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

Plant cell cultures:

Chemical factories of secondary metabolites

S. Ramachandra Raoa,*, G.A. Ravishankarb

aLaboratory of Biofunctional Materials, School of Materials Science, Japan Advanced Institute of Science and

Technology, 1-1, Asahidai, Tatsunokuchi, Ishikawa 923-1292, JapanbPlant Cell Biotechnology Department, Central Food Technological Research Institute, Mysore 570 013, India

Abstract

This review deals with the production of high-value secondary metabolites including

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: 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 metabolites, which are used as pharmaceuticals, agrochemicals, flavours, fragran-

ces, colours, biopesticides and food additives. Over 80% of the approximately 30,000 known

natural products are of plant origin (Phillipson, 1990; Balandrin and Klocke, 1988; Fowler

0734-9750/02/$ – see front matter D 2002 Elsevier Science Inc. All rights reserved.

PII: S0734 -9750 (02 )00007 -1

* Corresponding author. Tel.: +81-761-51-1668; fax: +81-761-51-1665.

E-mail addresses: [email protected], [email protected] (S. Ramachandra Rao).

Biotechnology Advances 20 (2002) 101–153

and Scragg, 1988). The number of known chemical structures is estimated to be 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

Balick, 1994). The advent of chemical analyses and the characterization of molecular

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

Cost

(US$ per kilogram)

Ajmalicine Antihypertensive Cath. roseus 37,000

Artemisinin Antimalarial Artemisia annua 400

Ajmaline – Ra. serpentina 75,000

Acinitine – Acotinum spp. n/a

Berberine Intestinal ailment C. japonica 3250

Camptothecin Antitumour Camptotheca acuminata 432,000

Capsaicin Counterirritant Ca. frutescens 750

Castanospermine Glycoside inhibitor Castanospermum australe n/a

Codeine Sedative P. somniferum 17,000

Colchicine Antitumour Colchium autumnale 35,000

Digoxin Heart stimulant Di. lanata 3000

Diosgenin Steroidal precursor Dioscorea deltoidea 1000

Ellipticine Antitumour Orchrosia elliptica 240,000

Emetine – Cephaclis ipecaccuanha 1500

Forskolin Bronchial asthma Coleus forskolii n/a

Ginsenosides Health tonic Panax ginseng n/a

Morphine Sedative P. somniferum 340,000

Podophyllotoxin Antitumour Podophyllum petalum n/a

Quinine Antimalarial Cinchon. ledgeriana 500

Sanguinarine Antiplaque Sanguinaria canadensis 4,800

P. somniferum

Shikonin Antibacterial L. erythrorhizon 4500

Taxol Anticancer Taxus brevifolia 600,000

Vincristine Antileukemic Cath. roseus 2,000,000

Vinblastine Antileukemic Cath. roseus 1,000,000

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

S. Ramachandra Rao, G.A. Ravishankar / Biotechnology Advances 20 (2002) 101–153102

still depend upon biological sources for a number of secondary metabolites including

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 of photosynthetic activity, produce secondary metabolites. Many of them are

unique to the plant kingdom and are not produced by microbes or animals. 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

S. Ramachandra Rao, G.A. Ravishankar / Biotechnology Advances 20 (2002) 101–153 103

by the combined efforts of cell biologists and chemical engineers. Chemical engineers are

currently developing improved and appropriate bioreactors for the improvement of produc-

tion systems by adopting techniques of growth and metabolite production coupled 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 engineered plants. Moreover, the need for safer drugs without side effects has led

to the use of natural ingredients with proven safety. These factors have laid emphasis on the

use of biotechnological methods to enhance the production of pharmaceuticals and 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 biosynthetically totipotent, which means that each cell in culture retains complete

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 independent of geographical and seasonal variations and various environmental

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 Alkaloids Terpenoids Quinones Steroids

1. Anthocyanins 1. Acridines 1. Carotenes 1. Anthroquinones 1. Cardiac glycosides

2. Coumarins 2. Betalaines 2. Monoterpenes 2. Benzoquinones 2. Pregnenolone

3. Flavonoids 3. Quinolizidines 3. Sesquiterpenes 3. Naphthoquinones derivatives

4. Hydroxycinnamoyl 4. Furonoqui nones 4. Diterpenes

derivatives 5. Harringtonines 5. Triterpenes

5. Isoflavonoids 6. Isoquinolines

6. Lignans 7. Indoles

7. Phenolenones 8. Purines

8. Proanthocyanidins 9. Pyridines

9. Stilbenes 10. Tropane

10. Tanins alkaloids

Adapted from Stockigt et al. (1995).


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.

There are a number of plant cell cultures producing a higher amount of secondary

metabolites than in intact plants (Table 5). However, there are still problems in the

production of metabolites by cell cultures resulting from the instability of cell lines, low

yields, slow growth and scale-up problems (Ravishankar and Venkataraman, 1993).

Table 4

Food additives from plant cell cultures

Product type Plant species Reference

Colours

Anthocyanins V. vinifera Pepin et al. (1995)

Euphorbia spp. Yamamoto et al. (1982)

D. carota Rajendran et al. (1994)

Pe. frutescens Zhong and Yoshida (1995)

Betalaines B. vulgaris Klebnikov et al. (1995)

Cheno. rubrum Berlin et al. (1986)

Crocin Crocus sativus Sujata et al. (1990)

Crocetins Gardenia jasminoides George and Ravishankar (1995)

Carotenoids Lycopersicon esculentum Fosket and Radin (1983)

Anthraquinones Cinchon. ledgeriana Robbins and Rhodes (1986)

M. citrifolia Kieran et al. (1993)

Naphthoquinones L. erythrorhizon Sim and Chang (1993)

Flavours

Vanillin Va. planifolia Dornenburg and Knorr (1996)

Garlic Allium sativum Ohsumi et al. (1993)

Onion Allium cepa Collin and Masker (1988)

Basmati Oryza sativa Suvarnalatha et al. (1994)

Citrus flavour Citrus spp. Cresswell (1990)

Cocoa flavour Theobromo cacao Townsley (1972)

Pungent food additive

Capsaicin Ca. frutescens Lindsey and Yeoman (1984)

Ca. annuum Johnson et al. (1990)

Sweeteners

Stevioside Stevia rebaudiana Swanson et al. (1992)

Glycyrrhizin Glycyrrhiza glabra Hayashi et al. (1988)

Thaumatin Thaumatococcus danielli van der Wel and Ledeboer (1989)

Essential oils

Mint oil Mentha piperata Chung et al. (1994)

Chamomile oil Ma. chamomilla Kireeva et al. (1978)

Jasmine oil Jasmine officinale Banthrope (1994)

Aniseed oil Pim. anisum Ernst (1989)

Adapted from Ramachandra Rao (1998).


Several strategies have been adopted for the enhancement of metabolites (Table 6).

There has been tremendous success in the production of high-value secondary metab-

olites 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 5

High yields of secondary products

Product Plant species Yield (% D.W.) Reference

Rosmarinic acid Sa. officinalis 36.0 Hippolyte et al. (1992)

Rosmarinic acid Col. blumei 21.4 Ulbrich et al. (1985)

Anthroquinones M. citrifolia 18.0 Zenk et al. (1975)

Shikonin L. erythrorhizon 12.4 Fujita (1988)

Berberine Th. minus 10.6 Kobayashi et al. (1988)

Jatrorhizine Berberis wilsonae 10.0 Breuling et al. (1985)

Anthocyanins Pe. frutescens 8.9 Zhong et al. (1994)

Berberine C. japonica 7.5 Matsubara et al. (1989)

Diosgenin Diosc. deltoidea 3.8 Sahai and Knuth (1985)

Sanguinarine P. somniferum 2.5 Park et al. (1992)

Serpentine Cath. roseus 2.2 Zenk et al. (1977)

Adapted from Ravishankar and Ramachandra Rao (2000).

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


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 high-

yielding 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 phenyl-

alanine, 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 accumula-

tion 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 concentra-

tions 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


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 concen-

trations 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 NH4+ 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 Chrys-

anthemum 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-


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.


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 purpureaglyco-

side 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.


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 (Ram-

achandra 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.


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. Cyclo-

dextrins 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 complex-

ation of flavours provides protection against the damaging factors of the environment (Qi

and Hedges, 1995).


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 trans-

formed 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 Artemesinin Park et al. (1989)

Atro. belladona Atropine Benjamin et al. (1987)

Begonia spp. – Takayama and Misawa (1981)

Cath. roseus Vindoline Staba and Chung (1981)

Cinchona spp. Vinblastine Krueger et al. (1982)

Quinine Hirata et al. (1987)

Di. lanata Cardenolides Lui and Staba (1979)

Di. purpurea Cardenolides Hagimori et al. (1982a,b)

Pelargonium tomentosum Essential oils Charlwood and Moustou (1988)

Picrprrhiza kurroa Kutkin Upadhyay et al. (1989)

Ste. rebaudiana Steviosides Akita et al. (1994)

Withania somniferum Withanolides Heble (1985)

Polygonum tinctorium Indirubin Shim et al. (1998)

Decentra pergrina Alkaloids Konishi et al. (1998)



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 8

Root cultures of medicinal plants


B. vulgaris Betalaines Hamill et al. (1986)

Bidens alba Polyacetylenes Norton and Towers (1986)

Calystegia sepium Tropane alkaloids Jung and Tepfer (1987)

Coreopsis tinctoria Phenylpropanoids Thron et al. (1989)

Hyoscamus albus Hyoscyamine Hashimoto and Yamada (1986)

Hyoscamus muticus Hyoscyamine Flores et al. (1987)

Hemidesmus indicus 2-hydroxy-4-methoxybenzaldehyde Sreekumar et al. (1998)

Artemisia absynthium Volatile oils Kennedy et al. (1993)

Polygonum tinctorium Indigo, Indirubin Shim et al. (1998)

Adapter from Payne et al. (1991).

Table 9

Hairy root cultures producing pharmaceutical products of interest


Bidens spp. Polyacetylenes McKinely et al. (1993)

Cinchon. ledgeriana Quinolene alkaloids Hamill et al. (1987)

Cichorium intybus Esculetin Bais et al. (1999)

Datura spp. Tropane Rhodes (1989)

Cassia spp. Anthroquinones Ko et al. (1988)

Duboisia leichhardtii Tropane alkaloids Mano et al. (1989)

Echinacea purpurea Alkaloids Trypsteen et al. (1991)

Glycyrrhiza uralensis Glycyrrhizin Ko et al. (1989)

Hy. albus Alkaloids Shimomura et al. (1991)

Panax ginseng Saponin Yoshikawa and Furuya (1987)

Salvia miltorrhiza Diterpenes Hu and Alfermann (1993)

Artemi. absynthium Volatile oil Kennedy et al. (1993)

L. erythrorhizon Shikonin Shimomura et al. (1986)

Ra. serpentina Ajmaline, serpentine Benjamin et al. (1994)

Rubia cordifolia Anthroquinones Shin and Kim (1996)

Gl. glabra Isoprenylated flavonoids Asada et al. (1998)

Panax ginseng Ginsenoside Kunshi et al. (1998)

Hy. muticus Hyoscyamine Sevon et al. (1998)



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 cul-

tures, 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 air-

sparging 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.


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)

Fig. 2. Configuration of hairy root bioreactors.


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

Table 10

Biotransformations using hairy root cultures for production of pharmaceuticals

Plant species Substrate Product Reference

Hy. niger Hyoscyamine Scopolamine Hashimoto and Yamada

(1983)

Cinchon. ledgeriana Tryptophan Quinine Hay et al. (1986)

Nicotiana spp. Lysine, cadaverine Nicotine Walton and Belshaw

(1988)

Putrescine, agmatine Anabasine Walton et al. (1988)

Duboisia myoporoides Putrescine Scopolamine Yoshioka et al. (1989)

Spermidine Hyoscyamine

Cadaverine

Panax ginseng 2-phenylpropanoic acid Sugar esters Furuya et al. (1989)

Panax ginseng Digitoxigenin Digitoxin Kawaguchi et al. (1990)

Atro. belladona Hyoscyamine Scopolamine Subroto et al. (1996)

Dubo. leichhardtii Hyoscyamine Scopolamine Subroto et al. (1996)

Panax ginseng 18b-glycyrrhetinic acid Glucosides Asada et al. (1993)

Bidens sulphureus Butylated hydroxytoluene Stilbene quinones Flores et al. (1994)

Panax ginseng Phenolic acid Glycosylated phenolic

compounds

Ushiyama and Furuya

(1989)


have also been used (Terashima et al., 1999a,b). Hiatt et al. (1989) estimated that plant-

derived 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 During 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


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).


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 biotransforma-

tions 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.


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 hollow-

fibre 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 Cathenamine Ajmalicine Agarose Felix et al. (1981)

Di. lanata Digitoxin Digoxin Alginate Brodelius et al. (1979)

D. carota Digitoxigenin Periplogenin Alginate Jones and Veliky (1981)

N. tabacum Keto esters Hydroxy esters Alginate Naoshima and Akakabe (1989)

P. somniferum Codeinone Codeine Polyurethane foam Furuya et al. (1984)

Furusaki et al. (1988)

Muc. pruriens L-Tyrosine L-DOPA Alginate Wichers et al. (1983)

Capsicum spp. Ferulic acid,

vanillylamine

Vanillin,

capsaicin

Alginate Johnson et al. (1996)

Synthesis from precursors

Cath. roseus Tryptamine,

secologanin

Ajmalicine Alginate, agarose Brodelius et al. (1979)

Ca. frutescens Isocapric acid, Capsaicin Polyurethane foam Brodelius and Nilsson (1980)

vanillylamine, Lindsey and Yeoman (1983b)

valine and Lindsey and Yeoman (1984)

ferulic acid

Coffea arabica Theobromine Caffeine Membrane Lang et al. (1990)

N. tabacum Phenylalanine Caffeoyl

putrescine

Alginate Berlin et al. (1989)

De novo synthesis

Ca. frutescens Capsaicin Polyurethane foam Johnson (1993)

Cath. roseus Ajmalicine Alginate, agarose Brodelius and Nilsson (1980)

Glycine max Phenolics Hollow fibres Prenosil and Pedersen (1983)

Lavandula vera Pigments Polyurethane foam Lindsey and Yeoman (1984)

Diosc. deltoidea Diosgenin Polyurethane foam Ishida (1988)

Th. minus Berberine Alginate Kobayashi et al. (1987)

Tagetes patula Thiophenes Alginate Ketel et al. (1987)

M. citrifolia Anthraquinones Alginate Brodelius et al. (1979)


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 me-

tabolites 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, glucosyla-

tion, isomerization, methylation, demethylation, epoxidation, etc. The presence of biotrans-

formation 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).

Table 12

Dramatic effects of immobilization on secondary metabolite production in plant cell cultures

Plant species Product Fold change

Type of

immobilization Reference

Ca. frutescens Capsaicin > 100 (I) Foam Lindsey and Yeoman (1984)

Ca. frutescens Capsaicin > 100 (I) Gel Ravishankar et al. (1988)

Tag. patula Thiophenes Ca 20 (I) Natural glass Hulst and Tramper (1989)

Coffea arabica Methylxanthin 13 (I) Gel Haldimann and Brodelius (1987)

Cath. roseus Ajamlicine 3.5 (I) Gel Asada and Shuler (1989)

Cath. roseus Total alkaloids No de novo

synthesis

Membrane Payne et al. (1988)

Muc. pruriens L-DOPA No de novo

synthesis

Gel Wichers et al. (1983)

Salvia miltiorrhiza Cryptotanshinone 2.5 (D) Gel Miyasaka et al. (1986)

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


(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

Table 13

Biotransformation of flavour compounds by plant cell culture systems


Citrus limon Valencene Nootkatone Drawert et al. (1984)

Citrus paradisi Valencene Nootkatone Drawert et al. (1984)

N. tabacum Linalool 8-hydroxylinalool Suga and Hirata (1990)

Mentha spp. Pulegone Isomenthone Aviv and Galun (1978)

Menthol Neomenthol Aviv et al. (1981)

Ste. rebaudiana Steviol Stevioside Furuya (1978)

Lavandula angustifolia Geranial Geraniol Lappin et al. (1987)

Neral Nerol

Citronellal Citronellol

Va. planifolia Ferulic acid Vanillin Romagnoli and Knorr

(1988)

Ca. frutescens Ferulic acid Vanillin Johnson et al. (1996)

Eucalyptus perriniana Isoeugenol

and eugenol

Isoeugenyl b-rutinoside and

eugenyl b-glucosideOrihara et al. (1992)

G. jasminoides Acetophenone (S)-aromatic alcohol Akakabe and Naoshima

(1993)

Curcuma zedoaria Germacrone Guaiane-type sesquiterpenes Sakui et al. (1992)

Gl. glabra Glycyrrhitinic acid Hydroxyglycoside esters Hayashi et al. (1992)

Coffea arabica Vanillin Vanillin glucosides Kometani et al. (1993a)

Papaver bracteatum Linalyl acetate Linalool, a-terpineol Hook et al. (1990)

Geraniol

Achillea millefolium Borneol, menthol

thymol and farnesol

Many products Figueiredo et al. (1996)

N. tabacum Carvone Dihydrocarvone

neodihydrocareol

Hirata et al. (1982)

Cath. roseus Piperitone Hydroxypiperitone Hamada and Furuya (1996)

N. tabacum Pulegone Menthone 4-hydroxymenthone Hamada and Furuya (1996)

Cath. roseus Geraniol 10-hydroxygeraniol Hamada and Furuya (1996)

Cath. roseus Glycyrrhizin Glycyrrhetic acid Hamada and Nakata (1992)

Coffea arabica Capsaicin Capsaicin glucoside Kometani et al. (1993b)

Euc. perriniana Menthol Menthol 3-O-b-gentiobiosides Orihara et al. (1991)

Euc. perriniana Camphor Camphor glucosides Orihara et al. (1994)

Euc. perriniana Borneol (�) Borneol b-gentiobioside Orihara and Furuya (1993)

Cymbidium spp. Menthyl acetate Menthol Mironowicz et al. (1987)

Adapted from Ramachandra Rao (1998).


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 14

Biotransformations using plant cell cultures for production of pharmaceuticals


P. somniferum Thebaine Codeine Wilhelm and Zenk (1997)

Di. purpurea Digitoxin Digoxin Alfermann et al. (1980)

Di. lanata Digitoxin Digoxin Alfermann et al. (1980)

Euc. perriniana Taxol Taxol derivatives Hamada et al. (1996)

Podo. hexandrum Coniferyl alcohol Podophyllotoxin Van Uden et al. (1995)

Muc. pruriens Tyrosine DOPA Pras et al. (1993)

Cath. roseus Vindoline Vincrisitne DiCosmo and Misawa (1995)

Vinblastine

Ca. frutescens Ferulic acid Capsaicin, vanillin Johnson et al. (1996)

Vanillylamine Capsaicin, vanillin

Ca. frutescens Protocatechuic aldehyde Ramachandra Rao and

Caffeic acid Ravishankar (2000a)

Ca. frutescens Digitoxin Digoxin,

purpureaglycoside A

Ramachandra Rao et al.

(2002)

Spirulina platensis Codeine Morphine Ramachandra Rao et al. (1999)

Ca. frutescens Isoeugenol Vanillin, capsaicin Ramachandra Rao and

Ravishankar (1999)

Table 15

Bioreactor types used for plant cell cultures

Reactor type Plant species Reference

Stirred tank Cath. roseus Drapeau et al. (1986)

N. tabacum Kato et al. (1977)

Bubble column N. tabacum Noguchi et al. (1977)

Airlift stirred M. citrifolia Wagner and Vogelmann (1977)

Aerated L. erythrorhizon Tanaka (1987)

Fluidized bed Cath. roseus Morris et al. (1984)

Tubular membrane Cath. roseus Shuler et al. (1983)

Disc turbine Ruta vulgaris Shuler and Hallsby (1985)


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 16

Large-scale suspension cultures reactors for plant cell cultures

Plant species Product Bioreactor capacity Reference

Cath. roseus Serpentine 100 l airlift Smart and Fowler (1981)

Col. blumei Rosmarinic acid 300 l airlift Rosevear (1984)

L. erythrorhizon Shikonin 750 l agitated Tabata and Fujita (1985)

N. tabacum Biomass 20,000 l agitated Kato et al. (1976)

Biomass 1500 l bubble column Noguchi et al. (1977)

Panax ginseng Saponins 20,000 l agitated Ushiama et al. (1986)

E. purpurea Biomass 750–75,000 l agitated Ritterhaus et al. (1990)

Ra. serpentina Biomass 750–75,000 l agitated Ritterhaus et al. (1990)

Panax ginseng Biomass 750–75,000 l agitated Ritterhaus et al. (1990)

Adapted from Sahai (1994).

Table 17

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

Precursor Productivity (g) % Conversion

b-methyldigitoxin added 17.24 100

Unconverted b-methyldigitoxin 2.04 11.8

b-methyldigoxin formed 14.36 81.7

By-product 0.28 1.4

Yield 94.9

Adapted from DiCosmo and Misawa (1995).


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, immobi-

lized 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.


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 CO2 + 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.


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


weight basis

11.3.3. Yields in SM+precursor (2.5-mM coumaric acid)

1. Immobilized cells: not done


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—protoca-

techuic 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).


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.

Table 18

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

Bioreactor Productivity

Bubble column

Biomass density 100 g fresh weight/l

Average productivity 1.7 mg/l/day

Average specific productivity 17 mg/g fresh weight/day

Maximum duration of continuous operation 21 days

Total production 35 mg/l in 21 days

Packed-bed reactor

Biomass density 380 g/l

Average productivity 7.4 mg/l/day

Average specific productivity 19.5 mg/g fresh weight/day

Maximum duration of continuous operation 28 days

Total production 207 mg/l in 28 days

Production under fungal elicitation 330 mg/l in 28 days

Adapted from Madhusudhan (1998).

Fig. 5. Chemical structure of vanillin.


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 Reference

Ferulic acid

Aspergillus niger Lesage-Messen et al. (1996)

Pycnoporous cinnabarinus

Pseudomonas fluorescens Andreoni et al. (1995)

Pseudomonas fluorescens AN 103 Narbad and Gasson (1998)

Corynebacterium glutamicum Labuda et al. (1993)

Ca. frutescens Ramachandra Rao (1998)

Spir. platensis Ramachandra Rao et al. (1996a)

Haemotococcus pluvialis Usha et al. (1999)

Va. planifolia Romagnoli and Knorr (1988)

Va. planifolia Funk and Brodelius (1990)

Eugenol

Corynebacterium glutamicum Tadasa and Kayahara (1983)

Pseudomomnas spp. Rabenhorst (1996)

Arthrobacter globiformis Cooper (1987)


Isoeugenol

Enterobacter spp. Rabenhorst (1991)

Serratia spp.


Ca. frutescens Ramachandra Rao and Ravishankar (1999)

Vanillylamine

Asper. niger Yoshida et al. (1997)

E. coli

Spir. platensis Ramachandra Rao (1998)

Ca. frutescens Sudhakar Johnson et al. (1996)

Coniferyl aldehyde

Ca. frutescens Ramachandra Rao (1998)


Coniferyl aldehyde Markus et al. (1992)

Vanillyl alcohol

Ca. frutescens Ramachandra Rao and Ravishankar (1998)


Penicillum simplicissimum Fraaije et al. (1997)



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.


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 wound-

healing 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 20

Effect of antioxidants on lipid peroxidation

Compound IC50

Butylated hydroxyanisole 0.125

Butylated hydroxytoluene 0.250

a-tocopherol 0.500

Anthocyanin from carrot cell cultures 0.005

Adapted from Narayan et al. (1999).

Table 21

Growth and production of anthocyanin in D. carota cell cultures

Parameters Value

Growth

Specific growth rate 0.217/day

Doubling time 3.2 days

Growth index 12.2

Maximum cell density 18.92 ± 1.17 g/l

Dry weight/fresh weight ratio 0.085

Packed cell volume 0.95 ml/g fresh weight

Anthocyanin production

Average content 15.58% (w/w) dry mass

Specific productivity ( qp) 10.52 mg/g/day

Total production 3.1 g/l in 15 days

Average productivity 207 mg/l/day

Adapted from Madhusudhan (1998).


(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 rate-

limiting 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.

Fig. 7. Chemical structure of anthocyanin.


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


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 (Schu-

macher 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. Down-

regulation of PPO increases the quality attributes of crop plant-derived commodities,

whereas overexpression reduces danger from pests. It has been concluded that no phenotypic


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-hydroxyben-

zoic 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 high-

cost, 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


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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