Plant Cell Cultures Chemical Factories of Secondary Metabolites %5BReview%5D

Biotechnology Advances 20 (2002) 101–153

Plant cell cultures: Chemical factories of secondary metabolites S. Ramachandra Rao a,*, G.A. Ravishankar b a

Laboratory of Biofunctional Materials, School of Materials Science, Japan Advanced Institute of Science and Technology, 1-1, Asahidai, Tatsunokuchi, Ishikawa 923-1292, Japan b Plant Cell Biotechnology Department, Central Food Technological echnological Research Institute, Mysore 570 013, India

Abstract

This This revi review ew deal dealss with with the the prod produc ucti tion on of high high-v -val alue ue seco second ndar ary y meta metabo bolit lites es incl includ udin ing g pharmaceuticals pharmaceuticals and food additives through plant cell cultures, shoot cultures, root cultures and transgenic roots obtained through biotechnological means. Plant cell and transgenic hairy root cultures are promising potential alternative sources for the production of high-value secondary metabolites of industrial importance. Recent developments in transgenic research have opened up the possibility of the metabolic engineering of biosynthetic pathways to produce high-value secondary metabolites. The production of the pungent food additive capsaicin, the natural colour anthocyanin and the natural flavour vanillin is described in detail. D 2002 Elsevier Science Inc. All rights reserved. Keywords: Keywords: Secondary metabolites; Pharmaceuticals; Plant cell cultures; Hairy root cultures; Biotransformation; Immobilization

1. Introduction

For centuries, mankind is totally dependent on plants as source of carbohydrates, proteins and fats for food and shelter. In addition, plants are a valuable source of a wide range of secondary secondary metabolites, metabolites, which are used as pharmaceu pharmaceuticals ticals,, agrochemic agrochemicals, als, flavours, flavours, fragranfragrances, colours, biopesticides biopesticides and food additives. Over 80% of the approximately approximately 30,000 known natural products are of plant origin (Phillipson, 1990; Balandrin and Klocke, 1988; Fowler

* Corresponding author. author. Tel.: +81-761-51-1668; fax: +81-761-51-1665. E-mail addresses: [email protected], [email protected] (S. Ramachandra Rao). 0734-9750/02/$ – see front matter D 2002 Elsevier Science Inc. All rights reserved. PII: S 0 7 3 4 - 9 7 5 0 ( 0 2 ) 0 0 0 0 7 - 1

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S. Ramachandra Rao, G.A. Ravishankar / Biotechnology Advances 20 (2002) 101–153

and Scragg, Scragg, 198 1988). 8). The number number of kno known wn chemic chemical al struct structure uress is estima estimated ted to be nearly nearly fourfold greater than that in the microbial kingdom. In 1985, of the 3500 new chemical structures identified, 2600 came from the higher plants. Worldwide, 121 clinically useful prescription drugs are derived from plants (Payne et al., 1991). The surveys of plant medicinal usage by the American public have shown an increase from just about 3% of the population in 1991 to over 37% in 1998 (Brevoort, 1998). The US market sales of plant medicinals have climbed to about US$3 billion per year (Glaser, 1999). Even today, 75% of the world’s population relies on plants for traditional medicine. In the US, where chemical synthesis dominates the pharmaceutical industry, 25% of the pharmaceuticals are based on plant-derived chemicals (Payne et al., 1991; Farnsworth, 1985). Plants will continue to provide novel products as well as chemical models for new drugs in the coming centuries, because the chemistry of the majority of plant species is yet to be characterized (Cox and Balic Balick, k, 19 1994 94). ). The The adve advent nt of chemi chemica call anal analyse ysess and and the the char charac acte teriz rizat ation ion of mol molec ecula ular r structures have helped in precisely identifying these plants and correlating them with their activity under controlled experimentation. Despite advancements in synthetic chemistry, we

Table 1 Plant-derived pharmaceuticals of importance Product

Use

Plant species

Ajmalicine Artemisinin Ajmaline Acinitine Berberine Camptothecin Capsaicin Cast astanosp nosper ermi mine ne Codeine Colchicine Digoxin Diosgenin Ellipticine Emetine Forskolin Ginsenosides Morphine Podophyllotoxin Quinine Sanguinarine

Antihypertensive Antimalarial – – Intestinal ailment Antitumour Counterirritant Gly Glycosid osidee inhi inhibi bito tor r Sedative Antitumour Heart stimulant Steroidal precursor Antitumour – Bronchial asthma Health tonic Sedative Antitumour Antimalarial Antiplaque

Shikonin Taxol Vincristine Vinblastine

Antibacterial Anticancer Antileukemic Antileukemic

Cath. roseus Artemisia annua Ra. serpentina Acotinum spp. C. japonica Camptotheca acuminata Ca. frutescens Castanospermum australe P. somniferum somniferu m Colchium autumnale Di. lanata Dioscorea Dioscorea deltoidea Orchrosia Orchrosia elliptica Cephaclis ipecaccuanha ipecaccuanha Coleus forskolii Panax ginseng P. somniferum somniferu m Podophyllum petalum Cinchon. ledgeriana Sanguinaria canadensis P. P. somniferum L. erythrorhizon erythrorhizon Taxus brevifolia Cath. roseus Cath. roseus

n/a: not available. Adapted from Ravishankar and Ramachandra Rao (2000).

Cost (US$ per kilogram) 37,000 400 75,000 n/a 3250 432,000 750 n/a 17,000 35,000 3000 1000 240,000 1500 n/a n/a 340,000 n/a 500 4,800 4500 600,000 2,000,000 1,000,000

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and Scragg, Scragg, 198 1988). 8). The number number of kno known wn chemic chemical al struct structure uress is estima estimated ted to be nearly nearly fourfold greater than that in the microbial kingdom. In 1985, of the 3500 new chemical structures identified, 2600 came from the higher plants. Worldwide, 121 clinically useful prescription drugs are derived from plants (Payne et al., 1991). The surveys of plant medicinal usage by the American public have shown an increase from just about 3% of the population in 1991 to over 37% in 1998 (Brevoort, 1998). The US market sales of plant medicinals have climbed to about US$3 billion per year (Glaser, 1999). Even today, 75% of the world’s population relies on plants for traditional medicine. In the US, where chemical synthesis dominates the pharmaceutical industry, 25% of the pharmaceuticals are based on plant-derived chemicals (Payne et al., 1991; Farnsworth, 1985). Plants will continue to provide novel products as well as chemical models for new drugs in the coming centuries, because the chemistry of the majority of plant species is yet to be characterized (Cox and Balic Balick, k, 19 1994 94). ). The The adve advent nt of chemi chemica call anal analyse ysess and and the the char charac acte teriz rizat ation ion of mol molec ecula ular r structures have helped in precisely identifying these plants and correlating them with their activity under controlled experimentation. Despite advancements in synthetic chemistry, we

Table 1 Plant-derived pharmaceuticals of importance Product

Use

Plant species

Ajmalicine Artemisinin Ajmaline Acinitine Berberine Camptothecin Capsaicin Cast astanosp nosper ermi mine ne Codeine Colchicine Digoxin Diosgenin Ellipticine Emetine Forskolin Ginsenosides Morphine Podophyllotoxin Quinine Sanguinarine

Antihypertensive Antimalarial – – Intestinal ailment Antitumour Counterirritant Gly Glycosid osidee inhi inhibi bito tor r Sedative Antitumour Heart stimulant Steroidal precursor Antitumour – Bronchial asthma Health tonic Sedative Antitumour Antimalarial Antiplaque

Shikonin Taxol Vincristine Vinblastine

Antibacterial Anticancer Antileukemic Antileukemic

Cath. roseus Artemisia annua Ra. serpentina Acotinum spp. C. japonica Camptotheca acuminata Ca. frutescens Castanospermum australe P. somniferum somniferu m Colchium autumnale Di. lanata Dioscorea Dioscorea deltoidea Orchrosia Orchrosia elliptica Cephaclis ipecaccuanha ipecaccuanha Coleus forskolii Panax ginseng P. somniferum somniferu m Podophyllum petalum Cinchon. ledgeriana Sanguinaria canadensis P. P. somniferum L. erythrorhizon erythrorhizon Taxus brevifolia Cath. roseus Cath. roseus

n/a: not available. Adapted from Ravishankar and Ramachandra Rao (2000).

Cost (US$ per kilogram) 37,000 400 75,000 n/a 3250 432,000 750 n/a 17,000 35,000 3000 1000 240,000 1500 n/a n/a 340,000 n/a 500 4,800 4500 600,000 2,000,000 1,000,000


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still depe still depend nd up upon on biolo biologic gical al sourc sources es for for a nu numbe mberr of seco second ndar ary y metab metaboli olites tes inclu includin ding g pharmaceuticals (Pezzuto, 1995). Their complex structural features are difficult to synthesize. We have listed some plant-derived pharmaceuticals in Table 1. Elaborative pathways from basic primary metabolites, which are synthesized immediately as a result result of pho photos tosynt ynthet hetic ic activi activity ty,, produc producee second secondary ary metabo metabolite lites. s. Many Many of them them are unique to the plant kingdom and are not produced produced by microbes microbes or animals. animals. However, However, with the advancement of transgenic research, it is possible to produce compounds and molecules, which were also not originally synthesized in plants.

2. Need for a biotechnological approach

Biotechnology offers an opportunity to exploit the cell, tissue, organ or entire organism by growing them in vitro and to genetically manipulate them to get desired compounds. Many facets of biotechnological approaches can be envisaged (Table 2). Since the world population is increasing rapidly, there is extreme pressure on the available cultivable land to produce food and fulfill the needs. Therefore, for other uses such as production of pharmaceuticals and chemicals from plants, the available land should be used effectively. Hence, it is appropriate to develop modern technologies leading to plant improvement for better utilization of the land to meet the requirements. The development of micropropagation methods for a number of medicinal plant species has been already reported and needs to be adopted (Naik, 1998). Cryopreservation of cells is an area of importance in the conservation of medicinal plants. It has already been used in many plant species. The development and adoption of plant cell culture and organ culture methods have lead to the production of plant products on a large scale. This has been possible Table 2 Aspects of biotechnological approaches to plant-derived secondary metabolites 1. Plant cell tissue and organ cultures Cell culture Shoot culture Root culture Scale-up of cultures 2. Transgenic plants/organisms Metabolic engineering Heterologous expression Molecular forming 3. Micropropagation of medicinal plants Endangered plants High-yielding varieties Metabolically engineered plants 4. Newer sources Algae Other photosynthetic marine forms 5. Safety considerations

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by the combined efforts of cell biologists and chemical engineers. Chemical engineers are currently developing improved and appropriate bioreactors for the improvement of production syst systems ems by adoptin adopting g techniq techniques ues of growth growth and metabo metabolite lite produc production tion couple coupled d with downstream processing of the products. The improvements in molecular biological research have given a new dimension to in vitro culture as well as for plant improvement, enhancing the yields of the product and resulting in multiple products or producing novel products from genetically genetically engineered engineered plants. Moreover Moreover,, the need for safer drugs without side effects effects has led to the use of natural ingredients with proven safety. These factors have laid emphasis on the use of bio biotec techno hnolog logica icall method methodss to enhanc enhancee the produc production tion of pharma pharmaceu ceutic ticals als and food food additives, both in quality and quantity.

3. Plant cell culture as a source of secondary metabolites

Plant cell cultures are an attractive alternative source to whole plant for the production of high-value secondary metabolites (Ravishankar et al., 1999; Dornenburg and Knorr, 1997; Scragg, 1997; Alfermann and Petersen, 1995; DiCosmo and Misawa, 1995; Stockigt et al., 1995; Endress, 1994; Ravishankar and Venkataraman, 1990) (Tables 3 and 4). Plant cells are are bios biosyn ynth thet etic ical ally ly tot totip ipote otent nt,, whic which h mean meanss that that each each cell cell in cultu culture re reta retains ins comp comple lete te genetic information and hence is able to produce the range of chemicals found in the parent plant. The advantages of this technology over the conventional agricultural production are as follows. It is indepen independen dentt of geogra geographic phical al and season seasonal al variat variations ions and variou variouss enviro environme nmenta ntall factors. It offers a defined production system, which ensures the continuous supply of products, uniform quality and yield. It is possible to produce novel compounds that are not normally found in parent plant. Table 3 Groups of natural products that were so far isolated from tissue and suspension cultures of higher plants Phenylpropanoids 1. 2. 3. 4.

Anthocyanins Coumarins Flav Flavon ono oids ids Hydrox Hydroxyci ycinna nnamoy moyll derivatives 5. Isof Isofla lavo vono noiids 6. Lignans 7. Phenolenones 8. Proant Proanthoc hocyan yanidi idins ns 9. Stilbenes 10. Tanins

Alkaloids

Terpenoids

1. Acridines 2. Betalaines 3. Quin uinoliz oliziidin dines 4. Furono Furonoqui qui n none oness 5. Harringtonines 6. Iso Isoquin quinol olin inees 7. Indoles 8. Purines 9. Pyridi Pyridines nes 10. Tropane alkaloids

1. 2. 3. 4. 5.

Adapted from Stockigt et al. (1995).

Quinones

Carotenes 1. Anthroquinones Monoterpenes 2. Benzoquinones Sesq Sesqui uite terp rpen enes es 3. Naph Naphth thoq oqu uinon inonees Diterp Diterpene eness Triterpenes

Steroids 1. Cardiac glycosides 2. Pregnenolone deri deriva vati tive vess


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Table 4 Food additives from plant cell cultures Product type Colours Anthocyanins

Betalaines Crocin Crocetins Carotenoids Anthraquinones Naphthoquinones Naphthoquinones Flavours Vanillin Garlic Onion Basmati Citrus flavour Cocoa flavour Pungent food additive Capsaicin Sweeteners Stevioside Glycyrrhizin Thaumatin Essential oils Mint oil Chamomile oil Jasmine oil Aniseed oil

Plant species

Reference

V. vinifera Euphorbia spp. D. carota Pe. frutescens B. vulgaris Cheno. rubrum Crocus sativus Gardenia jasminoides Lycopersicon Lycopersicon esculentum Cinchon. ledgeriana M. citrifolia L. erythrorhizon erythrorhizon

Pepin Pepin et al. (1995) (1995) Yamamoto et al. (1982) Rajendra Rajendran n et al. (1994) (1994) Zhong and Yoshida (1995) Klebnikov et al. (1995) Berlin Berlin et al. (1986) (1986) Sujata Sujata et al. (1990) (1990) George and Ravishankar (1995) Fosket Fosket and Radin Radin (1983) (1983) Robbins and Rhodes (1986) Kieran Kieran et al. (1993) (1993) Sim and Chang Chang (1993) (1993)

Va. planifolia Allium sativum Allium cepa Oryza sativa Citrus spp. Theobromo cacao

Dornenburg and Knorr (1996) Ohsumi Ohsumi et al. (1993) (1993) Collin and Masker (1988) Suvarnalatha et al. (1994) Cresswell (1990) Townsley (1972)

Ca. frutescens Ca. annuum

Lindsey and Yeoman (1984) Johnson Johnson et al. (1990) (1990)

Stevia rebaudiana rebaudiana Glycyrrhiza glabra Thaumatococcus danielli

Swanson Swanson et al. (1992) (1992) Hayashi Hayashi et al. (1988) (1988) van der Wel and Ledeboer Ledeboer (1989) (1989)

Mentha piperata Ma. chamomilla Jasmine officinale Pim. anisum

Chung Chung et al. (1994) (1994) Kireeva Kireeva et al. (1978) (1978) Banthrope (1994) Ernst (1989)

Adapted from Ramachandra Rao (1998).

It is independent of political interference. Efficient downstream recovery and product. Rapidity of production. In addition, plant cell can perform stereo- and regiospecific biotransformations for the production of novel compounds from cheap precursors. Ther Theree are are a nu numbe mberr of plan plantt cell cell cultu culture ress prod produc ucin ing g a hig highe herr amou amount nt of seco second ndar ary y meta metabo boli lite tess than than in inta intact ct plan plants ts (Tab (Table le 5). 5). Howe Howeve verr, ther theree are are stil stilll prob proble lems ms in the the production of metabolites by cell cultures resulting from the instability of cell lines, low yiel yields ds,, slo slow w grow growth th and and scal scalee-up up prob proble lems ms (Rav (Ravis isha hank nkar ar and and Venka enkata tara rama man, n, 19 1993 93). ).

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Table 5 High yields of secondary products Product

Plant species

Yield (% D.W.)

Reference

Rosmarinic acid Rosmarinic acid Anthroquinones Shikonin Berberine Jatrorhizine Anthocyanins Berberine Diosgenin Sanguinarine Serpentine

Sa. officinalis Col. blumei M. citrifolia L. erythrorhizon Th. minus Berberis wilsonae Pe. frutescens C. japonica Diosc. deltoidea P. somniferum Cath. roseus

36.0 21.4 18.0 12.4 10.6 10.0 8.9 7.5 3.8 2.5 2.2

Hippolyte et al. (1992) Ulbrich et al. (1985) Zenk et al. (1975) Fujita (1988) Kobayashi et al. (1988) Breuling et al. (1985) Zhong et al. (1994) Matsubara et al. (1989) Sahai and Knuth (1985) Park et al. (1992) Zenk et al. (1977)

Adapted from Ravishankar and Ramachandra Rao (2000).

Several strategies have been adopted for the enhancement of metabolites (Table 6). There has been tremendous success in the production of high-value secondary metabolites such as shikonin from cell cultures of Lithospermum erythrorhizon, berberine from Coptis japonica and sanguinarine from Papaver somniferum. These are produced at industrial levels.

4. Strategies to increase secondary metabolite production in plant cell cultures

During the past decade, a considerable progress has been made to stimulate formation and accumulation of secondary metabolites using plant cell cultures (Ravishankar and Ramachandra Rao, 2000; Dixon, 1999; Ravishankar and Venkataraman, 1993; Buitelaar and Tramper, 1992; Fowler and Stafford, 1992; Payne et al., 1991). The adopted strategies for enhancing the secondary metabolites of plant cell cultures have been described in detail.

Table 6 Strategies to enhance production of secondary metabolites in plant cell cultures 1. Obtaining efficient cell lines for growth 2. Screening of high-growth cell line to produce metabolites of interest a. Mutation of cells b. Amenability to media alterations for higher yields 3. Immobilization of cells to enhance yields of extracellular metabolites and to facilitate biotransformations 4. Use of elicitors to enhance productivity in a short period of time 5. Permeation of metabolites to facilitate downstream processing 6. Adsorption of the metabolites to partition the products from the medium and to overcome feedback inhibition 7. Scale-up of cell cultures in suitable bioreactors


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4.1. Screening and selection of highly productive cell lines

Strain improvement begins with the choice of a parent plant with high contents of the desired products for callus induction to obtain high-producing cell lines. Then, a screening of the heterogeneous population for variant cell clones containing the highest levels of desired product was employed. The heterogeneity in the biochemical activity existing within a population of cells has been exploited to obtain highly productive cell lines (Ogino et al., 1978). Selection can be easily achieved if the product of interest is a pigment. For example, in cultures of L. erythrorhizon, extensive screening of a number of clones resulted in a 13–20-fold increase in shikonin production (Fujita et al., 1984). Enhanced anthocyanin production by clonal selection and visual screening has been reported in Euphorbia milli and Daucus carota (Yamamoto et al., 1982; Dougall, 1980). Other techniques such as HPLC and RIA were also used to screen for high-yielding cell lines (Matsumoto et al., 1980; Zenk, 1978). Mutation strategies have also been employed in order to obtain overproducing cell lines. The use of selective agents has been employed as an alternative approach to select highyielding cell lines (Rhodes et al., 1988). In this method, a large population of cells is exposed to a toxic (or cytotoxic) inhibitor or environmental stress and only cells that are able to resist the selection procedures will grow. p-Fluorophenylalanine (PFP), an analogue of phenylalanine, was extensively used to select high-yielding cell lines with respect to phenolics (Berlin, 1980). Increased capsaicin and rosmarinic acid in PFP cell lines of Capsicum annuum and Anchusa were reported (Salgado-Garciglia and Ochoa-Alejo, 1990; Quesnell and Ellis, 1989). Other selective agents such as 5-methyltryptophan, glyphosate and biotin have also been used to select high-yielding cell lines (Amrhein et al., 1985; Wataneba et al., 1982; Widholm, 1974). 4.2. Manipulation of nutrients to improve yield

Manipulation of the culture environment must be effective in increasing the product accumulation. The expression of many secondary metabolite pathways is easily altered by external factors such as nutrient levels, stress factors, light and growth regulators. Many of the constituents of plant cell culture media are important determinants of growth and accumulation of secondary metabolites (Stafford et al., 1986; Misawa, 1985). 4.2.1. Sugar levels Plant cell cultures are usually grown heterotrophically using simple sugars as carbon source and inorganic supply of other nutrients. The level of sucrose has been shown to affect the productivity of secondary metabolite-accumulating cultures. Thus, sucrose concentrations of 2.5% (w/v) and 7.5% (w/v) in Coleus blumei media brought about rosmarinic acid yields of 0.8 and 3.3 g/l, respectively (Misawa, 1985). For indole alkaloid accumulation in cell culture as of Catharanthus roseus, 8% (w/v) sucrose was found to be optimal in the tested concentration range of 4 – 12% (w/v) (Knobloch and Berlin, 1980). Yields of benzophenanthridine alkaloids from suspension cultures of Eschscholtzia californica were

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increased 10-fold to around 150 mg/l by increasing the sucrose concentration to 8% (w/v) (Berlin et al., 1983). The osmotic stress created by sucrose alone and with other osmotic agents were found to regulate anthocyanin production in Vitis vinifera cell suspension cultures (Do and Cormier, 1990). A dual role of sucrose as carbon source and osmotic agent was observed in Solanum melongena (Mukherjee et al., 1991). However, higher concentrations of sucrose at 5% (w/v) reduced the anthocyanin production in cell suspension cultures of Aralia cordata, where 3% (w/v) favoured the anthocyanin accumulation (Sakamoto et al., 1993). 4.2.2. Nitrate levels Nitrogen concentration was found to affect the level of proteinaceous or amino acid products in cell suspension cultures. The plant tissue culture medium such as MS, LS or B5 has both nitrate and ammonium as sources of nitrogen. However, the ratio of the ammonium/ nitrate–nitrogen and overall levels of total nitrogen have been shown to markedly affect the production of secondary plant products. For example, reduced levels of NH 4 + and increased levels of NO3  promoted the production of shikonin and betacyanins, whereas higher ratios of NH4 + /NO3  increased the production of berberine and ubiquinone (Bohm and Rink, 1988; Nakagawa et al., 1984; Fujita et al., 1981; Ikeda et al., 1977). Reduced levels of total nitrogen improved the production of capsaicin in Capsicum frutescens, anthraquinones in Morinda citrifolia and anthocyanins in Vitis species (Yamakawa et al., 1983; Yeoman et al., 1980; Zenk et al., 1975). However, complete elimination of nitrate in cultures of Chrysanthemum cinerariaefolium induced twofold increases in pyrethrin accumulation in the second phase of culture (Rajasekaran et al., 1991). 4.2.3. Phosphate levels The phosphate concentration in the medium can have a major effect on the production of secondary metabolites in plant cell cultures. Higher levels of phosphate were found to enhance the cell growth, where it had negative influence on secondary product accumulation. Sasse et al. (1982) have given a number of examples to show that a medium limited in phosphate either induces or stimulates both the product and the levels of key enzymes leading to the product. Thus, reduced phosphate levels induced the production of ajmalicine and phenolics in Cath. roseus, of caffeoyl putrescines in Nicotiana tabacum and of harman alkaloids in Peganum harmala. Similar results were obtained in a separate study for the production of betacyanins in callus cultures of Beta vulgaris (Bohm and Rink, 1988). In contrast, increased phosphate was shown to stimulate synthesis of digitoxin in Digitalis purpurea and of betacyanin in Chenopodium rubrum and Phytolacca americana (Bohm and Rink, 1988; Hagimori et al., 1982a,b). 4.2.4. Growth regulators Growth regulator concentration is often a crucial factor in secondary product accumulation (DiCosmo and Towers, 1984; Deus and Zenk, 1982). The type and concentration of auxin or cytokinin or the auxin/cytokinin ratio alters dramatically both the growth and the product formation in cultured plant cells (Mantell and Smith, 1984). The growth regulator 2,4-


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dichlorophenoxyacetic acid (2,4-D) has been shown to inhibit the production of secondary metabolites in a large number of cases. In such cases, elimination of 2,4-D or replacement of 2,4-D by naphthalene acetic acid (NAA) or indole acetic acid (IAA) has been shown to enhance the production of anthocyanins in suspensions of Populus and D. carota, of betacyanins in suspensions of Portulaca, of nicotine in suspensions of N. tabacum, of shikonin in suspensions of L. erythrorhizon and of anthraquinones in M. citrifolia (Rajendran et al., 1992; Seitz and Hinderer, 1988; Tabata, 1988; Sahai and Shuler, 1984; Bohm and Rink, 1988; Zenk et al., 1975). However, stimulation by 2,4-D has been observed in carotenoid biosynthesis in suspensions of D. carota (Mok et al., 1976) and in anthocyanin production in callus cultures of Oxalis linearis (Meyer and van Staden, 1995). Cytokinins have different effects depending on the type of metabolite and species concerned. Thus, kinetin stimulated the production of anthocyanin in Haplopappus gracilus but inhibited the formation of anthocyanins in Populus cell cultures (Seitz and Hinderer, 1988; Mok et al., 1976). Gibberellic acid and abscisic acid are reported to suppress production of anthocyanins in a number of cultures (Bohm and Rink, 1988; Seitz and Hinderer, 1988). 4.2.5. Precursor feeding Precursor feeding has been an obvious and popular approach to increase secondary metabolite production in plant cell cultures. The concept is based upon the idea that any compound, which is an intermediate, in or at the beginning of a secondary metabolite biosynthetic route, stands a good chance of increasing the yield of the final product. Attempts to induce or increase the production of plant secondary metabolites, by supplying precursor or intermediate compounds, have been effective in many cases. The addition of phenylalanine as a precursor led to improvement in rosmarinic acid yield in Col. blumei cell cultures (Ibrahim, 1987). Addition of phenylalanine to Salvia officinalis suspension cultures stimulated the production of rosmarinic acid and decreased the production time as well (Ellis and Towers, 1970). Phenylalanine is also the precursor of the N -benzoylphenylisoserine side chain of taxol, and supplementation of Taxus cuspidata cultures with phenylalanine resulted in increased yields of taxol (Fett-Neto et al., 1994, 1993). Use of the distant precursor phenylalanine and a near precursor such as isocapric acid resulted in enhanced capsaicin content in cell cultures of Ca. frutescens (Lindsey and Yeoman, 1985; Yeoman et al., 1980). Feeding of ferulic acid to cultures of Vanilla planifolia resulted in increase in vanillin accumulation (Romagnoli and Knorr, 1988). Similarly, anthocyanin synthesis in carrot cultures was restored by the addition of a dihydroquarcetin (naringen). Furthermore, addition of leucine led to enhancement of volatile monoterpenes a- and b-pinine in cultures of Perilla frutescens, whereas addition of geraniol to rose cell cultures led to accumulation of nerol and citronellol (Mulder-Krieger et al., 1988). 4.3. Optimizing the culture environment

Culture environmental conditions such as light, temperature, medium pH and oxygen have been examined for their effect upon secondary metabolite accumulation in many types of cultures.

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4.3.1. Temperature A temperature range of 17–25  C is normally used for the induction of callus tissues and growth of cultured cells. However, each plant species may favour a different temperature. Toivonen et al. (1992) found that lowering the cultivation temperature increased the total fatty acid content per cell in dry weight. When the temperature was maintained at 19 C, biotransformation of digitoxin to digoxin is favoured, whereas 32  C favours purpureaglycoside A formation in Digitalis lanata cell cultures (Kreis and Reinhard, 1992). Ikeda et al. (1977) observed a higher yield of ubiquinone in tobacco cell cultures at 32 C when compared to either 24 or 28 C. Courtois and Guren (1980) reported a 12-fold higher production of crude alkaloids in cell cultures of Cath. roseus at 16 C as compared to the normal 27 C. 4.3.2. Illumination Accumulation of anthocyanin was strongly stimulated by light in cell cultures of D. carota and Vitis hybrids (Seitz and Hinderer, 1988). Illumination was found to affect the composition of sesquiterpenes in callus cultures of Marticaria chamomilla (Mulder-Krieger et al., 1988). Exclusion of light in callus cultures of Citrus limon prompted the accumulation of monoterpenes (Mulder-Krieger et al., 1988). 4.3.3. Medium pH The medium pH is usually adjusted between 5 and 6 before autoclaving and extremes of pH are avoided. The concentration of hydrogen ions in the medium changes during the development of the culture. The medium pH decreases during ammonia assimilation and increases during nitrate uptake (McDonald and Jackman, 1989). Photoautotrophic cell suspension cultures of Cheno. rubrum showed that the increase in the external pH from 4.5 to 6.3 increased the cytosolic pH by 3.0 units and the vacuolar pH by about 1.3 units (Husemann et al., 1992). 4.3.4. Agitation and aeration The importance of aeration and agitation is crucial for large-scale production. Kreis and Reinhard (1989) described that the influence of dissolved oxygen levels of 50% allowed an alkaloid yield of around 3-g/l culture after 20 days of growth in an airlift bioreactor. Higher aeration rates produced a dramatic decrease in alkaloid productivity. It is evident that airlift and stirred tank bioreactors can allow similar secondary product levels in cultured plant cells. However, in stirred tank vessels, the characteristics of the stirrer may be critical (Kreis and Reinhard, 1989). Ambid and Fallot (1981) investigated the effect of the composition of the gaseous environment on production of volatiles by fruit suspension cultures. They reported that the addition of carbon dioxide stimulated the synthesis of monoterpenes by Muscat grape suspensions and induced the formation of linalool. Kobayashi et al. (1991) reported that the use of carbon dioxide at the 2% level was critical to prevent cell browning and to sustain berberine production in suspension cultures of Thalictrum minus in bubble column reactors.


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4.4. Elicitation

Plants produce secondary metabolites in nature as a defense mechanism against attack by pathogens. Plants have been found to elicit the same response as the pathogen itself when challenged by compounds of pathogenic origin (elicitors). Elicitors are signals triggering the formation of secondary metabolites. Secondary pathways are activated in response to stress. Biotic and abiotic elicitors are used to stimulate secondary metabolite product formation in plant cell cultures, thereby reducing the process time to attain high product concentrations and increased culture volumes (Barz et al., 1988; Eilert, 1987; DiCosmo and Tallevi, 1985). Elicitors of fungal, bacterial and yeast origin, viz. polysaccharides, glycoproteins, inactivated enzymes, purified curdlan, xanthan and chitosan, and salts of heavy metals were reported for the production of various secondary metabolites (Ramachandra Rao et al., 1996a,b; Rajendran et al., 1994; Guo et al., 1992; Furze et al., 1991; Johnson et al., 1991; Robbins et al., 1991; DiCosmo et al., 1987; Funk et al., 1987). Treatment of P. somniferum cell suspensions with a homogenate of Botrytis mycelium resulted in a remarkable accumulation of sanguinarine of up to 3% of the cell dry weight (Constabel, 1990). The treatment of root cultures of Datura stramonium with copper and cadmium salts has been found to induce the rapid accumulation of high levels of sesquiterpenoid defensive compounds (Furze et al., 1991).

4.5. Permeabilization

In most cases, the products formed by plant cell cultures are stored in the vacuoles. In order to release the products from vacuoles of plant cells, two membrane barriers — plasma membrane and tonoplast — have to be penetrated. Cell permeabilization depends on the formation of pores in one or more of the membrane systems of the plant cell, enabling the passage of various molecules into and out of the cell. The permeability of the cells can be monitored by measuring the activity of enzymes of the primary metabolism, viz. hexokinase, glucose 6-phosphate dehydrogenase, isocitrate dehydrogenase, malic and citrate synthetase (Brodelius, 1988b). Attempts have been made to permeabilize the plant cells transiently, to maintain the cell viability and to have short time periods of increased mass transfer of substrate and metabolites to and from the cell (Parr et al., 1987; Brodelius and Nilsson, 1983). A wide variety of permeabilizing agents are used to enhance the accessibility of enzymes or to provoke release of intracellular stored product (Berlin et al., 1989; Knorr et al., 1985; Parr et al., 1984; Felix, 1982). Organic solvents such as isopropanol, dimethylsulfoxide (DMSO) and polysaccharides like chitosan have been used as permeabilizing agents (Van Uden et al., 1990; Beaumont and Knorr, 1987; Knorr and Teutonico, 1986). Other permeabilization methods include ultrasonication, electroporation and ionophoretic release, in which the cells are subjected to a low current in a specially designed device (Brodelius et al., 1988). In addition, using high electric field pulses and ultrahigh pressure (Dornenburg and Knorr, 1993) has been reported for the recovery of secondary metabolites.

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4.6. In situ product removal

A low accumulation of secondary compounds in cell cultures in a number of cases may not be due to a lack of key biosynthetic enzymes but rather due to feedback inhibition, enzymatic or nonenzymatic degradation of the product in the medium or volatility of substances produced. In such cases, it should be possible to increase the net production by the addition of an artificial site for product accumulation, for example, by use of second solid or liquid phase introduced into the aqueous medium. Such ‘‘three phase systems’’ accumulate traces of secondary metabolites from the culture medium. The use of in situ product removal of metabolites has a number of key potential advantages beyond promoting secretion. The removal and sequestering of the product in a nonbiological compartment may increase its total production (Beiderbeck and Knoop, 1988). Robbins and Rhodes (1986) reported that addition of amberlite XAD-7 resin stimulated the production of anthraquinones by 15 times compared to a medium without adsorbent. Addition of charcoal led to 20–60-fold improvements in yields of coniferyl aldehyde in Ma. chamomilla suspensions (Beiderbeck and Knoop, 1987). The addition of Miglyol or silica gel RP8 stimulated ethanol production in cell cultures of Pimpinella anisum (Mulder-Krieger et al., 1988). The addition of XAD-4 increased the vanilla flavour production in Vanilla fragrans cell suspension cultures (Knuth and Sahai, 1991).

4.7.  -Cyclodextrins

Many plant cell cultures hardly convert precursors in the presence of organic phases, often as a result of dramatic decrease of cell viability. Therefore, the enzymatic activities are reduced in these systems. A new approach to solve this problem of bioconversion of water-insoluble precursors is to combine the advantages of apolar systems (higher solubility if the substrate) and aqueous systems (maintenance of cell viability) by carrying out bioconversions in the presence of clathering agents such as cyclodextrins. Cyclodextrins have the ability to form stable inclusion complexes with natural spices and flavour substances in their cyclodextrin cavity (Haggin, 1992). Cyclodextrins may be modified through substituting various functional compounds on the primary or secondary phase of the molecule. The chemically modified cyclodextrins are more water soluble than native cyclodextrins (Eastburn and Tao, 1994; Szejtli, 1988). Through complexation, the physical properties of ligands are changed, including their solubility in aqueous media (Qi and Hedges, 1995; Van Uden et al., 1994; Szejtli, 1986, 1982). The hydroxylation of b-estradiol to 4-hydroxy and 2-hydroxy b-estradiol was enhanced in the presence of b-cyclodextrin in Mucuna pruriens cell cultures (Woerdenbag et al., 1990a) and multistep conversion of coniferyl alcohol into podophyllotoxin was demonstrated in Podophyllum hexandrum cell suspension cultures (Woerdenbag et al., 1990b). Cyclodextrin complexation of flavours provides protection against the damaging factors of the environment (Qi and Hedges, 1995).


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5. Organ cultures as a source of pharmaceuticals

Since production of secondary metabolites is generally higher in differentiated tissues, there are attempts to cultivate shoot cultures and root cultures for the production of medicinally important compounds. These organ cultures are relatively more stable (Roja, 1994). There are a number of medicinal plants whose shoot cultures have been studied for metabolites (Table 7). Similarly, root cultures are valuable sources of medicinal compounds (Table 8). Root systems of higher plants generally exhibit slower growth and are difficult to harvest. Hence, alternative methods need to be found for root-derived compounds. Until now, however, there is no commercial process as an alternative for root-derived compounds, except in case of utilizing hairy root culture systems, which has been described in this review.

6. Hairy root cultures for pharmaceuticals

The ability of Agrobacterium rhizogenes to induce hairy roots in a range of host plants has lead to studies on it as a source of root-derived pharmaceuticals (Flores et al., 1999). Tepfer (1990) summarized 116 plants belonging to 30 dicotyledonous families wherein hairy roots have been induced. Hairy roots are induced by transfer of T-DNA from the plasmid of Agr. rhizogenes (Ambros et al., 1986; Petit et al., 1983) to host tissue, resulting in root formation by virtue of auxin synthesis genes coded by bacterial DNA. The Ri plasmid of Agr. rhizogenes also elicits the synthesis of opines such as agropine or mannopine. The transformed nature of the roots is genetically checked by opine detection or Southern analysis. The interest in hairy roots is mainly due to their ability to grow fast without needing an external supply of auxins. Many times, they do not need incubation under light. They are fairly well stable in metabolite yield due to their genetic stability. Because of these Table 7 Shoot cultures of medicinal plants Plant species

Product

Reference

Artemi. annua Atro. belladona Begonia spp. Cath. roseus Cinchona spp.

Artemesinin Atropine – Vindoline Vinblastine Quinine Cardenolides Cardenolides Essential oils Kutkin Steviosides Withanolides Indirubin Alkaloids

Park et al. (1989) Benjamin et al. (1987) Takayama and Misawa (1981) Staba and Chung (1981) Krueger et al. (1982) Hirata et al. (1987) Lui and Staba (1979) Hagimori et al. (1982a,b) Charlwood and Moustou (1988) Upadhyay et al. (1989) Akita et al. (1994) Heble (1985) Shim et al. (1998) Konishi et al. (1998)

Di. lanata Di. purpurea Pelargonium tomentosum Picrprrhiza kurroa Ste. rebaudiana Withania somniferum Polygonum tinctorium Decentra pergrina


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Table 8 Root cultures of medicinal plants Plant species

Product

Reference

B. vulgaris Bidens alba Calystegia sepium Coreopsis tinctoria Hyoscamus albus Hyoscamus muticus Hemidesmus indicus Artemisia absynthium Polygonum tinctorium

Betalaines Polyacetylenes Tropane alkaloids Phenylpropanoids Hyoscyamine Hyoscyamine 2-hydroxy-4-methoxybenzaldehyde Volatile oils Indigo, Indirubin

Hamill et al. (1986) Norton and Towers (1986) Jung and Tepfer (1987) Thron et al. (1989) Hashimoto and Yamada (1986) Flores et al. (1987) Sreekumar et al. (1998) Kennedy et al. (1993) Shim et al. (1998)

Adapter from Payne et al. (1991).

advantages, many of the root-derived plant products once not considered feasible for production by cell culture are being reinvestigated for production using the hairy root culture technology. A number of such examples are given in Table 9. Several factors influence the yield of secondary metabolites of pharmaceutical interest in hairy root cultures. These are nutrients, elicitors and biotransformations of precursors to products and genetic manipulation through the Ri plasmid of Agr. rhizogenes. Several hairy roots have been put to scale-up studies in bioreactors. However, due to their structural features and metabolite localization characteristics, they need different type of reactors than the ones used for plant cell cultures. Hairy roots need anchorage for growth Table 9 Hairy root cultures producing pharmaceutical products of interest Plant species

Product

Reference

Bidens spp. Cinchon. ledgeriana Cichorium intybus Datura spp. Cassia spp. Duboisia leichhardtii Echinacea purpurea Glycyrrhiza uralensis Hy. albus Panax ginseng Salvia miltorrhiza Artemi. absynthium L. erythrorhizon Ra. serpentina Rubia cordifolia Gl. glabra Panax ginseng Hy. muticus

Polyacetylenes Quinolene alkaloids Esculetin Tropane Anthroquinones Tropane alkaloids Alkaloids Glycyrrhizin Alkaloids Saponin Diterpenes Volatile oil Shikonin Ajmaline, serpentine Anthroquinones Isoprenylated flavonoids Ginsenoside Hyoscyamine

McKinely et al. (1993) Hamill et al. (1987) Bais et al. (1999) Rhodes (1989) Ko et al. (1988) Mano et al. (1989) Trypsteen et al. (1991) Ko et al. (1989) Shimomura et al. (1991) Yoshikawa and Furuya (1987) Hu and Alfermann (1993) Kennedy et al. (1993) Shimomura et al. (1986) Benjamin et al. (1994) Shin and Kim (1996) Asada et al. (1998) Kunshi et al. (1998) Sevon et al. (1998)



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since they are highly branched (Fig. 1). Various configurations of hairy root bioreactors are given in Fig. 2. Certain hairy roots are tolerant to submerged cultivations. Taya et al. (1989) found that stirred tank reactors are poor for cultivation of B. vulgaris. In our experience, hairy roots of B. vulgaris do not produce betalaines when submerged totally. In shake flask cultures, they proliferate fast and produce betalaines after achieving a stationary phase of growth. Air-sparging without impeller-driven agitation may also be helpful as in the case of hairy roots of Nicotiana rustica (Rodriguez-Mendiola et al., 1991). We have found that use of airsparging system is suitable for chicory hairy roots for esculin and esculetins production (Bais et al., 1999). Therefore, any single design of reactor will not be suitable for all hairy roots. Most remarkable developments of scale-up in large vessels have been in the cultivation of Panax ginseng hairy root biomass in a 20-ton cultivation tank (Scheidegger, 1990). Hairy roots also produce valuable compounds by biotransformations (Table 10). Recently, an innovative approach of coculture of hairy roots of Atropa belladona and shooty teratomas of scopolamine-rich Duboisia plants was done, wherein hyoscyamine released by the former was bioconverted to scopolamine by the latter tissues (Subroto et al., 1996). However, these systems need to be studied carefully within parameters of uptake mechanisms, biosynthetic potential of the tissue and, if possible, the extracellular release of the final product. Thus, hairy root technology will be useful in not only producing root-derived compounds but also to produce novel compounds by biotransformations. The ability to engineer the genomic DNA of hairy root through tailormade Ri plasmid will be a tremendous use in producing novel compounds. Using this systems, it may be possible to

Fig. 1. Highly branched hairy root cultures of Cichorium intybus .

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Fig. 2. Configuration of hairy root bioreactors.

produce more than one product with a single type of hairy root. There already are attempts in this direction as exemplified by the introduction of cDNA of ornithine decarboxylase (ODC)


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Table 10 Biotransformations using hairy root cultures for production of pharmaceuticals Plant species

Substrate

Product

Reference

Hy. niger

Hyoscyamine

Scopolamine

Cinchon. ledgeriana Nicotiana spp.

Tryptophan Lysine, cadaverine

Quinine Nicotine

Putrescine, agmatine Putrescine Spermidine Cadaverine 2-phenylpropanoic acid Digitoxigenin Hyoscyamine Hyoscyamine 18b-glycyrrhetinic acid Butylated hydroxytoluene Phenolic acid

Anabasine Scopolamine Hyoscyamine

Hashimoto and Yamada (1983) Hay et al. (1986) Walton and Belshaw (1988) Walton et al. (1988) Yoshioka et al. (1989)

Duboisia myoporoides

Panax ginseng Panax ginseng Atro. belladona Dubo. leichhardtii Panax ginseng Bidens sulphureus Panax ginseng

Sugar esters Digitoxin Scopolamine Scopolamine Glucosides Stilbene quinones Glycosylated phenolic compounds

Furuya et al. (1989) Kawaguchi et al. (1990) Subroto et al. (1996) Subroto et al. (1996) Asada et al. (1993) Flores et al. (1994) Ushiyama and Furuya (1989)

from yeast into N. rustica. ODC enhances the formation of alkaloid nicotine since it is the first step of pyrrolidine alkaloid biosynthesis. Hyoscyamine 6b-hydroxylase (H6H) enzyme that catalyzes epoxidative conversion of hyoscyamine to scopolamine is an important step in the biogenetic pathway (Yamada and Hashimoto, 1988), which has been induced in Atro. belladona by the H6H gene from Hyoscyamus niger . The hairy roots of Atro. belladona produced high amounts of scopolamine (Hashimoto et al., 1993). Such approaches would enhance the utility of hairy roots to produce novel compounds. Most importantly, these compounds are produced at shorter periods of time than in intact plants since the growth cycle in vitro is shorter than in vivo conditions.

7. Production of foreign proteins in transgenic plants

Recently, transgenic plants are considered to be economically competitive production systems for the manufacture of variety of foreign proteins. These include recombinant antibodies and antibody fragments (James and Lee, 2001; Sharp and Doran, 2001; Doran, 2000; Smith and Glick, 2000; Wongsamuth and Doran, 1997; Tavladoraki et al., 1993; Hein et al., 1991; Hiatt et al., 1989), enzymes such as b-glucuronidase (Kurata et al., 1998) and invertase (Verdelhan des Molles et al., 1999) and proteins of therapeutic value such as human interleukin (IL)-2 and IL-4 (Magnuson et al., 1998), ribosome-inactivating proteins (Remi shih et al., 1998; Francisco et al., 1997), ricin (Sehnke and Ferl, 1999) and human a1-antitrypsin (Terashima et al., 1999a,b). The most commonly used host species for protein synthesis in suspension cultures is tobacco (Sharp and Doran, 1999; Liu and Lee, 1999; Kurata et al., 1998; Wongsamuth and Doran, 1997), although rice cell suspension cultures

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have also been used (Terashima et al., 1999a,b). Hiatt et al. (1989) estimated that plantderived antibodies are likely to cost less than the ones derived from hybridoma cells and that plant cell media are composed of simple sugars and salts less complex than mammalian media. Consequently, purification of secreted protein is simpler and more economical. Plant cell-derived proteins are likely to be safer than those derived from other systems, since plant cell pathogens are not harmful to humans. The expression of these heterologous proteins in plants has an advantage over their expression in the microbial system, since full-length antibodies have been produced in plants (De Wilde et al., 1996). On the other hand, Escherichia coli produced only a monovalent Fab fragment, the largest antibody found in a microbe (Smith, 1996). Hairy roots of tobacco ( N. tabacum) were used to produce full-length murine IgG1 monoclonal antibody and the maximum production of 18 mg/l was reported in 21-day grown shake flasks, and up to 14% of the antibody was secreted into the medium. The decline in antibody production was found after 14 days of cultivation and mainly due to its degradation. Antibody production by transgenic hairy roots has a negligible effect on growth compared with hairy roots of wild-type tobacco (Wongsamuth and Doran, 1997). The loss of protein due to product instability during plant cell culture and subsequent purification significantly influences the product yields due to occurrence of unfavourable environmental conditions. Protein stability following secretion improved with the addition of appropriate chemical stabilizers. Increased recovery of a mouse monoclonal antibody heavy chain by the addition of DMSO, gelatin and polyvinylpyrrolidone (Magnuson et al., 1998; Wongsamuth and Doran, 1997) was reported. The other new protein stabilizer, bacitracin, enhanced the cell growth (Sharp and Doran, 1999) and inhibit the degradation of plant-derived proteins (Bateman et al., 1997). Full-length antibodies were first expressed by Hiatt et al. (1989). They have expressed a monoclonal antibody 6D4 from a mouse hybridoma cell line in tobacco. The k and g chains of the IgG molecule was expressed with or without mammalian leader sequence. The transgenic lines upon crossing produced plants with expression of both the chains. However, the plants without leader sequence did not show assembled g – k complexes and hence lacked functional characteristics. The use of plant-derived endoplasmic reticulum (ER) targeting sequence has been studied by Du¨ring et al. (1990), with demonstration of accumulation of antibody in ER of transgenic tobacco. De Wilde et al. (1996) found that both full-length IgG molecules and Fab fragments accumulated in the intercellular spaces of mesophyll cells in leaves of the transgenic Arabidopsis thaliana. Their study with immunolocalization confirms the production of full-length antibody and its secretion by plant cells. Moreover, in plants, single cells are able to assemble secretory antibodies, whereas two different cell types are required in mammals. Ma et al. (1995) have obtained transgenic N. tabacum that expressed a murine monoclonal antibody k chain, a hybrid IgA–IgG heavy chain, a murine joining in chain and a rabbit secretory component. These chains were assembled into functional, high-molecular-weight secretory immunoglobulins that recognized the native Streptococcal antigen I/II, a surface adhesion molecule. Plant cells also possess the requisite mechanism for the assembly and expression of other complex recombinant protein molecules. The ability to produce monoclonal antibodies in


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plants also led to the opportunity to develop an inexpensive method of mucosal immunoprotection against genital herpes (Zetlin et al., 1998). The expression of the production of a full-length functional antibody in algal forms has also been reported (Conrad and Fiedler, 1994). Smith (1996) states that the folding of full-size immunoglo bulins in algae is possibly due to its less reducing cytoplasm. This cytoplasm contains chaperonins, which facilitate folding. Ma et al. (1994) have studied the prospects of using plant-produced antibodies in passive immunization against the cell surface antigen of Streptomyces mutans , which cause tooth decay. The fact that plant-derived antibodies have been approved for clinical trials (Ma and Hein, 1995) is an encouraging development. However, R&D of large-scale antibody production in plants should be pursued by developing systems of higher expression using easy and efficient downstream processing and the large-scale cultivation of transgenic plants in controlled greenhouses to drive this exciting research area into the realm of application. The storage of antibodies for extended periods can be made possible by expressing them in seeds (Fiedler and Conrad, 1995). 7.1. Vaccines

The ability to express genes encoding the antigens of bacterial and viral pathogens in plants, with retention of immunogenic properties, is bound to revolutionize vaccine production. The concept of edible vaccine stemmed from the landmark discovery made by Mason et al. (1992). Plant systems will be a choice, which may be relatively less expensive for production as well as storage of antigens. Strategies for candidate vaccine production are of two types: (1) stable genomic integration with foreign DNA introduced either by Agrobacterium T-DNA vectors or by microprojectile bombardment and (2) transient expression using viral vectors (Mason and Arntzen, 1995). The first system affords stable transformation, which may be heritable. Furthermore, the expression could be targeted to specific loci using appropriate promoters. The second system involves the use of viral vectors for transient expression in plants, a potentially useful means of producing high levels of recombinant antigens. Foreign protein expression could be achieved either by foreign gene transcription using a subgenomic promoter or the fusion of foreign proteins to peptides, which contain the capsid protein that normally coats the virus (Mason and Arntzen, 1995). High levels of a-trichosanthin, an antiviral protein in transformed plants (Kumagai et al., 1993), have been obtained by the foreign gene transcription method. Turpen et al. (1995) have developed a method of expressing the coat promoter of the tobacco mosaic virus (TMV) using peptides of epitopes derived from malarial sporozoites. The antigenicity measured by ELISA and Western blot demonstrate that the recombinant coat protein recognized appropriate monoclonal, antimalarial antibodies. Similarly, Cowpea mosaic virus has also been used for the expression of foreign peptides such as antigens from the rabies virus and HIV-1 (Yusibov et al., 1997). Stable genomic transformation encoding foreign antigens has been demonstrated in case of hepatitis B surface antigens as well as E. coli and Vibrio cholerae (Thanavalla et al., 1995).

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One can produce edible vaccines by cloning the genes of fruit plants and expressing the vaccines in the edible parts of the fruit. However, it is necessary to address the problem such as oral tolerance (Mahon et al., 1998) and the standardization of dosage requirement for the scale of protection. These topics need to be studied along with ecological considerations on a case-by-case basis to get approval for each vaccine production and administration to target groups.

8. Immobilization of plant cells for the production of secondary metabolites

Improvement in the secondary metabolite production of cell cultures is often associated with the organization and differentiation of plant cells. The concept of organization and differentiation led to the use of immobilization technology, which has long been used for microbes and enzymes. Immobilization is defined as a technique, which confines a catalytically active enzyme or cells on a fixed support and prevents its entry into liquid phase (Yeoman et al., 1990; Yeoman, 1987; Fowler, 1986; Lindsey and Yeoman, 1985, 1983a,b). Immobilized plant cells have been used for single and multistep biotransformations of precursors to desired products as well as for the de novo biosynthesis of secondary metabolites. Ever since, Brodelius et al. (1979) described the entrapment of viable cells of Cath. roseus , M. citrifolia and Di. lanata in calcium alginate gel and this technique has received much attention. Extensive reviews on various aspects of plant cell immobilization are available (Williams and Mavituna, 1992; Payne et al., 1991; Scragg, 1991; Hulst and Tramper, 1989; Brodelius 1988a; Hall et al., 1988; Lindsey and Yeoman, 1987). Immobilization of plant cells has distinct advantages as biocatalyst over the immobilized enzyme system. It is necessary to provide the immobilized enzyme with proper pH, the flow of reaction mixture temperature and as supply of cofactors. Immobilized enzymes are generally applied to single-step reactions. Furthermore, there will be loss in activity during isolation of enzymes from the organism. The advantage of immobilized enzyme is the high rate of activity. In contrast to immobilized enzymes, immobilized cells have distinct advantages: (a) it can carry out multienzyme operations; (b) by selecting highly biosynthetic cells, catalytic activity can be enhanced; (c) there is no need to provide cofactors since cells themselves produce them and (d) immobilized cells can be easily handled as compared to immobilized enzymes. Thus, immobilized cells are gaining much importance as biocatalysts. The following prerequisites are essential to adopt immobilization for secondary metabolite production (Payne et al., 1991). The product of interest should not be strictly growth associated. Growth of cells should be suppressed to prevent disintegration of the immobilization matrix, which may lead to disruption of the process. Immobilized cells should maintain prolonged viability and biosynthetic capacity with high rates of sustainable secondary metabolite production. The product should leach out of the cells and beads into the medium.


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The most widely used technique involves the entrapment of cells, in some kind of gel or combination of gels, which are allowed to polymerize around them (Novais, 1988). Calcium alginate is the most widely used matrix. Besides this, other gels such as agar, agarose, gelatin, carrageenan and polyacrylamide have also been used (Nilsson et al., 1983). However, gels of alginate are most widely used because of their simplicity and relatively lack of toxicity. The other alternative supports are polyurethane foam and hollowfibre membranes. Table 11 gives a number of examples of the systems of immobilization, which have been used with plant cells together with the associated plant species and their products.

Table 11 Immobilized plant cell systems used for production of secondary metabolites Plant species

Substrate/precursor Product

Immobilization method Reference

Bioconversions Cath. roseus Di. lanata D. carota N. tabacum P. somniferum

Cathenamine Digitoxin Digitoxigenin Keto esters Codeinone

Ajmalicine Digoxin Periplogenin Hydroxy esters Codeine

Agarose Alginate Alginate Alginate Polyurethane foam

L-Tyrosine Ferulic acid, vanillylamine

L-DOPA Vanillin, capsaicin

Alginate Alginate

Felix et al. (1981) Brodelius et al. (1979) Jones and Veliky (1981) Naoshima and Akakabe (1989) Furuya et al. (1984) Furusaki et al. (1988) Wichers et al. (1983) Johnson et al. (1996)

Ajmalicine

Alginate, agarose

Brodelius et al. (1979)

Capsaicin

Polyurethane foam

Brodelius and Nilsson (1980) Lindsey and Yeoman (1983b) Lindsey and Yeoman (1984)

Caffeine Caffeoyl putrescine

Membrane Alginate

Lang et al. (1990) Berlin et al. (1989)

Capsaicin Ajmalicine Phenolics Pigments Diosgenin Berberine Thiophenes Anthraquinones

Polyurethane foam Alginate, agarose Hollow fibres Polyurethane foam Polyurethane foam Alginate Alginate Alginate

Johnson (1993) Brodelius and Nilsson (1980) Prenosil and Pedersen (1983) Lindsey and Yeoman (1984) Ishida (1988) Kobayashi et al. (1987) Ketel et al. (1987) Brodelius et al. (1979)

Muc. pruriens Capsicum spp.

Synthesis from precursors Cath. roseus Tryptamine, secologanin Isocapric acid, Ca. frutescens vanillylamine, valine and ferulic acid Coffea arabica N. tabacum

Theobromine Phenylalanine

De novo synthesis Ca. frutescens Cath. roseus Glycine max Lavandula vera Diosc. deltoidea Th. minus Tagetes patula M. citrifolia

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Table 12 Dramatic effects of immobilization on secondary metabolite production in plant cell cultures Plant species

Product

Fold change

Ca. frutescens Ca. frutescens Tag. patula Coffea arabica Cath. roseus Cath. roseus

Capsaicin Capsaicin Thiophenes Methylxanthin Ajamlicine Total alkaloids

Muc. pruriens

L-DOPA

Salvia miltiorrhiza

Cryptotanshinone

> 100 (I) > 100 (I) Ca 20 (I) 13 (I) 3.5 (I) No de novo synthesis No de novo synthesis 2.5 (D)

Type of immobilization

Reference

Foam Gel Natural glass Gel Gel Membrane

Lindsey and Yeoman (1984) Ravishankar et al. (1988) Hulst and Tramper (1989) Haldimann and Brodelius (1987) Asada and Shuler (1989) Payne et al. (1988)

Gel

Wichers et al. (1983)

Gel

Miyasaka et al. (1986)

(I): increase, (D): decrease. Adapted from Payne et al. (1991).

Immobilization can have a dramatic impact on cellular physiology and secondary product formation. The more dramatic responses are summarized in Table 12.

9. Biotransformations for the production of secondary metabolites

Biotransformation can be defined as a process through which the functional groups of organic compounds are modified either stereo- or regiospecifically by living cultures, entrapped enzymes or permeabilized cells to a chemically different product. The production of high-value food metabolites, fine chemicals and pharmaceuticals can be achieved by biotransformations using biological catalysts in the form of enzymes and whole cells (Ravishankar and Ramachandra Rao, 2000; Krings and Berger, 1998; Meyer et al., 1997; Scragg, 1997; Berger, 1995; Cheetham, 1995). The range of flavour metabolites and pharmaceuticals produced by plant cell cultures through biotransformation is shown in Table 13. Cell suspension cultures, immobilized cells, enzyme preparations and hairy root cultures can be applied for the production of food additives or pharmaceuticals by biotransformation process (Table 14). There are two main reasons to choose plant cells for biotransformation purposes. Firstly, these cells are generally able to catalyze the reactions stereospecifically, resulting in chirally pure products. Secondly, they can perform regiospecific modifications that are not easily carried out by chemical synthesis or by microorganisms (Hamada and Furuya, 1996; Pras et al., 1995; Stockigt, 1993; Pras, 1992; Stepan-Sarkissan, 1991). These reactions include reduction, oxidation, hydroxylation, acetylation, esterification, glucosylation, isomerization, methylation, demethylation, epoxidation, etc. The presence of biotransformation potential in plant cells is a necessary condition for practical application. However, for a successful and viable process, the following prerequisites must be met (Steck and Constabel, 1974).


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Table 13 Biotransformation of flavour compounds by plant cell culture systems Plant species

Substrate

Product

Reference

Citrus limon Citrus paradisi N. tabacum Mentha spp.

Valencene Valencene Linalool Pulegone Menthol Steviol Geranial Neral Citronellal Ferulic acid

Nootkatone Nootkatone 8-hydroxylinalool Isomenthone Neomenthol Stevioside Geraniol Nerol Citronellol Vanillin

Drawert et al. (1984) Drawert et al. (1984) Suga and Hirata (1990) Aviv and Galun (1978) Aviv et al. (1981) Furuya (1978) Lappin et al. (1987)

Ste. rebaudiana Lavandula angustifolia

Va. planifolia

Ca. frutescens Ferulic acid Eucalyptus perriniana Isoeugenol and eugenol Acetophenone G. jasminoides Curcuma zedoaria Gl. glabra Coffea arabica Papaver bracteatum

Germacrone Glycyrrhitinic acid Vanillin Linalyl acetate

Achillea millefolium

Borneol, menthol thymol and farnesol Carvone

N. tabacum Cath. roseus N. tabacum Cath. roseus Cath. roseus Coffea arabica Euc. perriniana Euc. perriniana Euc. perriniana Cymbidium spp.

Piperitone Pulegone Geraniol Glycyrrhizin Capsaicin Menthol Camphor Borneol Menthyl acetate

Vanillin Isoeugenyl b-rutinoside and eugenyl b-glucoside (S)-aromatic alcohol Guaiane-type sesquiterpenes Hydroxyglycoside esters Vanillin glucosides Linalool, a-terpineol Geraniol Many products Dihydrocarvone neodihydrocareol Hydroxypiperitone Menthone 4-hydroxymenthone 10-hydroxygeraniol Glycyrrhetic acid Capsaicin glucoside Menthol 3-O- b-gentiobiosides Camphor glucosides () Borneol b-gentiobioside Menthol

Romagnoli and Knorr (1988) Johnson et al. (1996) Orihara et al. (1992) Akakabe and Naoshima (1993) Sakui et al. (1992) Hayashi et al. (1992) Kometani et al. (1993a) Hook et al. (1990) Figueiredo et al. (1996) Hirata et al. (1982) Hamada and Furuya (1996) Hamada and Furuya (1996) Hamada and Furuya (1996) Hamada and Nakata (1992) Kometani et al. (1993b) Orihara et al. (1991) Orihara et al. (1994) Orihara and Furuya (1993) Mironowicz et al. (1987)

Adapted from Ramachandra Rao (1998).

(1) The culture must have the necessary enzymes. (2) The substrate or precursor must not be toxic to the culture. (3) The substrate must reach the cellular compartment of the cell. (4) The rate of product formation must be faster than its further metabolism. 9.1. Advantages of biotransformations

The advantages include the production of novel compounds, enhancement in the productivity of desired compound and overcoming the problems associated with chemical synthesis. Importantly, the studies on biotransformation lead to basic information to elucidate

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Table 14 Biotransformations using plant cell cultures for production of pharmaceuticals Plant species

Substrate

Product

Reference

P. somniferum Di. purpurea Di. lanata Euc. perriniana Podo. hexandrum Muc. pruriens Cath. roseus

Codeine Digoxin Digoxin Taxol derivatives Podophyllotoxin DOPA Vincrisitne

Wilhelm and Zenk (1997) Alfermann et al. (1980) Alfermann et al. (1980) Hamada et al. (1996) Van Uden et al. (1995) Pras et al. (1993) DiCosmo and Misawa (1995)

Capsaicin, vanillin Capsaicin, vanillin

Johnson et al. (1996)

Ca. frutescens

Thebaine Digitoxin Digitoxin Taxol Coniferyl alcohol Tyrosine Vindoline Vinblastine Ferulic acid Vanillylamine Protocatechuic aldehyde Caffeic acid Digitoxin

Spirulina platensis Ca. frutescens

Codeine Isoeugenol

Ca. frutescens Ca. frutescens

Digoxin, purpureaglycoside A Morphine Vanillin, capsaicin

Ramachandra Rao and Ravishankar (2000a) Ramachandra Rao et al. (2002) Ramachandra Rao et al. (1999) Ramachandra Rao and Ravishankar (1999)

the biosynthetic pathway, and catalysis can be carried out under mild conditions, thus reducing undesired by-products, energy, safety and costs.

10. Scale-up of plant cell cultures

Since plant cells produce unique pharmaceuticals, which can be harnessed, they need to be produced in large-scale bioreactors. Configuration of bioreactors used for microbial cells cannot always be utilized directly for plant cells, owing to distinctive features, which are not favourable for plant cell cultivation. Plant cells are less stable in productivity, highly shear sensitive, exhibit low oxygen requirements (ca. 1-mmol O2 at 10  6 cells) slow growth (doubling time 25–110 h) and often occur as cell clumps of 2–4-mm diameter. Different Table 15 Bioreactor types used for plant cell cultures Reactor type

Plant species

Reference

Stirred tank

Cath. roseus N. tabacum N. tabacum M. citrifolia L. erythrorhizon Cath. roseus Cath. roseus Ruta vulgaris

Drapeau et al. (1986) Kato et al. (1977) Noguchi et al. (1977) Wagner and Vogelmann (1977) Tanaka (1987) Morris et al. (1984) Shuler et al. (1983) Shuler and Hallsby (1985)

Bubble column Airlift stirred Aerated Fluidized bed Tubular membrane Disc turbine


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Table 16 Large-scale suspension cultures reactors for plant cell cultures Plant species

Product

Bioreactor capacity

Reference

Cath. roseus Col. blumei L. erythrorhizon N. tabacum

Serpentine Rosmarinic acid Shikonin Biomass Biomass Saponins Biomass Biomass Biomass

100 l airlift 300 l airlift 750 l agitated 20,000 l agitated 1500 l bubble column 20,000 l agitated 750 – 75,000 l agitated 750 – 75,000 l agitated 750 – 75,000 l agitated

Smart and Fowler (1981) Rosevear (1984) Tabata and Fujita (1985) Kato et al. (1976) Noguchi et al. (1977) Ushiama et al. (1986) Ritterhaus et al. (1990) Ritterhaus et al. (1990) Ritterhaus et al. (1990)

Panax ginseng E. purpurea Ra. serpentina Panax ginseng

Adapted from Sahai (1994).

configuration of bioreactors has been adopted depending on the nature of cells. Few representative references are made in Table 15. Several studies are available for large-scale cultivation of plant cells (Table 16). Cell/tissue types such as cell suspension cultures, immobilized cells and hairy roots have been very ideal for scale-up. However, the configuration of reactors will be different for different cell or tissue types. No one design could be recommended as a common one. The most important contribution, however, is to produce not only the biomass but also the metabolite in an economical manner. In the present state of art, any compound less than US$ 1000 per kilogram is difficult to produce. Hence, it is advantageous to utilize bioreactor technology for compounds, which are costlier and has a higher market demand. 10.1. Scale-up of biotransformations

So far, a few reports are available on scale-up of biotransformation. In the food area, the development of a process for making high-fructose corn syrup by immobilized glucose isomerase was reported. In the pharmaceutical industry, the first biotransformation was the production of modified penicillin by immobilized penicillin acylase using single-step biotransformation (West, 1996). Biotransformation of 12b-hydroxylation of methyldigitoxin to methyldigoxin and other metabolites such as deacetayllanatoside C by Di. lanata cell

Table 17 Biotransformation of b-methyldigitoxin to b-methyldigoxin by Di. lanata cells in 20-l reactor Precursor

Productivity (g)

% Conversion

b-methyldigitoxin added Unconverted b-methyldigitoxin b-methyldigoxin formed By-product Yield

17.24 2.04 14.36 0.28

100 11.8 81.7 1.4 94.9

Adapted from DiCosmo and Misawa (1995).

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cultures was reported (Kreis and Reinhard, 1992; 1990; Reinhard et al., 1989). A biotransformation process for the production of digoxin was developed using Di. lanata in cell suspension cultures using 20- and 300-l airlift bioreactors. The results of the study indicated that maximum digoxin was reached in 8% (w/v) glucose medium, and about 80% of the digoxin produced was found in the medium (Table 17). Panda et al. (1992) demonstrated the alkaloid production by precursor feeding using stirred tank bioreactors in plant cell cultures of Holarrhena antidysentrica.

11. Food additive production from plant cell cultures studied in the authors’ laboratory

11.1. Capsaicin

Capsaicin (Fig. 3) is a pungent principle of green pepper ( Capsicum spp.). Capsaicin preparations are used as a counterirritant in lumbago, neuralgia and rheumatic disorders. Taken internally, Capsicum preparation has a tonic and carminative action especially useful in dyspepsia. Capsicum oleoresin is added to tannin or rose gargles for pharyngitis and relaxed sore throat. Capsaicin is administered as a bacteriostatic (Gal, 1965) or fungistatic compound in the form of powder, tincture, liniment, plaster, ointment and medicated wool. In the US, two commercial creams, viz. Zostrix and Axsain, are formulated with purified capsaicin for alleviation of arthritis pain (Anonymous, 1991). Commercial production of capsaicin by separation from Capsicum oleoresin and its subsequent purification would involve several steps. Capsaicin can be produced by immobilized cell cultures, which leach out to the medium, facilitating easy separation and purification. The cultivation of the Capsicum crop takes 4 –5 months and therefore cannot offer a continuous production procedure. In contrast, immobilized cell cultures of Capsicum offer a continuous method of producing capsaicin under in vitro controlled conditions. There are several reports of capsaicin production in immobilized cell cultures (Johnson et al., 1990, 1991; Ravishankar et al., 1988; Mavituna et al., 1987; Lindsey and Yeoman, 1984). Our attempts to enhance the level of capsaicin by treating immobilized cell cultures of Ca. frutescens with elicitors (Johnson et al., 1990) and permeability agents (Johnson et al., 1991) or by manipulating culture conditions (Johnson, 1993) have been successful. An innovative method of capsaicin production by immobilizing the placenta, the site of synthesis of capsaicin (Iwai et al., 1979), was reported by Johnson and Ravishankar (1996). Capsaicin production increased several folds higher in immobilized

Fig. 3. Chemical structure of capsaicin.


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placenta than in chilly fruit. With precursor biotransformation using coumaric acid, it registered an 11-fold increase (Johnson and Ravishankar, 1996). Here, we provide capsaicin production in 2-l airlift culture vessel using immobilized cells/ placenta. An airlift culture vessel of cylindrical shape was constructed with Corning glass of the configuration given in Fig. 4. 11.2. Inputs of operation

1. 20-day-old Capsicum cells to placenta (100-g fresh weight). 2. Alginate and calcium chloride solution required to envelop 100-g cells/placenta in beads. One litre of 2.5% (w/v) sodium alginate with cells extruded into 2 l of 0.9% (w/v) calcium chloride dihydrate. 3. Beads washed with water were transferred into the vessel containing 1 l of MS medium supplemented with 3% sucrose, 2-mg/l 2,4-D and 0.5-mg/l kinetin [standard medium (SM)]. 4. Airflow (2:1 mixture of CO 2 + air for the initial 7 days of culture and 4:1 for the latter 7 days of production) at a rate of 4 V.V.M. 5. Incubation at 25 ± 2 C under continuous light of 2000 lx. 6. pH adjustment to 5.8 during culture. 7. Replenishment of entire medium after 7 days with fresh medium. 8. Capsaicin recovery and analysis in 2 weeks of culture with two harvests at 7-day intervals.

Fig. 4. Schematic representation of column reactor process for capsaicin production using immobilized cell cultures of Ca. frutescens .

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11.3. Yield components

The airlift culture vessel was run with immobilized cells or placenta for 2 weeks under three different conditions using (1) SM, (2) SM + elicitor or (3) SM + precursor ( p-coumaric acid). The results are presented below. 11.3.1. Yields in SM

1. Immobilized cells: 35 mg/100-mg fresh cells/15 days or 0.4% on dry weight basis 2. Immobilized placenta: 300 mg/100-g fresh placenta/15 days or 0.1% on dry weight basis 11.3.2. Yields in SM+elicitor Elicitors used were curdlan at 8-mg/l concentration for immobilized cells and Rhizopus oligosporus mycelial extract equivalent to 2.5 g of mycelium/l of medium administered for placental immobilized cultures.

1. Immobilized cells: 76 mg/100-g fresh cells/15 days or 0.95% on dry weight basis 2. Immobilized placenta: 300 mg/100-g fresh placenta/15 days or 1.25% on dry weight basis 11.3.3. Yields in SM+precursor (2.5-mM coumaric acid)

1. Immobilized cells: not done 2. Immobilized placenta: 1148 mg/100-g fresh placenta/15 days or 7.6% on dry weight basis By our method, we are able to get a maximum production of 1.15 g/l. However, increases can be obtained using continuous cultivation with adsorption of capsaicin and recycling of medium to minimize the cost. The plant may not be viable if it is less than 10,000-l capacity. We have recently studied capsaicin production in bubble column and packed-bed reactor at 1-l level, which needs further scale-up and its performance to be evaluated vis-a` -vis alginate immobilized system (Table 18). Apart from the production of capsaicin from immobilized cell cultures of Ca. frutescens , attempts have been made in our laboratory to produce vanilla flavour metabolites from Capsicum-immobilized cultures upon feeding of phenylpropanoid intermediates — protocatechuic aldehyde, caffeic acid (Ramachandra Rao and Ravishankar, 2000a), ferulic acid, coniferyl aldehyde and veratraldehyde (Ramchandra Rao and Ravishankar, 1998). The feeding of phenylpropanoid precursors not only produced the vanilla flavour metabolites but also increased the yields of capsaicin (Ramachandra Rao, 1998; Ramachandra Rao and Ravishankar, 2000a). Feeding of clove principle, isoeugenol, to immobilized cultures of Ca. frutescens showed formation of vanilla flavour metabolites (Ramachandra Rao and Ravishankar, 1999).


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Table 18 Comparison of continuous production of capsaicin from bubble column and packed-bed reactor Bioreactor

Productivity

Bubble column Biomass density Average productivity Average specific productivity Maximum duration of continuous operation Total production Packed-bed reactor Biomass density Average productivity Average specific productivity Maximum duration of continuous operation Total production Production under fungal elicitation

100 g fresh weight/l 1.7 mg/l/day 17 mg/g fresh weight/day 21 days 35 mg/l in 21 days 380 g/l 7.4 mg/l/day 19.5 mg/g fresh weight/day 28 days 207 mg/l in 28 days 330 mg/l in 28 days

Adapted from Madhusudhan (1998).

11.4. Vanillin

Vanillin (Fig. 5) is the most universally accepted aroma chemical used in processed foods, pharmaceutical products and perfumeries (Prince and Gunson, 1994; Anonymous, 1993; Webster, 1995). It occurs in the vanilla bean at a level of 2% dry weight, and it is associated with many other compounds. Approximately 12,000 tons of vanillin is consumed annually, from which only 20 tons are extracted from the vanilla beans (Dornenburg and Knorr, 1996; Feron et al., 1996; Berger, 1995). The cost of the pure natural vanillin flavour is priced at US$ 4000 per kg, while the synthetic equivalent costs about 12 US$ per kg (Krings and Berger, 1998; Berger, 1995). Other than flavour qualities, use of vanillin as antimicrobial against molds and yeast has been described (Lopez-Malo et al., 1995; Cerrutti et al., 1997; Cerrutti and Alzamora, 1996). Vanillin was found to be an antioxidant (Burri et al., 1989) and will be useful in the development of health foods. Vanillin is also known for its antimutagenic activity (Kometani et al., 1993a) and glucosylvanillin has been found to impart distinctive flavour note, hence would be useful in tonics and syrups. Interestingly, at the 8th FAOBMB Congress, Iekhsan et al. (1998) reported the discovery of use of vanillin as antidote for neutralizing the toxic effect of toxin from Chinorex fleckeri (boxjellyfish). There are a number of cases of poisoning by boxjellyfish, and often, it is fatal to individuals. The findings of Iekhsan et al. (1998) hence provide newer utility of vanillin as a pharmaceutically important compound.

Fig. 5. Chemical structure of vanillin.

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Production of vanillin by conventional method by cultivating Vanilla plants is tedious and expensive. Though vanilla flavour has its own utility as flavouring, the use of vanillin obtained through biotechnology assumes significance since there is worldwide demand for natural compounds. In this context, vanillin and its other flavour metabolites are being produced through biotransformation to provide alternate method of production of biovanillin through cheaper substrates (Table 19). The increase in production of vanillin and its other flavour metabolites has been made possible by several factors using optimization by enhanced substrate availability mediated by cyclodextrin (Ramachandra Rao and Ravisha-

Table 19 Biotransformations leading to vanillin formation through microbial and plant cell cultures Organism bioconverting the substrate Ferulic acid Aspergillus niger Pycnoporous cinnabarinus Pseudomonas fluorescens Pseudomonas fluorescens AN 103 Corynebacterium glutamicum Ca. frutescens Spir. platensis Haemotococcus pluvialis Va. planifolia Va. planifolia Eugenol Corynebacterium glutamicum Pseudomomnas spp. Arthrobacter globiformis Spir. platensis Isoeugenol Enterobacter spp. Serratia spp. Spir. platensis Ca. frutescens Vanillylamine Asper. niger E. coli Spir. platensis Ca. frutescens Coniferyl aldehyde Ca. frutescens Spir. platensis Coniferyl aldehyde Vanillyl alcohol Ca. frutescens Spir. platensis Penicillum simplicissimum Adapted from Ravishankar and Ramachandra Rao (2000).

Reference Lesage-Messen et al. (1996) Andreoni et al. (1995) Narbad and Gasson (1998) Labuda et al. (1993) Ramachandra Rao (1998) Ramachandra Rao et al. (1996a) Usha et al. (1999) Romagnoli and Knorr (1988) Funk and Brodelius (1990) Tadasa and Kayahara (1983) Rabenhorst (1996) Cooper (1987) Ramachandra Rao et al. (1996a) Rabenhorst (1991) Ramachandra Rao et al. (1996a) Ramachandra Rao and Ravishankar (1999) Yoshida et al. (1997) Ramachandra Rao (1998) Sudhakar Johnson et al. (1996) Ramachandra Rao (1998) Ramachandra Rao et al. (1996a) Markus et al. (1992) Ramachandra Rao and Ravishankar (1998) Ramachandra Rao et al. (1996a) Fraaije et al. (1997)


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nakar, 1998), elicitation of bioconversion process using microbial elicitors (Ramachandra Rao, 1998) and in situ adsorption of products using adsorbents, viz. activated charcoal (Knuth and Sahai 1991; Westcott et al., 1994) and amberlite XAD-4 and XAD-7 (Ramachandra Rao and Ravishankar, 2000b), to overcome the feedback inhibition, thereby enhancing yield and recovery. The formation of vanilla flavour metabolites from various phenylpropanoids in cell cultures of C. frutescens is shown in Fig. 6 11.5. Anthocyanin

Anthocyanins are the most ubiquitous pigments seen in nature, widely distributed in the pericarps of several fruits, flowers and vegetables. They are glycosylated polyhydroxy and polymethoxy derivatives of flavylium (2-phenylbenzopyrrolium) salts belonging to the group

Fig. 6. Vanilla flavour metabolites formation from various phenylpropanoid precursors in Ca. frutescens cell cultures.

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Table 20 Effect of antioxidants on lipid peroxidation Compound

IC50

Butylated hydroxyanisole Butylated hydroxytoluene a-tocopherol Anthocyanin from carrot cell cultures

0.125 0.250 0.500 0.005

Adapted from Narayan et al. (1999).

of two or three portions — the aglycone base, a group of sugars and a group of acyl acids. The aglycone moiety is referred to as anthocyanidin. There are more than 15 anthocyanidins (Francis, 1982). Anthocyanins are widely used a colourants. However, they are known to have several pharmacological attributes such as antiinflammatory, antiulcer and woundhealing properties (Koichi and Hasashi, 1990; Vega et al., 1987). We have recently established the antioxidant properties of anthocyanin obtained through cell culture (Narayan et al., 1999). Culture-derived anthocyanin effectively prevented autooxidation of lipids as well as lipid peroxidation in biological systems (Table 20). Our studies have shown high anthocyanin production in mutated cell cultures of D. carota (Table 21). Chromatographic analyses of anthocyanin extract showed the presence of cyanidin xylosyl galactose, cyanidin monoglucose and cyanidin galactose in the molar ratio of 5:3:2. The chemical structure of cyanidine-3-glucoside and delphinidin-3-glucoside is shown in Fig. 7. Presently, the commercial source of anthocyanins is the grape peel in view of its quoted price of US$ 1200–1500 per kilogram (Ilker, 1987) and estimated market of US$ 200 million. Plant cell cultures are an alternative source of anthocyanin. The possibility of obtaining novel anthocyanin with two to three times greater stability at slightly acidic pH has been demonstrated using D. carota cells (Vunsh et al., 1986) Research in our laboratory has shown that anthocyanin production in carrot cultures is stimulated by indole-3-acetic acid Table 21 Growth and production of anthocyanin in D. carota cell cultures Parameters Growth Specific growth rate Doubling time Growth index Maximum cell density Dry weight/fresh weight ratio Packed cell volume Anthocyanin production Average content Specific productivity ( q p) Total production Average productivity Adapted from Madhusudhan (1998).

Value 0.217/day 3.2 days 12.2 18.92 ± 1.17 g/l 0.085 0.95 ml/g fresh weight 15.58% (w/w) dry mass 10.52 mg/g/day 3.1 g/l in 15 days 207 mg/l/day


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Fig. 7. Chemical structure of anthocyanin.

(Rajendran et al., 1992). Elicitation of anthocyanin by fungal elicitor has been useful to enhance the yields by 1.25-fold using Aspergillus flavus mycelial extract (Rajendran et al., 1994). Similarly, its elicitation by phycocyanin was also reported (Ramachandra Rao et al., 1996a,b). The cell aggregate size of 500–850 mm was critical for maximum anthocyanin production (Madhusudhan and Ravishankar, 1996). The high yields obtained by several researchers have resulted in exciting possibility of scale-up of anthocyanin production systems. In the future, anthocyanins will be used for antioxidant effect as well as their value as colours. Developments in genetic engineering of anthocyanin production have been elegantly reviewed by Guttason (1994). There is a possibility of obtaining plants with flowers of different hues. This aspect may be adopted for cell culture production. However, to date, no enhancement of hue has been demonstrated.

12. Metabolic engineering of biochemical pathways

It is envisaged that the use of plant cell culture approach for pharmaceutically important compounds can be made industrially applicable only upon the overproduction of metabolites or the production of novel compounds that too with the expression of more than one compounds of interest in a plant. This is not realized by routine cell line selection and other parameters of growth/production media development. Therefore, there is a need to study the metabolic pathways and its regulatory steps for overexpressing the regulatory genes for ratelimiting steps. The recent advancement in plant cell and tissue culture techniques and the genetic transformation of plants using Agrobacterium-mediated or direct gene transfer make possible the incorporation of foreign genes into plants producing new genotypes of desirable traits. Thus, genetically engineered or modified plants can be used as ‘‘green bioreactors’’ for the production of value-added chemicals (Saito et al., 1992). The progress of genetic manipulation largely depends on (a) the ability to obtain genes encoding desirable traits, (b) the proper alignment of the expressed gene with its endogenous substrate and the compartmentation/transport of its product, (c) the successful transformation and reproducible regeneration of transgenic plants, (d) the proper site of gene integration combined with the level and patterns of gene expression and (e) the evaluation of the altered metabolic pathways.

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12.1. Molecular strategies

Strategies for overproduction of metabolites by genetic manipulation can be as follows.

1. Overproduction of precursors of secondary metabolites (e.g. ‘‘A’’). 2. Overexpression of the gene product, which limits the specific pathway (e.g. ‘‘B’’). 3. Creating a new branch for a preexisting pathway (e.g. ‘‘F to C’’). 4. Down-regulation of existing reaction using antisense methods (blocking, e.g. ‘‘C to E’’). 5. Manipulation of regulatory genes whose products may bind DNA and function as transcriptional activators or repressors. 6. Selection of regulatory mutants with increased tissue-independent expression for in vitro production of metabolites. 7. Sometimes, the tissue/organ-specific expression by using appropriate promoters is required to target the metabolite production. This would result in metabolic energy saving by localized production of metabolites. Studies related to the importance of plants by metabolic engineering or transgenic protocols are presented here, where success has been obtained for enhancement of yields. 12.2. Tryptophan decarboxylase (EC 4.1.1.28)

Tryptophan decarboxylase catalyses the decarboxylation of L-tryptophan to tryptamine. Tryptamine is a substrate for strictosidine synthase (Stockigt and Zenk, 1977). Interestingly, cDNA clone from Cath. roseus encoding tryptophan decarboxylase has been expressed in tobacco plants (Songstad et al., 1991), which resulted in the increased levels of tryptamine and tyramine as predicted. This study has been extended to metabolically engineer Brassica napus, wherein tryptophan, which is generally bioconverted to glucosinolate (an undesirable product of oil seed cake, which renders the product unpalatable by cattle), is diverted away by expressing tryptophan decarboxylase gene of Cath. roseus. In such a situation, transgenic B. napus produced low amount of glucosinolates, making the oil seed cake palatable to cattle (Chavadej et al., 1994). 12.3. Strictosidine synthase (EC 4.3.3.2)

Strictosidine synthase catalyzes the stereospecific condensation of primary amino group of tryptamine and aldehyde group of iridoid glycoside secologanin to form the indole


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alkaloid strictosidine. Strictosidine synthase is of biotechnological interest in the biomimetic synthesis of monoterpenoid indole alkaloids because the reaction product is a mixture of the diastereomers. The cDNA encoding strictosidine synthase was isolated from Rauwolfia serpentina and it has been functionally expressed in E. coli, Saccharomyces cerevisiae and insect cells using a baculovirus vector (Kutchan, 1989; Kutchan et al., 1988). Strictosidine synthase from Cath. roseus has been expressed in tobacco (McNight et al., 1990). 12.4. H6H (EC 1.14.11.11)

The biosynthesis of scopolamine from hyoscyamine is catalyzed by H6H. This enzyme catalyzed hydroxylation and epoxidation reactions (Robbins et al., 1994). The cDNA encoding H6H from Hy. niger was introduced into Atropa belladona using either Agrobacterium tumefaciens - or Agr. rhizogenes-mediated transformation. The resultant Atropa transgenic plants contained high levels of scopolamine (Yun et al., 1992). This is a first example of metabolic engineering to produce medicinally useful compound. 12.5. Berberine bridge enzyme (EC 1.5.3.9)

Berberine bridge enzyme catalyses the stereospecific conversion of N -methyl group (S )-reticuline into (S )-scoulerine (Kutchan, 1995). Direct conversion of N -methyl group into a methylene bridge moiety is not easily achievable presently by synthetic chemistry. It is of interest to note that berberine bridge enzyme is an elicitor-induced yeast cell wall (Schumacher et al., 1987), thereby providing possibility of overproduction of berberine in cell cultures. Moreover, cDNA encoding the berberine bridge enzyme has been isolated by Dittrich and Kutchan (1991) in elicited cell suspensions of Es. californica (California poppy) and expressed heterologously into insect cultures using baculovirus expression vector, which resulted in the production of sufficient quantities. 12.6. Berbamunine synthase

Berbamunine synthase (EC 1.1.3.34) catalyzes the formation of the ether linkage between one molecule of ( R)- N -methylcoclaurine and one molecule of ( S )- N -methylcoclaurine to form the bisbenzylisoquinoline alkaloid berbamunine. The cDNA encoding berbamurine synthase was isolated from B. stolonifera cell suspension cultures and expressed in insect culture using a baculovirus expression vector (Kraus and Kutchan, 1995). 12.7. Polyphenol oxidase (PPO)

PPO is ubiquitous in higher plants and its regulation provides several advantages. Downregulation of PPO increases the quality attributes of crop plant-derived commodities, whereas overexpression reduces danger from pests. It has been concluded that no phenotypic

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effects are visible in transgenic tomato or tobacco plants with high or low PPO levels (Steffens et al., 1994). 12.8. p-Hydroxycinnamoyl-CoA hydratase/lyase (HCHL)

Higher plants possess the phenylpropanoid pathway for the production of lignin and some secondary phenolic compounds. Coumaric acid and ferulic acids and their CoA esters are intermediates in these routes. Expression of the bacterial gene for the side chain cleavage enzyme in plants may offer the potential to generate vanillin and related flavour compounds in a range of crops by diversion of phenylpropanoid metabolism. At IFR, attempts have been made to produce vanillin in hairy root cultures of Dat. stramonium by expressing the HCHL gene from Pseudomonas fluorescens AN103. The expression of the HCHL gene in Dat. stramonium hairy root cultures caused substantial changes in the content of p-hydroxybenzoic acid in the form of glucosides and glucose esters and along with traces of glucosides of p-hydroxybenzyl alcohol and vanillic acid. The potential of this line to biotransform added ferulic acid to vanillin is being studied (Mitra et al., 1999). 12.9. 4-coumarate-CoA ligase (4CL)

4CL (EC 6.2.1.12) catalyses a branch-point reaction (conversion of hydroxycinnamic acids into the corresponding CoA esters) between the central phenylpropanoid pathway and pathways leading to various secondary metabolites such as flavonoids, lignans, lignin and related phenylpropanoid compounds (Holton and Cornish, 1995; Douglas, 1996). Brodelius and Xue (1997) isolated and characterized 4CL from Va. planifolia cell suspension cultures. Based on studies using the inhibitors of 4CL (Funk and Brodelius, 1990), they concluded that the expression of antisense mRNA for 4CL gene would redirect the flow of phenylpropanoid precursors from lignin biosynthesis into flavour compound biosynthesis in Va. planifolia . Thus, genetic engineering for metabolic alteration or overexpression of pathway has much to offer. There are exciting possibilities to produce high-value, low-volume pharmaceutical products such as the ones described here. Moreover, one can imaginatively produce novel compounds by expression of foreign genes. The questions regarding environmental study of transgenic plants and regulatory considerations of administration of the phytopharmaceuticals derived from them has not been dealt since it is beyond the scope of this review.

13. Conclusion

Though plant cell cultures can produce a whole range of secondary metabolites, there are few success stories of production at commercial scale. The prospect of production of highcost, low-volume products such as anti-HIV and anti-cancer compounds is very high, thus putting this technology to a position of being able to make a commercial impact in a few selected pharmaceuticals. The advancement of knowledge in phytochemistry, regulation of secondary pathways and ability to express desired traits by transgenics is expected to drive


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technology to produce a range of pharmaceutical and healthcare products. Vaccines and antibodies from plants are already fast-advancing fields, with investments from multinational companies. They aim to provide protection against diseases of all sections of society, since the poor have been deprived of expensive immunization schedules. Use of transgenic plants for the production of pharmaceuticals will be bound by the IPR protection. Those who can invest in the R&D will benefit in the era of processes and patents. Developing countries, which are still behind in recombinant DNA research, may not be able to develop their own complete technologies. Nations rich in biodiversity and knowledge base of their local vegetation may protect their plants and develop cultivation methods. The technique of micropropagation by in vitro culture methods will be a handy tool, which does not need high levels of biotechnology. The R&D of production of secondary metabolites through biotechnology has made a beginning and is expected to provide disease-remedial or disease-preventive molecules at affordable costs for the benefit of mankind.

Acknowledgments

The authors would like to thank the Council of Scientific and Industrial Research (CSIR), Department of Science and Technology (DST) and the Department of Biotechnology (DBT), Government of India, New Delhi for sponsoring the research grants on plant cell cultures for secondary metabolites. They also like to thank B. Suresh for his help in the preparation of this review.

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