Production of plant secondary metabolites: A

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Production of plant secondary metabolites: A historical perspective Article in Plant Science · October 2001 DOI: 10.1016/S0168-9452(01)00490-3

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Plant Science 161 (2001) 839– 851 www.elsevier.com/locate/plantsci

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Production of plant secondary metabolites: a historical perspective F. Bourgaud *, A. Gravot, S. Milesi, E. Gontier Laboratoire Agronomie et En6ironnement, UMR INRA-INPL-ENSAIA, 2 A6enue de la foreˆt de Haye, 54500, Vandoeu6re, France Received 10 April 2001; received in revised form 28 June 2001; accepted 28 June 2001

Abstract Studies on plant secondary metabolites have been increasing over the last 50 years. These molecules are known to play a major role in the adaptation of plants to their environment, but also represent an important source of active pharmaceuticals. Plant cell culture technologies were introduced at the end of the 1960s as a possible tool for both studying and producing plant secondary metabolites. Different strategies, using in vitro systems, have been extensively studied with the objective of improving the production of secondary plant compounds. Undifferentiated cell cultures have been mainly studied, but a large interest has also been shown in hairy roots and other organ cultures. Specific processes have been designed to meet the requirements of plant cell and organ cultures in bioreactors. Despite all of these efforts of the last 30 years, plant biotechnologies have led to very few commercial successes for the production of valuable secondary compounds. Compared to other biotechnological fields such as microorganisms or mammalian cell cultures, this can be explained by a lack of basic knowledge about biosynthetic pathways, or insufficiently adapted reactor facilities. More recently, the emergence of recombinant DNA technology has opened a new field with the possibility of directly modifying the expression of genes related to biosyntheses. It is now possible to manipulate the pathways that lead to secondary plant compounds. Many research projects are now currently being carried out and should give a promising future for plant metabolic engineering. © 1999 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Secondary metabolite; Metabolic engineering; Plant; Cell; Tissue; Culture; Hairy root; Shoot; Bioreactor

1. Introduction Two hundred years of modern chemistry and biology have described the role of primary metabolites in basic life functions such as cell division and growth, respiration, storage, and reproduction. In biology, the concept of secondary metabolite can be attributed to Kossel [1]. He was the first to define these metabolites as opposed to primary ones. Thirty years later an important step forward was made by Czapek [2] who dedicated an entire volume of his ‘plant biochemistry’ series to what he named ‘endproduckt’. According to him, these products could well derive from nitrogen metabolism by what he called ‘secondary modifications’ such as deamination. Compared to the main molecules found in plants, these secondary metabolites were soon defined * Corresponding author. E-mail address: [email protected] (F. Bourgaud).

by their low abundance, often less than 1% of the total carbon, or a storage usually occurring in dedicated cells or organs. In the middle of the 20th century, improvement of analytical techniques such as chromatography allowed the recovery of more and more of these molecules, and this was the basis for the establishment of the discipline of phytochemistry. Paper chromatography obviously revealed that some of these molecules were pigments, however, other possible implications of such secondary compounds in plant life were still largely mysterious as stated by Czapek’s term ‘end products’. Thanks to the improvement of biochemical techniques and the rise of molecular biology, it has been clearly demonstrated that secondary products play a major role in the adaptation of plants to their environment. These molecules largely contribute to plant fitness by interacting with the ecosystems. They have been described as being antibiotic, antifungal and an-

0168-9452/01/$ - see front matter © 1999 Elsevier Science Ireland Ltd. All rights reserved. PII: S 0 1 6 8 - 9 4 5 2 ( 0 1 ) 0 0 4 9 0 - 3

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F. Bourgaud et al. / Plant Science 161 (2001) 839–851

tiviral, and therefore able to protect plants from pathogens (phytoalexins), and also anti-germinative or toxic for other plants (allelopathy). Besides, they constitute important UV absorbing compounds, thus preventing serious leaf damage from the light [3]. They also act on animals, such as insects (anti-feeding properties) or even cattle for which forage grasses such as clover or alfalfa can express estrogenic properties and interact with fertility (see [4,5] for reviews on the chemical ecology of secondary compounds). Plant secondary compounds are usually classified according to their biosynthetic pathways [5]. Three large molecule families are generally considered: phenolics, terpenes and steroids, and alkaloids. A good example of a widespread metabolite family is given by phenolics: because these molecules are involved in lignin synthesis, they are common to all higher plants. However, other compounds such as alkaloids are sparsely distributed in the plant kingdom and are much more specific to defined plant genus and species. This narrower distribution of secondary compounds constitutes the basis for chemotaxonomy and chemical ecology. Due to their large biological activities, plant secondary metabolites have been used for centuries in traditional medicine. Nowadays, they correspond to valuable compounds such as pharmaceutics, cosmetics, fine chemicals, or more recently nutraceutics. Recent surveys have established that in western countries, where chemistry is the backbone of the pharmaceutical industry, 25% of the molecules used are of natural plant origin [6]. A good example could be aspirin (acetylsalicylate) which derives from salicylate. The genuine molecule can be isolated in large quantities from many plants (Spiraea ulmaria, Betula lenta…), but the chemical is synthesized as an acetyl-derivative in order to lower secondary effects (stomachaches). Chemical synthesis apart, the production of plant secondary metabolites has, for a long time, been achieved through the field cultivation of medicinal plants. However, plants originating from particular biotopes can be hard to grow outside their local ecosystems. It also happens that common plants do not withstand large field cultures due to pathogen sensitiveness (anthracnose on Hypericum perforatum or Arnica montana). This has led scientists and biotechnologists to consider plant cell, tissue and organ cultures as an alternative way to produce the corresponding secondary metabolites.

2. Plant cell cultures Evidence that plant cell cultures are able to produce secondary metabolites came quite late in the history of in vitro techniques. It had been considered for a long

time that undifferentiated cells, such as callus or cell suspension cultures were not able to produce secondary compounds, unlike differentiated cells or specialized organs [7]. Zenk and co-workers experimentally demonstrated that this theory was wrong, as they could observe dedifferentiated cell culture of Morinda citrifolia yielding 2.5 g of anthraquinones per liter of medium [8]. This finding opened the door to a large community of vitro culturists who extensively studied the possible use of plant cultures for the production of secondary compounds of industrial interest (mainly pharmaceutics and dyes).

2.1. Prospecting genetic 6ariability The main research programs for the production of secondary metabolites from plant cell cultures are represented in Fig. 1. At the origin, pharmacognosists discover active principles from plants [9]. Once interesting compounds are identified from plant extracts, the first part of the work consists in collecting the largest genetic pool of plant individuals that produce the corresponding substances. This work allows the screening of hyper-producing plants that present the most valuable secondary metabolites. However, a major characteristic of secondary compounds is that their synthesis is highly inducible. Hence, it is not sure that a given extract is a good indicator of the plant potential for producing the compounds, because it is always possible that the corresponding metabolism was repressed prior to quantifying the molecules.

2.2. Establishment of in 6itro cell lines After choosing the most promising individual plants, begins the real work of in vitro cultures with callus initiation. This work consists mainly in determining the medium that will be best adapted for cultivation. This medium optimization plays on mineral composition and organic constituents with special attention to hormonal balances that govern dedifferentiation/differentiation mechanisms. Although still largely empirical and often fastidious, this work is now facilitated by the use of incomplete factorial experiments or surface response methods [10]. Once calli are obtained, it is well known that they can undergo somaclonal variation [11], usually during several subculture cycles. This is a critical period where, due to this vitro-variation, secondary metabolite production is often variable from one subculture cycle to another. After a period of time (from several weeks to several years) genetic stability occurs and each callus can be considered as homogeneous cell aggregate, just as if it was derived from single cell cloning. There is little doubt that more than one work describing an


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Fig. 1. Guidelines for the production of secondary metabolites from plant cell.

erratic production of secondary metabolites with dedifferentiated cell cultures has been carried out with nonstabilized cell lines. This argument is generally poorly discussed in the literature (with the exception of specialized work on gene silencing [12]) because markers are missing to evaluate the state of genetic stability of a culture. In a recent work conducted in our laboratory, it was decided that a cell line could not be regarded as stabilized until growth parameters could be repeated during three consecutive subculture cycles in stable culture conditions. It is clear that this condition is necessary but not sufficient to conclude that there is genetic stability. However, because growth is a strong polygenic character it was considered as being ‘better than nothing’ to evaluate the ending of erratic phenomena. Therefore, we closely monitored growth parameters such as length of lag, of log phases, and growth speed during the log phase, for each of the 217 different callus lines obtained from Psoralea plants (Leguminosae) and capable of producing isoflavones [13]. Results showed that an increasing number of the 217 callus lines had stable growth parameters over time. Moreover, once considered as stable a cell line never went back to erratic growth parameters which strengthened the validity of our hypothesis. In our experiments 90% of the collection was ‘growth stabilized’ after 16 subculture cycles (48 weeks). Very few authors indicate the time necessary to stabilize cell cultures. However, the period we determined is rather short compared to the 2 years reported by Fett Neto et al. [14].

2.3. Cell suspension cultures When genetic stability is reached, it is necessary to screen the different callus lines according to their aptitudes to provide an efficient metabolite production (Fig. 1). Hence, each callus must be assessed separately for its growth speed as well as intracellular and extracellular metabolite concentrations. This allows an evaluation of the productivity of each cell line (mg of products g − 1 of cell day − 1 or mg of products l − 1 day − 1) so that only the best ones will be taken to cell suspensions and reactor studies. Compared to cell growth kinetics, which is usually an exponential curve, most secondary metabolites are produced during the plateau phase. This lack of production during the early stages can be explained by carbon allocation mainly distributed for primary metabolism (building of cell structures and respiration) when growth is very active. On the other hand, when growth stops, carbon is no longer needed in large quantities for primary metabolism and secondary compounds are more actively synthesized. It has been frequently observed that many new enzymatic activities, absent during lag or log phases, appear during this plateau phase. This has led many authors to speak of a possible biochemical differentiation of the cells when growth stops [15]. However, some secondary plant products are known to be growth-associated with undifferentiated cells, such as betalains and carotenoids.

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Table 1 Effect of environmental conditions and elicitor treatments on furocoumarin content in higher plants Environmental factor or elicitor Plant diseases Ceratocystis fimbriata Sclerotinia sclerotiorum Unknown Erwinia caroto6ora Rhodotula rubra Phoma companata Pseudomonas cichorii Insect damages Effect of light UV UV UV Air quality Acidic fog Ozone Temperatures Cold (−15° C, control 26° C) Hot (32° C, 21° C) control 21° C) Chemicals CuSO4 CuSO4 NaCl NaCl H2SO4 Ca(OCl)2

Plant species

Effect on furocoumarin content

Authors

Pastinaca sati6a (root apex) Apium gra6eolens (stalks) Daucus carota (roots) Apium gra6eolens (stalks) Ruta gra6eolens (hydroponic) Pastinaca sati6a (leaves) Glehnia littoralis (roots) Pastinaca sati6a (leaves)

×20 (8-MOP)

[18]

×235 (Psoralen) ×24 (Total furocoumarins) ×77 (8-MOP)

[19]

×24 (Total furocoumarins)

[21]

No modification

[22]


[23]

×9 (psoralen)

[24]

×2.2 (8-MOP) ×1.8 (Psoralen)

[25]

×3.4 (Linear furocoumarin)

[26]

×2.5–10 (Total furocoumarin)

[27]

×2 (Psoralen)

[24]

×5.4 (Linear furocoumarins)

[28]


[29]


[26]

×11 (Psoralen)

[30]


[26]

×2.8 (Psoralen)

[30]

Decrease but higher percentage on leaf surface ×2 (Psoralen)

[31]

Apium gra6eolens (stalks) Ruta gra6eolens (leaves) Glehnia littoralis (roots) Apium gra6eolens (leaves) Petroselinum crispum (leaves) Apium gra6eolens (leaves) Psoralea cinerea (leaves) Apium gra6eolens (leaves) Psoralea cinerea (fruits) Ruta gra6eolens (leaves) Psoralea cinerea (fruits) Ruta gra6eolens (leaves) Psoralea cinerea (fruits)

Decrease but higher percentage on leaf surface ×1.5 (Psoralen)

[20]

[30] [31] [30]

Different molecules from this family were investigated. Linear furocoumarins refer to a mixture of various molecules derived from psoralen. 8-MOP is for 8-methoxypsoralen (also called xanthotoxin).

Cell suspensions constitute a good biological material for studying biosynthetic pathways. Indeed, compared to callus cultures they allow the recovery of large quantities of cells from which enzymes can be more easily isolated [16]. These biosynthetic studies enable

the researcher to spot limiting enzyme activities (or genes) in the production of valuable metabolites. These bottleneck steps can sometimes be overlapped by feeding the cell cultures with a precursor metabolite that corresponds to the product of a limiting enzymatic


activity although there is always the risk of activating a feedback inhibition somewhere else on the pathway [17]. Many traditional strategies can be used to increase the production of secondary metabolites but elicitation is usually one of the most successful. This consists in applying chemical or physical stresses to the cell suspension cultures that will trigger the production of secondary metabolites that are normally not produced. This is currently done with biotic elicitors (chitosan, autoclaved mycelium of pathogenic fungi, various protein extracts) or abiotic factors (temperature, UV light, heavy metal salts, pH, etc.). Elicitation can be very efficient at increasing secondary metabolite production as shown in Table 1. This table gives the results that were obtained, in the literature with furanocoumarins, for various plant species and elicitor treatments. These molecules are well-known phytoalexins and their synthesis can be greatly multiplied, using adequate methods. Table 1 clearly shows that pathogen fungus or bacterial attacks are the most efficient for increasing the furanocoumarin content. This statement is also true for most other secondary metabolites, which explains that fungus or bacterial autoclaved preparations are frequently used for elicitation. Other methods than elicitation have been developed with cells in liquid systems such as immobilization [32]. In this case plant cells or micro aggregates are encapsulated in polymers (alginate, carraghenans, etc.), and this usually enhances the production of secondary metabolites [33]. The main explanations for this come from a possible matrix effect of the polymers around the cells which could mimic a tissue organization between them. This is supposed to give rise to the so-called biochemical differentiation which favors the synthesis of secondary products [34].

2.4. Bioreactor cultures Bioreactor studies represent the final step that leads to a possible commercial production of secondary metabolites from plant cell cultures. This is an important phase as numerous problems arise when scaling up the work realized on Erlenmeyer flasks. For example growth is considerably modified when cells are cultivated in large tanks and the production of cell biomass remains a critical point for bioreactor productivity. This is mainly due to mass transfer limitations of oxygen [35] and nutrients [36], as well as inhomogeneous culture systems which cause cell sedimentation and death. Recent studies have confirmed the low percentage of viable cells, approximately 50%, generally present in such liquid systems, except for the first days of culture [37]. Another strong limitation of growth is due to plant cell sensitiveness to shear stress which is responsible for extensive cell death. This lysis is a consequence of the agitation of the culture medium. It tends to increase with cell size when the culture is

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becoming older [38]. Many studies describe alternative agitation processes that tend to lower this lysis such as air-lift or bubble reactors instead of traditional propeller helixes [39]. This allows biomass production to increase to a level compatible with industrial processes. After successful optimization of the biomass production in a bioreactor, plant cell cultures must undergo well-adapted processes to achieve a good production of secondary metabolites. Traditional processes described for microorganisms can be used for plant cell cultures such as batch, fed-batch (semi-continuous), perfusion and continuous fermentations. Basically, the process design is governed by (1) the relationships between growth and secondary metabolite synthesis; and (2) the possibility for the secondary products to be excreted or not in the medium. (1) If secondary products are produced at the end of the growth phase, it is logical to consider a two-step process where a first reactor is used for building up the biomass, and a second one for metabolite production [40]. On the other hand, when the production of a given metabolite is growth-associated, a single-step reactor is sufficient to grow the cells and recover the molecules at the same time [40]. (2) If the metabolites remain intracellular, it is usually necessary to kill the biomass, so that the chemicals can be extracted from the cells. This leads to a batch or fed-batch process. Conversely, extracellular production avoids destruction of the biomass for the extraction of the compounds as they can be directly recovered from the medium. This characteristic allows the building of a continuous system with an improved productivity, compared to a standard batch. When a compound tends to be intracellular, it is sometimes possible to force the excretion with permeabilization methods. This can be obtained following different treatments such as pH shock [41] or the addition of various chemicals (detergents, oligosaccharides, [42]) bearing in mind that they must preserve cell viability. Perfusion systems have also been designed with encapsulated cells [15]. In addition to the effect previously described on metabolite production, this treatment usually ensures protection for the cells against shear stress. Among the hundreds of secondary plant products that have been investigated with undifferentiated cell cultures, many works have been published on indole alkaloids [43], tropane alkaloids [44], and taxanes [45], for which the reader is invited to refer to the bibliographical reviews cited herein. However, the development of the shikonin production by Tabata and co-workers has a special place in the recent plant cell culture history ([46], and for a review, see [40]). Indeed, it was one of the first and most successful stories of an industrial scale-up associating plant cell culture and bioreactor technologies. Another well-known plant cell culture success is represented by the production of ginseng at a very large scale (20 000–25 000 l, see [47]).


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Fig. 2. Guidelines for the production of secondary metabolites from plant organ cultures.

3. Organ cultures Plant organs represent an interesting alternative to cell cultures for the production of plant secondary products. Two types of organ are generally considered for this objective: hairy roots and shoot cultures (Fig. 2).

3.1. Hairy root cultures Hairy roots are obtained after the successful transformation of a plant with Agrobacterium rhizogenes. They have received considerable attention from plant biotechnologists, for the production of secondary compounds. They can be subcultured and indefinitely propagated on a synthetic medium without phytohormones [48,49] and usually display interesting growth capacities owing to the profusion of lateral roots. This growth can be assimilated to an exponential model, when the number of generations of lateral roots becomes very large [50]. The specific growth rates are generally comparable, if not superior, to the parameters observed with undifferentiated cells. Biomass doubling times ranging from less than 1 day [51] to almost 1 week [52] have been reported. Small exogenous auxin concentrations usually increase both elongation rate and lateral branching [53]. Most authors working on hairy root cultures have reported the long-term genetic stability of this material [54,55]. This was clearly stated in a comprehensive study conducted by MaldonadoMendoza et al. [51] on more than 500 hairy root lines from Datura stramonium. They demonstrated that

growth and tropane alkaloid production was stable over a period of 5 years. However, some experimental results have been reported that do not match the hypothesis of a straightforward genetic stability of transformed roots. Yukimune et al. [56] observed a decrease in growth rate during repeated selection cycles when trying to increase tropane alkaloid production of Duboisia hairy root lines. These changes can probably be attributed to the variation of the expression of Ri tDNA oncogenes in the transformed roots [57,58]. In addition to their growth capacities, hairy roots display interesting properties regarding the production of secondary compounds. The metabolite pattern found in hairy roots is similar, if not always identical to that of plant roots [59,60]. In most cases, the differences are only on trace compounds [61]. A major characteristic of hairy roots is that they are able to produce secondary metabolites concomitantly with growth. Hence it is possible to get a continuous source of secondary compounds from actively growing hairy roots [62], unlike the usual results obtained with cell suspension cultures. The same strategies, as developed for cell cultures, have been used to increase the metabolite production from hairy roots. Successful results have been obtained by modifying the nutrient composition of the medium [63], or applying elicitors [64]. It is also possible to cultivate roots under light, in order to get green tissues. These light-adapted hairy roots usually demonstrate different biosynthetic capacities, compared to dark-grown ones [65], due to the presence of chloroplastic enzymes. However, growth rates can be severely depressed with hairy roots under light exposure [66].


3.2. Shoot cultures As with roots, it is possible to cultivate plant aerial parts (shoots) for the production of secondary metabolites (Fig. 2). Shoot cultures can be transgenic, the so-called shooty teratomas, if they are obtained after infection with Agrobacterium tumefaciens [67], or nontransgenic through the simple use of appropriate hormonal balance [68]. Like organ cultures, shoots exhibit some comparable properties to hairy roots, namely genetic stability and good capacities for secondary metabolite production, and also the possibility of gaining a link between growth and the production of secondary compounds [65,68]. However, there are some differences in the metabolic pattern, as some syntheses are specifically located in either roots or shoots [69]. Other differences concern a somewhat slower growth rate as the fastest doubling time reported is approximately 3 days [70], and also the necessity to expose shoot cultures to light, which can be a problem with large tank reactors made from steel.

3.3. Organ cultures in bioreactors Compared to cell suspension cultures, organ cultures generally display a lower sensitivity to shear stress although some hairy root lines have been described to be susceptible too. This happens with Catharanthus roseus hairy roots, which need to be cultivated in an air-sparged bioreactor [71]. Other methods have been reported to efficiently reduce the stress associated with medium agitation. Immobilization of hairy roots into a polymer matrix is a well-known technique [72] but immobilization can also be reached spontaneously, as hairy roots usually grow actively and rapidly colonize the entire reactor [62]. It is also possible to protect the roots from agitation by using screens [73] or wire meshes [74]. Other less sensitive organs can be cultivated in stirred bioreactors [75]. One of the major problems encountered with organ cultures in bioreactors is due to the inhomogeneous character of the biomass compared to thin cell suspensions. Hairy roots in liquid systems grow in approximately spherical clumps but display a high degree of spatial heterogeneity. In a study conducted on Atropa belladonna hairy roots in a liquid-dispersed reactor, Williams and Doran [76] demonstrated the occurrence of both vertical and radial gradients of root density, with higher biomass levels near the top of the root bed, and in the inner part of the root pellets. This heterogeneity can be partially attributed to inhomogeneous and limiting mass transfers to the roots, regarding oxygen [77,78] and nutrients [76]. As a general rule, and due to their genetic stability, organs are less submitted to erratic metabolite production than undifferentiated cells. However, a spatial

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heterogeneity is always possible along the growing organ. In the case on hairy roots, each root tip grows linearly, and the older part of the roots are still present in the culture system. As a consequence, the total root biomass is always composed of young and older tissues. These tissues present various possibilities for the synthesis of secondary compounds as stated by Bourgaud et al. [79] with flavonoids. Young tips of Psoralea roots were demonstrated as more capable of synthesizing isoflavones (daidzein) whereas old roots accumulated isoflavone-derivatives like coumestrol. In most organ cultures, the production of secondary plant products is usually concomitant with growth. As a consequence, and unlike most secondary metabolites in cell suspension systems, it is possible to use a single-stage bioreactor for both growing the biomass and producing the compounds. However, like for cell cultures, most of the secondary metabolites tend to remain intracellular [62,79] especially when growth is still active [76]. Specific bioreactors have been designed for hairy root cultures in order to overcome the limiting factors existing for biomass and secondary metabolite production. Submerged cultures have successfully been replaced by dispersed liquid systems such as nutrient mist reactors [80] or drip-tube techniques [62]. Two-phase systems have also been used to facilitate the release and recovery of the secondary compounds in the medium [81,82]. This technology helps to continuously remove the compounds from the medium, and helps to prevent the feedback repression of the synthesis [62,83]. Despite all the improvements that have been made to reach a better understanding of plant organ cultures in bioreactors, this technology has led to even fewer commercial successes than cell suspension cultures for the production of secondary metabolites. Yet, plant organ cultures are still regarded as promising and many projects are under development [84,85].

4. Need for breakthroughs for the production of secondary compounds Industrial interest emerged quite early in the story of plant vitro cultures and secondary metabolites. Indeed, at least six large-scale industrial productions were engaged between 1976 and 1986 [86].1 However, since that time, this technology has led to only a few applications for the production of commercial compounds. This lack of success can be attributed to several bottlenecks. First of all, it is obvious that industrial processes were started at a time when basic knowledge was

1

Ubiquinone-10 (Japan Tobacco & Salt Pub. Co.), ginseng (Svoboda Co.; Nitto Denko Co.); shikonine (Mitsui Petrochemical Ind. Ltd), berberine (Yamaguchi Co.), rosmarinic acid (Natterdam Co.).

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severely lacking on both plant vitro cultures and secondary compounds. It is astonishing to realize that a key-problem like cell viability, and its assessment, has been studied only recently with plant cells [37,38], whereas this topic has been considered for a long time with animal cell cultures [87]. Also, secondary metabolites are produced following long biosynthetic pathways that can involve dozens of enzymes. This synthesis is much more complex than for recombinant proteins produced with mammalian or prokaryotic biotechnologies, which usually involve one or two genes. This can partially explain the lack of success of plant cell and tissue cultures compared to other expression systems. A second bottleneck has to do with the economic feasibility of plant cell and organ cultures. Indeed, this technology requires high-cost bioreactors associated with aseptic conditions that are expensive to maintain [88]. Besides, unlike mammalian cell cultures which produce high-value molecules, plant vitro cultures have also been considered for the production of low or moderate-value molecules such as food ingredients. As a consequence, economic feasibility is even more difficult to reach. There have been many attempts in the last decade to address the problem of cost-effectiveness of plant vitro culture technologies. Several new routes have been investigated. The first one is the design of low-cost bioreactors associated with a reduction of online controls and probes, and a simplified sterilization process [88]. Another possible strategy could be the use of plastic bags as already in use for the cultivation of microalgae [89]. Such bags are available in varying volumes (20– 1000 l) and an aeration system can be adapted. These plastic bags are obviously much cheaper to use than culture reactors. However, they do not address all the problems encountered with cell cultures such as cell autotrophy or sedimentation. Other authors have tried to change more radically the concept of plant cell tissue and organ cultures in bioreactors without the use of a traditional culture chamber. A new system has been proposed by Borisjuk and co-workers [90] although it was designed for the production of recombinant proteins from genetically modified plants. The basic concept is to grow entire plants in a system where roots are maintained in sterile conditions and separated from the aerial parts. The recombinant proteins are recovered in the nutrient solution that surrounds the roots with the help of the rhizospheric excretion from the plant. This concept is interesting because it uses whole plants. It addresses the problem of biomass production encountered with cell suspension cultures that are hard to develop. Another system has been recently proposed by Gontier and co-workers [91]. This last system has been designed specifically for the production of secondary compounds. The basic principle is to get rid of sterility but to keep a culture medium that allows the

manipulation of secondary compound production (elicitation, addition of precursors, etc.). Sterility is essential because a traditional in vitro culture medium is constituted of organic compounds, especially sugars, that would be altered by microorganisms if maintained in non-sterile conditions. If we use whole plants that are photosynthetically active, it is possible to avoid the use of organic compounds in a mineral-based medium. In this case, the medium is less amenable to contamination in open conditions. The other basic concept of this device is to yield secondary compounds in a non-destructive manner for the plants, therefore allowing repeated and regular harvests from the same biomass. In practice, this is achieved by growing the plants in hydroponic or aeroponic non-sterile conditions and by treating the medium in order to elicit the synthesis and to allow the excretion of the desired compounds, out of the roots. The last step is the recovery of the compounds from the medium with the help of purification processes. As a conclusion, this system uses the plants for what they can do best in natural conditions: they grow quite quickly when they are photosynthetically active. It also uses the concept of culture medium for what it is most useful in reactor culture technology: it allows a close control of secondary compound production. This technology has been has been successfully used at pilot scale on three different model plants: Datura innoxia for tropane alkaloids, Ruta gra6eolens for furocoumarins, and Taxus baccata for paclitaxel. Many attempts have been made to invent new devices that could lead to breakthroughs for the production of secondary compounds. However, the major changes in the area of plant secondary metabolites have probably been achieved thanks to the rise of molecular genetics in recent years, through the so-called metabolic engineering approach.

5. Plant metabolic engineering The last 15 years have produced a large quantity of results regarding the biosynthetic pathways leading to secondary metabolites. Concomitantly, at the beginning of the 1990s, a new discipline called metabolic engineering appeared. According to Bailey [92], metabolic engineering is: ‘the improvement of cellular activities by manipulation of enzymatic, transport, and regulatory functions of the cell with the use of recombinant DNA technology’. In many cases this approach relies on the identification of limiting enzyme activities, after successful pathway elucidation and metabolite mapping (metabolomics). Such limiting steps can be improved with an appropriate use of genetic transformation. Most of the strategies developed so far have played on the introduction of genes isolated from more efficient organisms, promoters that enhance the expression of a


target gene, or antisens and co-suppression techniques for the obtainment of knock-down plants for the desired traits. Besides their own biosynthetic capacities, plants can also be used as host organisms for the production of recombinant proteins. This corresponds to the so-called ‘molecular pharming’ when the technology utilizes fieldgrown transgenic plants. This last topic is outside the scope of this review as it is not typically focused on the production of secondary compounds. However, many research projects relevant to this category are currently being developed to which the reader is invited to refer [93,94].

5.1. Flower colors Amongst the first attempts to apply metabolic engineering to plant secondary metabolites is the modification of flower colors. Flower colors are principally given by flavonoids, betalains and carotenoids but flavonoids have undoubtedly been the most studied with the use of molecular genetics. This has represented a pioneer research in the field of antisens RNA technology [95], the study of plant promoters, and gene expression [96]. Spectacular results have been obtained by ‘sensing’ or ‘antisensing’ the chalcone synthase gene that led to flower color modification in different plants such as petunia [96], rose [97], gerbera [98], carnation [99], or chrysanthemum [100]. Metabolic engineering has also been extensively used for the modification of the carotenoid pathway, especially in the framework of the ‘golden rice’ project [101]. Three novel genes were introduced in ‘golden rice’ to re-route the precursor geranylgeranyl-diphosphate to the desired b-carotene: phytoene synthase, and lycopene b-cyclase from daffodil, and a desaturase from Erwinia [102]. More recently, it has been possible to change the coloration of tobacco chromoplasts and nectaries by altering the astaxanthin pathway [103]. These examples illustrate that it is now possible to manipulate the production of carotenoids and other food ingredients by the use of plant metabolic engineering.

5.2. Lignin synthesis Down-regulation of lignin synthesis is another subject of considerable interest for which the metabolic engineering of secondary compounds is currently being investigated. Indeed, in the case of the paper industry, lignin must be separated from cellulose at a considerable cost for the environment. Therefore, it would be an improvement of the chemical process to reduce the lignin content or to modify the lignin composition so that it would become more easily extractable. Promising results have been obtained by Hu et al. [104]. They produced transgenic aspen that was down-regulated by

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antisens inhibition of the 4-coumarate:coA ligase activity. They demonstrated up to a 45% reduction of lignin, associated with a 15% cellulose increase in the transformants. This compensatory effect is particularly relevant for the paper industry as cellulose is the most valuable part of wood pulp. Other results of considerable interest have been reached with Arabidopsis lignification mutants that accumulate lignin syringyl monomers to the detriment of guaiacyl units [105]. This was obtained by the overexpression of ferulate-5-hydroxylase, a major P450 gene from the pathway, and was also verified in other model plants such as tobacco and poplar [106]. This strategy could well lead to an improved chemical degradability of lignin as syringyl-based lignin is more easily processed. More detailed results about lignin genetic engineering can be found in dedicated bibliographical reviews [107,108].

5.3. Pharmaceutical compounds Plant metabolic engineering has also been successfully applied to the production of pharmaceutically useful secondary metabolites. Many pathways are currently being investigated but alkaloids have probably received more attention because of their pharmaceutical relevance. Pioneer investigations on medicinal plants and metabolic engineering were conducted by Yun et al. [109] on tropane alkaloids. These authors used a hyoscyamine 6b-hydroxylase (H6H) gene from Hyoscyamus niger, controlled by a 35s promoter, and succeeded in overexpressing h6h activity in Atropa belladonna. Transgenic Atropa plants displayed an enhanced conversion of hyoscyamine into scopolamine, which is a more pharmaceutically useful compound. These results clearly demonstrate that it is possible to considerably modify secondary metabolite patterns, playing on enzymes located downstream to the synthesis. Other experiments based on single gene transformations have been carried out by the same group on various alkaloid producing plants [110]. Overexpression of tobacco putrescine-N-methyl-transferase (PMT) in Atropa belladonna led to unmodified alkaloid profiles in the transformants whereas the same transformation carried out on Nicotiana syl6estris gave a 40% increase in leaf nicotine. Unlike H6H, PMT is placed upstream in alkaloid synthesis. PMT experiments demonstrate that it is possible to increase the metabolite flux so that it can benefit a downstream compound (nicotine). However, this strategy is not always successful, as in the case of Atropa belladonna, as other enzymes possibly limit the synthesis. Another strategy has been put forward by Sato and co-workers [110] with experiments on scoulerine 9-O-methyltransferase (SMT). It consists in playing with pathways that derive from the same branching point. SMT converts scoulerine in successive compounds that lead to berberine, but scoulerine can

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also be transformed into sanguinarine by a parallel pathway. SMT overexpression allowed an increase in the berberine content of Coptis transgenic cells (20%) to the detriment of the competitive pathway. The above results, obtained by the Yamada group, demonstrates the value of playing on a well-defined gene, for which the corresponding enzyme has been identified as a limiting step in a synthesis. However, multiple gene families are usually involved in the synthesis of a single secondary metabolite, and therefore it is important to think about multiple gene transformation and coordinate gene expression for the future. The first encouraging results were described by Beck von Bodman et al. [111]. They succeeded in expressing a polygenic construction driven by a single promoter in tobacco plants. Two genes placed on the construct were co-ordinately expressed in the same secondary pathway (mannopine synthesis). Although this biosynthesis has no pharmaceutical relevance, this basic research demonstrates that it is possible to create a new metabolic channeling in the case of plant secondary compounds and is full of promise for research to come.

6. Prospects Many other examples could be presented with plant metabolic engineering as this research area is developing actively, but being comprehensive on this topic is outside the scope of this review. For further readings, a first book entirely dedicated to this approach in the field of plant secondary metabolism has been recently published [112], confirming that the discipline is healthy. Metabolic engineering is probably a large step forward but playing on the genes will not solve all the problems that have prevented the development of commercial success in the field of plant secondary metabolites. To cite just a few ‘non-scientific’ problems, the so-called ‘molecular pharming’ has not gained public acceptance yet, due to environmental concerns about transgenic plants, especially in Europe. A possible alternative to field grown plants could be to use transformed cells or organs in reactors, as they are by definition cultivated under closed conditions, and therefore do not exhibit any environmental risk. The long-awaited breakthrough to reach an industrial accomplishment is probably in our hands with the combination of plant cell, tissue, and organ culture technologies and metabolic engineering.

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