems not to occur in one cell, but the rst part and the second part of the biosynthetic pathway occur in different cells (St-Pierre et al., 1999). Our efforts were rst of all focussed on the early part of the pathway leading to strictosidine, and from there to ajmalicine (Figure 1). Genes of this part of the pathway have been cloned in several laboratories. The tryptophan decarboxylase (TDC) (DeLuca, 2000; Verpoorte et al., 1997) and strictosidine synthase (STR)(Kutchan 1995; Verpoorte et al., 1997) has most extensively been studied. The enzyme TDC channels tryptophan from primary metabolism into the terpenoid indole alkaloid pathway. The enzyme STR couples tryptamine with secologanin, a product of the iridoid pathway. Both enzymes have been overexpressed in various plants and plant cells (Table 1). In case of C. roseus the overexpresion of these endogenous genes was accomplished, using the CaMV 35S promoter to obtain a constitutive overexpression in cell cultures (Canel et al., 1998; Whitmer et al., 1998; Whitmer, 1999). After TDC overexpression only more tryptamine was found, but the alkaloid production was not increased in the cell cultures. These results were expected, since TDC activity can be induced when cells are transferred to alkaloid production medium (Knobloch and Berlin, 1980), but generally no correlation between TDC activity and terpenoid indole alkaloid production is observed (Knobloch and Berlin, 1983; Mrillon et al., 1986; Eilert et al., 1987; Facchini and DiCosmo, 1991). A number of transgenic cell lines overexpressing STR showed a higher production of alkaloids, reaching total alkaloid levels of about 300 mg/l, with strictosidine, ajmalicine, serpentine, catharanthine and tabersonine as the major alkaloids in all the transgenic C. roseus cultures studied. Unfortunately, the higher alkaloid levels obtained went gradually down to the original levels of the wild type cells after one year, though the levels of the overexpressed enzymes remained high. Apparently the ux through the pathway is not only regulated through activity of the individual enzymes. Feeding experiments showed that these transgenic cells have a high capacity for alkaloid production. By feeding loganin levels of more than 600 M (ca. 300 mg/l) could be obtained for an STR overexpressing cell line (Whitmer, 1999). The cells seemed to be capable of producing tryptamine in considerable amounts, as in the STR-line up to 0.8 mM of loganin can be fed, without causing a lack of tryptamine, and yielding about 253 M of alkaloid. When tryptamine is fed in combination with loganin (1.6 mM) levels of 688 M of alkaloid could be obtained. At concentrations above 1.6 mM (up to 6.4 mM) no further increase of alkaloid production is observed. In the TDC overexpressing cell line the feeding of tryptophan or tryptamine in combination with loganin (6.4 mM) results in an alkaloid production of 1250 M (ca. 600 mg/l). But the strictosidine formed is rapidly converted and most of the alkaloids subsequently formed are eventually also catabolized. Serpentine is the alkaloid still accumulating at the end of the experiments. Consequently, by repeated feeding much higher levels of alkaloid can be obtained. Though secologanin is the direct precursor for strictosidine, feeding of this compound is not as efcient as feeding with loganin. After single feeding

19

Figure 1. Biosynthesis terpenoid indole alkaloids in Catharanthus roseus.

20
Table 1. Characteristics of some transgenic plant cell cultures overexpressing TDC, STR, or TDC and STR (Canel et al., 1998; Geerlings et al., 1999; Hallard et al., 1997; Hallard, 2000; Whitmer et al., 1998; Whitmer, 1999). Abbreviations: L loganin, S secologanin, T tryptamine, TDC tryptophan decarboxylase, STR strictosidine synthase. TDC activity pkat/mg protein Nicotiana tabacum cell suspension (TDC and STR) Morinda citrifolia cell suspension (TDC and STR) Cinchona ofcinalis hairy roots (TDC and STR) 1226 STR activity pkat/mg protein 28104 Tryptamine Strictosidine M 40 (after feeding 0.5 mM S+T) 10 (after feeding 0.31 mM S and 0.44 mM T) 1950 g/g FW Other alkaloids g/g DW

12.6 g/g FW

0.060.09

11.045.7

0.761.37 g/g FW

4.1

450

1200 g/g DW

Quinine 500 Quinidine 1000 Cinchonine + cinchonidine 400 Ajmalicine 1.4 Serpentine 0.2

Weigela Styriaca hairy roots (TDC and STR) Catharanthus roseus cell suspension (TDC) (T21)

Not detectable

148497

20 g/g DW

16.550.0

5.7167.9

0.25.7 mg/l

0.27 mg/l Up to 1200 M total alkaloid after feeding 6.4 mM L +T 0.945.3 mg/l Up to 688 M total alkaloid after feeding 1.6 mM L +T

Catharanthus roseus cell suspension (STR) (S1)

7.011.9

63.1273.9

0.11.7 mg/l

Catharanthus roseus cell suspension, wild type (CRPM)

4.512.4

8.523.2

0.11.1 mg/l

of secologanin the maximum level of alkaloids was about 25% of that after loganin feeding. But even in case of feeding of loganin in combination with tryptamine, only 45% of loganin is converted into alkaloid, when endogenous loganin (>0.4 mM) or tryptamine (>0.3 mM) is present in sufcient amounts. Apparently the iridoids are also channelled into other pathways. Obviously by overexpression of a few genes out of a pathway, the increase in ux is not very large. The plant cell, however, has quite a large capacity for producing alkaloids, but this capacity is not fully utilised. Apparently there are other regulatory mechanisms that control the ux. Like other secondary metabolites, the biosynthesis of terpenoid indole alkaloids in C. roseus

is also induced by stress conditions (fungal elicitor, UV-B light, jasmonate). Expression of TDC and STR was coordinately regulated by elicitation, jasmonate being the intermediate signal required. This indicated that these genes are controlled by common regulators (Menke, 1999; Menke et al., 1999a, b; van der Fits, 2000; van der Fits et al., 2000). As TDC makes that the plant cells can survive on a medium containing the toxic 5-methyltryptophan, one can search for regulatory genes by using a transposon that contains a constitutive promoter. In a cell, any time that this promoter comes in front of a regulatory gene that upregulates TDC, the cell will survive on the selection medium. Subsequently the surviving cell lines can be studied for which gene is upregulated, having the

21 ones in which the Tdc-gene itself is upregulated to be excluded. Eventually two regulatory genes were obtained. As both are inducible by jasmonate, they were named octadecanoid-responsive Catharanthus AP2domain transcription factors (ORCAs) (Menke, 1999; Menke et al., 1999a, b; van der Fits, 2000; Memelink et al., 2000, 2001). Further studies of these genes revealed that they each have a partly overlapping set of genes of the indole alkaloid biosynthesis that they regulate. But again, the total ux is not increased through the pathway. Though upon feeding tryptophan and loganin a two-fold increase of alkaloid production is observed in a C. roseus cell line overexpressing these two regulatory genes if compared with the wild type cell line (van der Fits et al., 2000). The search for regulatory genes thus needs to be continued, particularly looking for genes that control the early parts of the pathway, and in particular the iridoid pathway that in all experiments so far has been shown to be the rate limiting factor. Little is yet known about the iridoid part of the pathway. It includes several oxidations, for two of which now the genes have been identied. In both cases it concerns oxidations by a cytochrome P450 enzyme. Geraniol-10 hydroxylase converts geraniol into 10-hydroxygeraniol (Meijer et al., 1993; Collu, 1999; Collu et al., 2001) and secologanin synthase converts loganin into secologanin (Irmler et al., 2000; Yamanoto et al., 2000). Many steps are not yet fully elucidated, like the ones from the MEP pathway leading to IPP, the dephosphorylation of GPP to geraniol, the formation of iridodial from 10-hydroxygeraniol, the glucosylation of the iridoid moiety, the hydroxylation of deoxyloganic acid and/or deoxyloganin and the methylation of loganic acid and/or secologanic acid (Contin, 1999). These steps and their regulation might be identied by means of a proteomics approach. Production in other plants The Tdc- and Str-gene has also been introduced into other plants (Table 1). In tobacco plant cells both have been expressed, resulting in the production of tryptamine (up to to about 12.5 g/g FW). Feeding these cells with secologanin results in the production of strictosidine, but in contrast with C. roseus this glucoalkaloid is excreted to the medium, whereas in C. roseus cells it is stored in the vacuole. Apparently also on the level of cell physiology the biosynthesis of the alkaloids requires certain conditions (Hallard et al., 1997). The levels of strictosidine production As the slow growth of plant cells is an important constraint for an economically feasible production, the production of plant compounds in microorganisms would be an interesting alternative. However, this requires that all the genes of a pathway are available, that the necessary precursors are available in the microorganism, and that the products formed are not toxic for the microorganism. In general one can also say that this approach is only reasonable for relatively short pathways. As achieving a concerted action of all enzymes of for example the biosynthesis of vinblastine to function in a microorganism is not a realistic goal. in tobacco were about 20 mg/l, also considerably less than in the transgenic C. roseus cells. The C. roseus Tdc- and Str-genes where introduced with Agrobacterium rhizogenes in Cinchona ofcinalis, the hairy root culture obtained had considerable activity of these enzymes (Geerlings et al., 1999; Hallard et al., 1997). The STR enzyme activity was not able to utilise 5methoxytryptamine as substrate, indicating that it is due to the transgene. The hairy roots did produce 1.2 mg/g DW tryptamine, 1.9 mg/g strictosidine and about 1.9 mg/g of quinoline alkaloids. The quinoline alkaloid production is about 3 times higher than reported by Hamill et al. (1989) for a Cinchona hairy root culture. As the iridoid biosynthetic pathway is quite common in the plant kingdom, it could be interesting to overexpress the Tdc- and Str-genes in plants that are capable of producing secologanin. We thus initiated cell cultures of a series of plants that is capable of producing this compound (Hallard, 2000). Some of these indeed also produced secologanin in vitro. It was found that Weigelia Styriaca also had a glucosidase capable of converting strictosidine. Transformation with Agrobacterium rhizogenes harbouring a binary vector with the Tdc- and Str-genes resulted in a hairy root line that indeed produced a small amount of indole alkaloids, identied as tryptamine (20 g/g DW), ajmalicine (1.4 g/g DW) and serpentine (0.2 g/g DW) (Hallard, 2000). Another valuable example is the transformation of Brassica napus (canola) with the TDC gene of C. roseus. The mature seeds of transgenic plants produce reduced levels of indole glucosinolates, compounds that make the crop less palatable; thus increasing the economic value of the plant (Chavadej et al., 1994). Production in microorganisms

22 A short pathway or a bioconversion of some readily available precursors should thus be the targets for such an approach. We tried the feasibility of this approach by overexpressing the Str-gene in combination with the gene encoding strictosidine glucosidase (SGD) from C. roseus in yeast (Geerlings et al., 1998, 2001). Both enzymes indeed can be expressed in active form in yeast. The STR activity is mainly found in the medium of the transgenic yeast, whereas the SGD is mainly found in the cells. When fed with secologanin and tryptamine, this transgenic yeast is capable of producing 2 g/l of strictosidine in the medium in 3 days. By breaking the cells the glucoalkaloid was converted by the SGD activity into cathenamine. To overcome the problem of availability of secologanin, the transgenic yeast was grown on the juice of Symphoricarpus albus berries which contains about 1% of secologanin. In this way the indole alkaloids mentioned could be produced from a cheap and readily available source of both carbohydrates and secologanin (Geerlings et al. 1998, 2001). The STR-gene has also been overexpressed in E. coli (Kutchan et al., 1989) and in 22 bacterial strains where strictosidine was converted to vallesiachotamine and isovallesiachotamine (Shen et al., 1998). used both for plants and plant cell cultures, and even short pathways (bioconversions) can be introduced into microorganisms. However, it needs more basic knowledge about the pathways and their regulation. As many steps in biosynthetic pathways involve physiological aspects (e.g., transport, accumulation) and are not directly linked with a chemical reaction, the use of regulatory genes may be an important tool to upregulate a whole pathway.

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Conclusions Plant cell culture on a large scale has been shown to be feasible for industrial production. However, for the presently used phytochemicals only for a few the process has been successfully developed. For the others the production is too low to compete with these existing production methods. As the markets for most of the products are well established, and the prots small, little money is available to invest in a new biotechnological production. With the enormous capacity of high throughput screening for new biologically active compounds, plants are a very interesting source of chemodiversity for such screening programmes. This will certainly result in new plant derived drugs. Plant cell cultures offer here the possibility for production during the rst phases of drug development, during which also other production methods can be considered. This would avoid problems such as encountered with the supply of paclitaxel. Metabolic engineering is in this context an important tool to improve the plant cell factory for the production of the desired phytochemicals. It can be

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