Tabela Legal Plant Cell and Tissue Culture - Alternatives for Metabolite Production

Pergamon Biotechnology Advances, Vol. 13, No. 3, pp. 425--453, 1995

C, opyrisht © 1995 Elsevier Science lno. Printed in Great Britai~ All rishts reserved

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PLANT CELL AND TISSUE CULTURE: ALTERNATIVES FOR METABOLITE PRODUCTION

FRANK DICOSMO 12 and MASANARU MISAWA 2'3

1Department of Botany and ZlnJtitute of Biomedical Engineering, University of Toronto, Toronto, Canada MSS 3B2

JBIO INTERNATIONAL INC., Suite 420, 151 Bloor Street West, Toronto, Ontario, Canada MSS 1S4

ABSTRACT

Plant cell culture systems represent a potential renewable source of valuable medicinals, flavours, essences and colourants that cannot be produced by microbial cells or chemical syntheses. However, only a few cultures produce these compounds in commercially useful amounts. The low productivities are associated with our poor understanding of the biochemistry of these systems. Recent advances in molecular biology, enzymology, physiology and fermentation technology of plant cell cultures suggest that these systems will become a viable source of important natural products. This review examines the sate of the art of production of medicinal plant secondary metabolites by plant cell cultures.

KEY WORDS: Plant cell culture technology, secondary metabolites, pharmaceuticals, medicinal compounds.

INTRODUCTION

Plants produce biochemicals that are of importance in the healthcare, food, flavour and

cosmetics industries. Many pharmaceuticals are produced from the secondary metabolites

of plants. Examples are digitalis, L-DOPA, morphine, codeine, reserpine, and the

anticancer drugs vincristine, vinblastine and taxol used in treatment of ovarian and breast

cancers. Currently, these and many other natural products are produced solely from

massive quantities of whole plant parts. Often the source plants are cultivated in tropical

or subtropical, geographically remote, areas which are subject to political instability,

drought, disease and changing land use patterns and other environmental factors. In

425

426 F. DICOSMO and M. MISAWA

addition, the long cultivation periods between planting and harvesting make selection of

high-yielding strains difficult, thus resulting in expensive drugs. Cultivation periods may

range from several months to decades for the taxol yielding, Taxus brevifolia trees. In spite

of these difficulties and costs, the extraction of medicinals from cultivated plants or plants

in the wild continues because of a lack of credible alternatives.

Clearly, the development of alternative and complimentary methods to whole plant

extraction for the production of clinical medicines is an issue of considerable socio-

economic importance. These factors have generated considerable interest in the use of

plant cell culture technologies for the production of pharmaceuticals [2,11,13,17-

19,22,30,42,48,59] and other plant derived biochemicals [2,10,32,41]. Indeed, the plant cell

culture technology is now sufficiently advanced to allow for large quantities of relatively

homogeneous, undifferentiated cells to be produced. Plant cell and tissue culture systems

are complementary and may provide competitive metabolite production systems when

compared to whole plant extraction. Commercial production of plant natural products via

cell and tissue culture systems becomes viable when the product commands a price

exceeding about $1000/kg [75].

Several types of high-yielding tissue culture systems have been developed. These

include the cultivation of specific organ cultures, suspension cultured cells selected for high

productivity on production media and high-density culture [52,65,86,92]. Certain inorganic

and organic adjuvants stimulate product synthesis and/or accumulation, but product

accumulation is transient [16]. Immobilized cells show promise for increasing synthesis of

biochemicals; however, their benefits are only realized when the product is excreted into

the medium [43,67,80]. Such systems offer the possibility of commercialization for some

very expensive products only. For some high-value-added products the culture systems may

have to compete with total chemical and/or enzyme-mediated syntheses. For example, the

fascile synthesis of a-3', 4'-anhydrovinblastine can be achieved in industrially attractive

yields, with either ferric chloride mediated synthesis or by using peroxidase enzymes [cited

in 16]. The detailed analysis of the biochemistry of plant cell cultures will certainly allow

for improved manipulation of natural product biosynthesis [50]. The production of

secondary metabolites in large bioreactors is therefore an attractive alternative method for

high-value added biochemicals production, with the possibility of controlled production

according to demand [49,68]. Despite the obvious possibilities many problems remain to

PLANT CELL AND TISSUE CULTURE 427

be solved before plant cell culture methods are used for the industrial production of plant

metabolites.

Mitsui Petrochemical Industry Company in Japan was the first to produce a

pharmaceutical and dyestuff on commercial scale with the production of shikonin, valued

at $4,500/kg (Table 1). The company succeeded also in using plant cell culture

technologies to produce the pharmaceutical berberine at a yield of 3.5 g per litre of

culture fluid. In both these systems cultivation of the cells as high density cultures was

essential for productivity. Nitto Denko Company, also in Japan, has successfully cultivated

Panaxginseng cells in 20 m 3 bioreactors for biomass production. Other applications of cell

culture technologies for pharmaceutical production are highlighted in Table 1.

While large scale plant cell suspension cultures are expected to be suitable for

industrial production of plant-derived biochemicals, the production technology needs to

be further developed. Significant differences between microbial and plant cell cultures

mean that the methods developed for microbial systems may have to be modified [4].

Plant cells are sensitive to shear stresses because of their large size and the relatively

inflexible cell wall. For this and other reasons turbine impellers may not be suitable for

plant cell cultures; instead, airlift bioreactors are often recommended. Plant cell cultures

show relatively long growth cycles. Typical growth rates may range from 0.12 d -1 to 0.05

d-l ; thus, the typical doubling time of plant cell cultures is measured in days, as compared

to hours for microbial systems. In plant cells, the vacuole is usually the site of product

accumulation and extracellular product secretion is rare. This creates special problems for

downstream processing, as the cells must be collected and the product extracted. Indeed,

it would be beneficial to develop methods that would allow for product excretion directly

into the growth medium; this would facilitate recovery, permit recycling of the biomass and

perhaps recycling of some media.

Cultured plant cells usually produce low amounts and altered profiles of secondary

metabolites when compared with the intact plant. Production and product profiles may not

be stable, deteriorating ultimately to inactive cultures. The low product yields are yet to

be explained satisfactorily, but indicate metabolic blocks concerning specific enzyme

repression and activity. Nonetheless, significant progress has been made in culture yield

improvements as illustrated in Table 1.


Table 1. Examples of plant cell culture methodologies for the production of metabolites.

Products Production System Reference

Vincamine, 3.3 g'L -1 Cell culture of Vinca minor Equivincamine, 0.9 g.L -t

Shikonin, 4 g.L -1

Berberine, 3.5 g.L -1

Ajmaline, 0.4 g.L -1 Raucaffricine, 1.2 g.L-1

Strictosidine, gram amounts

a-3',4'-anhydrovinblastine, about 70% yield

a-3',4'-anhydrovinblastine, about 70% yield; vinblastine, about 20% yield

Methyldigoxin, 505 g produced in 3 months

Cell culture of Lithospermum erythrohizon

Cell culture of Coptis japonica

Cell culture of Rauwolfia serpentina

Immobilized strictosidine synthase

Glucose oxidase and peroxidase mediated dimerization

FeCl3-mediated dimerization

Biotransformation of methyldigitoxin by Digitalis lanata cells

Farmitalia, Ger. Often. DE 3,902,980 August 17, 1989; Chem. Abs. 196670S, 1990.

Mitsui Petrochem., (see Fujita, Y. and Tabata, M. in Plant Cell and Tissue Culture, Alan R. Liss Inc. pp. 169- 185, 1987).

Mitsui Petrochem. J. Chem. Tech. Biotech. 46: 61-69, 1989.

Schiibel et al. Phytochem. 28: 419-494, 1989.

Pfitzner and Zenk, Planta Med. 46: 10-14, 1982.

DiCosmo, F. Progress bz Plant Celhdar and Molecular Biology [16].

Allelix Inc., Vukovic, J. and Goodbody, A.E., U.S. Pat. 4,778,885.

Alferman, A.W. et al. (in Primary and Secondary Metabolism in Plant Cell Cultures, Springer-Verlag, pp. 316-322, 1985).

The increased use of plant cell culture systems in recent years is perhaps due to an

improved understanding of the biochemistry of secondary metabolic pathways in

economically important plants. This trend is expected to increase as advances in plant

biotechnology surge on. This review is not intended to be exhaustive, rather specific

reference is made to certain areas with demonstrated potential for industrial application.


APPROACHES TO INCREASE PRODUCTIVITY

For plant cell culture systems to become economically viable, it is important to develop

methods that would allow for consistent generation of high yields of products from

cultured cells [9]. Several products are accumulated in cultured cells at a higher level than

found in intact plants. This is due to careful selection of productive cells and cultural

conditions (Table 2). For example, berberine by Coptis japonica [54], ginsenosides by

Panax ginseng [83], rosmarinic acid by Coleus blumei [82], shikonin by Lithospermum

etythrorhizon [79], diosgenin by Dioscorea [55], ubiquinone-10 by Nicotiana tabacum were

accumulated in much higher levels in cultured cells than in the intact plants [30]. The

general experience, however, is that plant cell cultures usually lose the ability to produce

secondary metabolites that are characteristic of the intact plant [18]. In order to obtain

yields in concentrations high enough for commercial exploitation, efforts have focused on

the stimulation of biosynthetic activities of cultured cells using various methods.

Table 2. Secondary metabolites produced in high levels by plant cell cultures.

Compound Plant Species Yield (% dry wt) Cell Culture Whole Plant

Shikonin Ginsenoside Anthraquinones Ajmalicine Rosmarinic acid Ubiquinone-10 Diosgenin Benzylisoquinoline

alkaloids Berberine Berberine Anthraquinones Anthraquinones Nicotine Bisoclaurine Tripdiolide

Lithospermum erythrorhizon 20 (s*) 1.5 Panax ginseng 27 (c) 4.5 Morinda citrifolia 18 (s) 2.2 Catharanthus roseus 1.0 (s) 0.3 Coleus blumeii 15 (s) 3 Nicotiana tabacum 0.036 (s) 0.0003 Dioscorea deltoides 2 (s) 2

Coptisjaponica 11 (s) 5-10 Thalictrurn mb~or 10 (s) 0.01 Coptis japonica 10 (s) 2--4 Galium verum 5.4 (s) 1.2 Galium aparbze 3.8 (s) 0.2 Nicotiana tabacum 3.4 (c) 2.0 Stephania cepharantha 2.3 (s) 0.8 Tripterygium wilfordii 0.05 (s) 0.001

*s = suspension; c = callus


Optimization of Cultural Conditions

Physical optimization. Several chemical and physical parameters affecting culture growth

have been tested extensively with various plant cells. These parameters include media

components, phytohormones, pH, temperature, pO2, pCO2, agitation and light among

others. Manipulation of the physical aspects as well as the nutritional elements in a culture

is perhaps is the most fundamental approach for optimization of culture productivity.

Many reports and patents have discussed optimization of cultural conditions for improved

growth rates of cells and/or higher yield of desirable products. A few notable examples are

highlighted here. DiCosmo and Towers [18] summarized the effects of various culture

manipulations on secondary metabolite biosynthesis. Various well-known basal media for

the production of indole alkaloids have been tested; the results indicate that serpentine

accumulation depends on the composition of the medium used, as might be expected.

Among them, Murashige and Skoog (MS) formulation was recognized as superior for the

production of serpentine by Catharan thus roseus suspension. This is not always true for

other species in culture.

Sucrose and glucose are the preferred carbon source for plant tissue cultures;

although other carbohydrate sources are often used. The concentration of the carbon

source affects cell growth and yield of secondary metabolites in many cases. In general,

sucrose appears to be the preferred carbon source for cultured plant cell systems.

DiCosmo and Towers [18] provided a review of the effects of nutritional factors on plant

cell culture growth and product yield. Hormone, auxin and kinetin levels have shown the

most remarkable effects on growth and productivity of plant metabolites. Increased auxin

levels in cultures, such as 2,4-dichlorophenoyacetic acid (2,4-D) in the medium promotes

dedifferentiation of cells and diminishes the accumulation of secondary metabolites.

Indeed, auxins added to solid media stimulate callus induction, but for product synthesis

it may be advisable to use low levels or remove auxins. For example, cytokinins stimulated

alkaloid synthesis when auxin was removed from the medium of a cell line of Catharan thus

roseus. However, accumulation of L-DOPA by M a c u n a pruriens [11] was stimulated by

relatively high amounts of 2,4-D. In Catharan thus roseus increased levels of alkaloids are

produced by cells transferred to production media. Accumulation of alkaloids begins a few

days after cells are transferred to production media and the greatest specific yields are

reached after approximately 12 days. Berlin et al. [10] compared various strategies

designed to improve indole alkaloid biosynthesis in C. roseus.


Selection of high-producing strains. Plant cells in culture represent a heterogeneous

population in which physiological characteristics of individual plant cells are different. The

techniques used in selection and screening plant cell cultures for improved product

synthesis are described by Berlin and Sasse [9]. Cell cloning methods provide a promising

way of selecting cell lines yielding increased levels of product. Most selection methods are

based on production of pigments, such as anthocyanins, betacyanins and others. A strain

of Euphorbia milli also accumulated about 7-fold the level of anthocyanins produced by

the parent culture after 24 selections [89].

Cell selection and somatic cloning provide useful methods for recovering cells that

produce increased levels of secondary metabolites. However, the lack of rapid screening

methods often impedes application of the method. Deus and Zenk [15] pointed out that

a fluorescence assay could be used to select cells yielding ajmalicine and other major

heteroyohimbine alkaloids from C. roseus cultures. The method was dearly more viable

than the use of radioimmunoassay which can be time-consuming and costly. Indeed, the

technique of cell selection, based on somatic cloning is especially useful when coloured

products can serve as visual selection cues.

Addition of precursors. It has been possible to increase the biosynthesis of specific

secondary metabolites by feeding precursors to cell cultures. For example, amino acids

have been added to cell suspension culture media for production of tropane alkaloids,

indole alkaloids and other products. It is likely that the precursors may be either

incorporated directly into the product, or precursors may enter a specific product indirectly

through degradative metabolism and entry into interrelated pathways. Phenylalanine is a

precursor of rosmarinic acid [23]; addition of phenylalanine to Salvia officialis suspension

cultures stimulated the production of rosmarinic acid and decreased the production time

as well. Pbenylalanine is also the precursor of the N-benzoylphenylisoserine side chain of

taxol; supplementation of Taxus cuspidata cultures with phenylalanine resulted in increased

yields of taxol [27,28]. The literature is replete with examples of the effects of precursor

feeding on the production of secondary metabolites by cultured cells [18]. The evidence

clearly indicates that the timing of precursor addition is critical for an optimum effect. The

effects of feedback inhibition must surely be considered when adding products of a

metabolic pathway to cultured cells.


BIOTRANSFORMATIONS

Often, plant cells will biotransform a suitable substrate to a more desirable product.

Endress et aL [25] found Rauwolfia serpentina to yield a new indole alkaloid, 6-

hydroxytaumacline, in good yield when the cells were grown in the presence of ajmalicine.

Arbutin, a skin depigmentation agent, is produced by biotransformation of hydroquinone

using C. roseus cells [91]. Addition of the precursor into the liquid culture medium of C.

roseus yields arbutin.

Dombrowski and Alfermann [21] observed the glycosylation of salicylic acid and its

derivatives. They found that cultured Salb¢ matsudana cells glucosylated salicylic acid to

2-O-salicylic acid-13-D-glucoside. The cells also converted salicyl alcohol to salicin (2-0-

salicyl alcohol- 13-D-glucoside) and isosalicylicin (1-O-salicylalcohol- 13-D-glucoside). Salicylic

aldehyde was converted to the 13-D-glucosides of salicylic acid and salicyl alcohol.

Biotransformation of 13-methyldigitoxin to 13-methyldigoxin using D. lanata cells has

been studied extensively by Alfermann (see Table 1). About 600-700 mg of 13-

methyldigoxin per litre was obtained using a 200 L reactor; the process was evaluated for

commercialization by Boehringer Mannheim Company.

Allelix Inc. in Canada [56] established a processes for production of the antitumor

drug, vinblastine, from catharanthine and vindoline using biotransformation, as well as a

simple chemical synthesis. The process is currently being developed for commercialization

by Mitsui Petrochemical in Japan [31]. It is believed that biotransformation processes

including addition of substrates into cultures of suitable cell lines represents one of the

most commercially realistic approaches using plant tissue cultures for product synthesis.

However, the availability of expensive precursors is a key issue and limits economic

viability in some cases.

IMMOBILIZED CELLS FOR SECONDARY METABOLITE PRODUCTION

Compared with plant cell suspension cultures, it is widely held that adherent plant cells

(i.e., immobilized biocatalysts) provide a range of advantages [19,70]. The advantages

include: (1) the extended viability of cells in the stationary (and producing) stage, enabling

maintenance of biomass over a prolonged time period; (2) simplified downstream

processing (if products are secreted); (3) high cell density within relatively small

bioreactors showing reduced costs and risk of contamination; (4) reduced shear stress; (5)

increased product accumulation; (6) flow-through reactors can be used enabling greater


flow rates; and (7) minimization of fluid viscosity which in cell suspension causes mixing

and aeration problems.

However, the procedures most frequently used for plant cell immobilization, which

are based on entrapment of cells with gels, foams or membranes appear to be either

expensive for large scale application or technically unsound, and consequently none of the

methods has yet been applied commercially for the production of plant derived

biochemicals. The main drawbacks of these methods of immobilization are: (1) gels and

foams are too soft and deformable for use in large scale reactors; (2) membrane systems

are costly; (3) mass transfer is limited and creates undesirable nutrient gradients; (4)

trapped cells are not easily recovered; (5) growing cells tend to rupture the confining

matrix during extended cultivation; and (6) increased hydrodynamic pressures in flow-

through reactors.

In contrast to immobilization by entrapment which has been extensively reviewed

by others [43,69,80], we believe that immobilization by adsorption [19] may represent an

improved approach to cell culture. The advantages are: (1) cultured cells are induced to

adhere to selected polymeric, glass fibre, carbon fibre or ceramic fibre substrates by

controlling the surface tension and electrostatic properties of the liquid medium; (2)

improved mass transfer because no matrix surrounds the cells; (3) improved downstream

processing for excreted products such as proteins and enzymes; (4) immediate reuse of the

media as the cells are totally immobilized, and lower operating costs; (5) the ability of the

cell population to grow to exceedingly high densities with no destruction of the support

(a major advantage as high density cultures usually produce increased levels of

pharmaceuticals); (6) rapid immobilization of large-scale cultures is readily achieved under

physiological conditions; and (7) inexpensive matrices of any geometric configuration can

be used. Furthermore, and possibly fundamentally more important, it seems likely that the

biosynthetic potential of normally slow growing plant cells may be better exploited with

immobilized systems, in terms of both yield and subsequent isolation of the products.

Gel Entrapment Methods

Gel entrapment has been the preferred method of cell immobilization for cultured plant

cells. Calcium alginate gel is the most prevalent gel agent for immobilization. The first

report of immobilization of cultured plant cells described the use of a calcium alginate gel

for the incorporation of spherical clusters of cells of Morinda citrifolia, Digitalis purpurea,


and Catharanthus roseus [12]. Cell viability was maintained for a 22 day ctilture period in

an appropriate nutrient medium. Calcium alginate entrapped cells are usually prepared

by suspending the cells in a solution of sodium alginate which is subsequently extruded

through an appropriate small diameter opening. The resulting beads 'gel' on contact with

a solution of (50-100 I~M) calcium chloride. Cell distribution within the bead is more or

less homogeneous. The entrapped or immobilized cells can be recovered by chelation of

the calcium ion using EDTA or polyphosphates [53].

The effects of different gel immobilization agents for cells of C. roseus was

evaluated by Brodelius and Nilsson [13]. Cell viability was poor when in the presence of

acrylamide and glutaraldehyde, alginate and carrageenin were non-detrimental and the

best results, vis-a-vis growth and yield of alkaloids were reported for calcium alginate.

Continued growth of cells in the beads ultimately disrupts the gel and free cells emerge.

Consequently, gel entrapment is perhaps most useful with non-dividing cells in a nutrient-

or hormone-limited liquid medium [66].

Surface Immobilization

Surface immobilization takes advantage of the propensity of cultured plant cells to adhere

to inert surfaces immersed in the liquid. The attachment of plant cells to glass culture

vessel surfaces is a feature common to cell suspension cultures and complicates process

control. Recently, several groups have exploited 'surface immobilization' of plant cells

[2,46]. DiCosmo et al. [19] provide a review of plant cell adsorption to surfaces and

immobilization on glass fibres.

The surface immobilization of Catharanthus roseus, Nicotiana tabacum and G&cine

max cultured cells has also been reported [1-3]. A porous ceramic matrix retained cells

effectively. Also a rigid, but porous material of acrylic or rayon fibres bound by phenolic

resin, and a series of non-woven polyester or polypropylene textile materials were effective

immobilization substrates. Archambault et al. [2] suggested that the surface immobilization

of cultured plant cells is advantageous to gel entrapment methods. The surface

immobilization method is simple and gentle; the matrix is inexpensive and retains the

growing biomass efficiently and establishes a well-separated, two-phase system. The

polyester material has been incorporated into rolled column fitted within a laboratory-scale

bioreactors containing C. roseus cells [3].


DiCosmo et al. [19] discussed the process of surface immobilization of plant cells.

Theoretical considerations as well as some practical examples highlighting the use of glass

fibre mats as the immobilization matrix were provided. The use of glass fibre mats for cell

immobilization was tested in a 5 L bioreactor. The production of alkaloids by C. roseus,

and berberine and columbamine by Thalictrum rugosum cells immobilized on mats was

examined. Immobilization was found to be relatively rapid and occurred efficiently under

physiological conditions. The immobilization was firm and irreversible, and the system was

maintained for an extended period. The accumulation of indole alkaloids and tryptamine,

by glass fibre-immobilized cells of C. roseus was lower than in freely suspended cells. In

contrast, the accumulation of berberine, by immobilized and suspension cultured cells of

T. ntgosum was similar. The growth rate of immobilized cells of C. roseus and T. ntgosum

was lower than freely suspended cells. The increase in the accumulation of indole alkaloids

(Table 3) by cells of C. roseus when entrapped in alginate beads may arise as a response

to phosphate limitation, rather than due to the immobilization in alginate beads. Glass

fibre-immobilized C. roseus cells yield decreased production of indole alkaloids; this is in

accord with other non-alginate immobilized systems.

The process of cell immobilization by adsorption to the glass fibre mat is a

generally applicable phenomenon. The process involves non-specific, thermodynamic and

electrostatic interactions, as well as passive entrapment. The immobilization demonstrates

the industrial potential of the system, as well as being a technique of evaluating the

physiology and metabolism of adherent plant cells free of the constraints of an

encapsulating gel.

Other Approaches

The regulation of plant secondary metabolism at the biochemical and genetic levels will

undoubtedly lead to improved production systems in the near future [19a], particularly for

phenylpropanoid-derived substances. Selection of variant cell lines with increased

biosynthetic abilities for secondary metabolites will also provide improvements to cell

culture technology [8a]. These topics have been detailed authoritatively elsewhere [8a] and

will not be discussed here.


Table 3. Reported effect of various immobilization strategies on the accumulation of indole alkaloids by cultured cells of Catharanthus roseus.

Immobilization Product Product accumulation Reference system relative to suspensions

Alginate Ajmalicine (+)* Brodelius eta/- [8] Alginate Tryptamine ( - )* Maierus and Pare~ux I531

Ajmalicine Serpentine

Alginate Ajmalicine (+) Asada and Shuler [1] Agar/carrageenin Ajmalicine (-) Brodelius et at [8] Dialysis tubing Total alkaloids (-) Payne ct al. [63] Non-woven polyester Strictosidine lactam (-) Archambault eta/. [3]

Ajmalicine Tabersonine Serpentine

Glass fibres Tryptamine (-) DiCosmo et a/- [19] Catharanthine Ajmalicine

* (+) = increase; (-) = decrease

EXAMPLES OF SECONDARY METABOLITES AS CELL CULTURE PRODUCTS

A variety of plant derived alkaloids are used as pharmaceuticals. The typical tropane

alkaloids, atropine, hyoscyamine, scopolamine and cocaine, are widely used as antagonists

of the parasympathetic nervous system. Research on cell culture production of these

compounds has been active for more than 25 years. Industrial production has yet to

succeed because of the low yield from cultured cells. Nevertheless, the bisindole alkaloid

vinblastine, will likely be produced commercially by Mitsui Petrochemical in Japan using

a combination of plant cell culture and chemical synthesis.

Morphinan Alkaloids

Codeine is an analgesic and cough-suppressant from Papaver somni f emm, that is also the

traditional commercial source of morphine which can be converted to codeine. Mature

capsules of P. bracteatum also accumulate up to 3.5% thebaine which is convertible to


codeine. Production of codeine by undifferentiated cells ofPapaver spp. has met with little

success. Kamimura et al. [45] reviewed alkaloid production and indicated that

morphogenetic differentiation of cultured cells of P. bracteamm was required for

stimulating thebaine biosynthesis. Other researchers have confirmed similar findings with

P. bracteamm and P. somniferum [75,76].

The efficient production of thebaine and codeine using cell culture systems by de

novo synthesis has not been successful; however, the biotransformation of codeinone to

codeine with immobilized cells of P. somnifen~m has been shown to be possible [33]. The

conversion yield was 70.4%, and about 88% of the codeine converted was excreted into

the medium. Eilert et al. [22] showed that fungal mycelium (elicitor) ofBottytis sp. induced

production of sanguinarine by cultured P. sornnifentm cells. The presence of the elicitor

increased alkaloid production 26-fold in comparison with the elicitor-free culture.

Berberine

Berberine is an isoquinoline alkaloid which is found in roots of Coptis japonica and the

cortex of Phellondendron amurense. Berberine chloride is used for intestinal disorders in

the Orient. Five to six years are required to produce Coptis roots as the raw material.

Furuya et aL [34] investigated the production of berberine by Coptisjaponica cell cultures

while Yamada and Sato [88] at Kyoto University selected a high berberine producing cell

line of C. japonica. The latter cell line was subsequently transferred to Mitsui

Petrochemical where the yield of berberine was further improved. Hara et al. [39]

determined that addition of 10 -8 M gibberellic acid into the medium stimulated berberine

accumulation to 1.66 g'L -1 of medium. By careful selection several high yielding cell lines

were isolated. The Mitsui group produces berberine at a large scale with a productivity of

1.4 g.L -1 over 2 weeks. A high-density cell culture process for producing berberine more

efficiently has been developed at Mitsui. Addition of spermidine to Thalictrum mbzus is

known to stimulate the production of berberine in suspension culture [38]. Other

polyamines such as cadaverine, putrescine and spermine are ineffective [38]. Maximum

yield of berberine was obtained in the presence of 2 mM spermidine.

Tropane Alkaloids

Studies on the production of tropane alkaloids by plant tissue cultures have been ongoing

since the discovery of tropanes in Atropa belladonna callus tissue. However, the


concentrations of scopolamine and hyoscyamine in cultured cells are generally low and

plant cell culture technology has not yet enabled commercial production of these alkaloids.

Atropine, scopolamine and hyscyamine have been produced by cultured roots of Atropa

and Dubosia [30].

Cardenolides

Cardiac glycosides or cardenolides are derived commercially from Digitalis species. Some

cardenolides are used in the treatment of heart diseases. Cultivation of Digitalis plants in

fields of several countries, including the Netherlands, Hungary and Argentina yields the

commercial quantity of clinical drugs. Lanoxin is the Burroughs Wellcome digoxin product

that has major markets in the United States and Italy; total sales being about 6000 kg per

year with a market value of US$50 million. Other producers of cardenolides are

Boehringer Mannheim, Merck Darmstadt and Beiersdorf AG in Germany.

There are many studies concerning the production of cardiac glycosides by Digitalis

tissue cultures [66]. Generally, the yield of product is very low and decreases during

successive culture transfers. Many researchers have concluded that morphological

differentiation causes an increase in productivity of cultured cells. For example, Lui and

Staba [51] showed that organ cultures ofD. lanata leaves and roots produced cardenolides;

the amount of digoxin in the tissues increased with age [52]. Hirotani and Furuya [41]

found that differentiation of callus tissues of D. purpurea led to formation of cardinolides.

Hagimori et al. [37] of Japan Tobacco Inc. cultivated shoot-forming tissues of D, purpurea

in a 3 L jar fermenter and detected high concentrations of cardiac glycosides, including

digitoxin. The culture of differentiated tissue is necessary for secondary metabolites

production in some cases. Secondary metabolites such as digitoxin and digoxin have been

found in cell tissues cultures of D. lanata and D. purpurea. Other such compounds include

cholesterol, campesterol, stigmasterol, [l-sitosterol and 4-hydroxy-digitolutein. As might be

expected, the production of such products decrease with the total length of time the cells

have been in culture [47].

Biotransformation reactions using Digitalis plant cells appear to present a viable

commercial process. D. lanata and D. purpurea callus cultures have been shown to rapidly

yield pregnane from progesterone [35]. Organ cultures (leaf and root) of D. lanata and

shoot-forming callus tissues of D. purpurea produced increased amounts of digoxin and/or

digitoxin when progesterone was an adjuvant in the culture medium [37,52]. Indeed, the


biotransformation of digitoxin to digoxin iasing D. lanata cells provides a commercially

interesting approach to the production of digoxin. Digitalis leaves contain a relatively large

amount of digitoxin used as a substrate in the biotransformation. The biotransformation

is a 12 I~-hydroxylation reaction of digitoxin. The 13-methyldigitoxin has been found to be

the best substrate in the biotransformation since methyldigoxin is the major product.

Immobilized cells of D. lanata have been employed in semi-continuous culture. The results

of a typical biotransformation after 17 days in a 20 liter bioreactor are shown in Table 4.

The process was tested in large scale reactors by Boehringer Mannheim Company in

Germany; however, a commercial operation has not been attempted.

Table 4. Biotransformation of 13-methyldigitoxin to 13-methyldigoxin by Digitalis lanata cells in a 20 L reactor.

13-Methyldigitoxin added 17.24 g (100%) Unconverted 13-methyldigitoxin 2.04 g (11.8%) 13-Methyldigoxin formed 14.36 g (81.7%) By-product 0.28 g (1.4%) Yield 94.90%

L-DOPA

L-3,4-dihydroxyphenylalanine (L-DOPA) is an important intermediate in plant metabolism

and is a precursor of alkaloids, betalain, melanin, and other metabolites. It is used as a

potent drug in the treatment of Parkinson's disease. Callus tissue of Mucana pruriens can

accumulate 25 mg.L -1 DOPA, in medium containing a relatively high concentration of

2,4-D [11]. Cells of M. pruriens immobilized within alginate beads produced DOPA from

tyrosine in yields of up to 2% of the dry cell weight and the product was secreted into the

medium. Teramoto and Komamine [81] used callus tissues of Stizolobium hassjoo (Mucuna

hassjoo), M. pntriens and M. deeringiana, to study L-DOPA production. The highest yield

(80 ismol.g -1 fresh weight) was obtained when S. hassjoo cells were cultivated in MS

medium containing 0.025 mg.L -1 2,4-D and 10 mg.L -1 kinetin.


Valepotriates

Plants in the Valerianaceae genus have been used extensively as folk medicines [8]. For

example, Nardostachys jatamansi, Valeriana wallichii and F. officinalis L var. angustifolia

have been used in India and N. chbzensis has been employed in China for hundreds of

years. Partrinia sp. are also being used as sedatives in the former Soviet Union. Although

the active principles in these plants have not been identified, a group of compounds with

sedative, tranquillizer, and cytotoxic properties have been termed 'valepotriates.'

Callus tissues of nine different species of Valerianaceae were studied. Fedia

cornucopiae and Valeriana locusta cells yielded more valepotriates than found in the intact

plants. L-leucine was considered as a precursor of the valepotriates; hence, Baker and

Chavadej [8] isolated cell lines of F. wallichii resistant to trifluoroleucine, a leucine analog.

However no increase in the production of valepotriates was noted. One strain of F.

wallichii resistant to nystatin yielded 88 mg.g -1 (dry weight) valepotriates. A suspension

culture of F. wallichii treated with colchicine, which was expected to induce polyploid cells

yielded higher amount valepotriates than untreated cultures [8]. A two-phase culture,

developed by addition of RP-8 (Merck) into the culture medium induced secretion of

valepotriates into the medium thereby increasing the total yield of valepotriates. Certain

plant bioregulators, such as dimethyl-morpholinium-bromide, dimethyl-piperidinium-

bromide, dimethyl-piperidinium-chloride as well as 2-(3,4-dichloro-phenoxy)-triethylamine

added to cultures ofF. comucopiae and E wallichff at concentrations of 0.01 to 0.04 mmol,

during the early exponential growth stages of the cells, induced valepotriate accumulation.

Antitumor Compounds

Plants are an attractive source of novel antitumor compounds. A recent example being

taxol. The National Cancer Institute in the United States, for example, has conducted

extensive screening of plants since 1955 and has identified various potent compounds from

these [77] . These antitumor compounds include maytansine, tripdiolide,

homoharringtonine, bruceantin, ellipticine, thalicarpine, indicine-N-oxide, and baccharin.

In addition, other higher plant products including vinblastine, vincristine, the

podophyllotoxin derivative etoposide and camptotbecin and its derivative have already

been marketed as very important anticancer drugs. Taxol from Taxus brevifolia and related

species is now used in treating ovarian and breast cancer and is in clinical trials for other

neoplasms. Generally, plants produce relatively low amounts of secondary metabolites


showing antitumor activity (Table 5). An alternative to whole plant extraction of plant

natural products is through plant cell culture [57,58]. In the following we cite examples

were plant cell cultures have been used to study the possible production of antitumor

agents.

Table 5. Antitumor compounds isolated from higher plants [58].

Antitumor compounds Yield (% of plant dry weight)

Baccharin 2.0 x 10 -2 Bruceantin 1.0 x 10 -2 Camptothecin 5.0 x 10 -3 Ellipticine 3.2 x 10 -5 Homoharringtonine 1.8 x 10 -5 Maytansine 2.0 x 10 -5 Podophyllotoxin 6.4 x 10 -1 Taxol 5.0 x 10 -1 Tripdiolide 1.0 x 10 -3 Vinblastine, vincristine 5.0 x 10 -3

Camptotheein. Camptotheca acumb~ata, a plant native of North China, produces a potent

antitumor alkaloid, camptothecin. It is highly active in Walker rat carcinosarcoma and

mouse leukemia, p388 and L1210; clinical trials in patients with gastrointestinal cancer

demonstrated toxicity. Sakato and Misawa [71] induced callus from the stem of C.

acum#zata on MS solid medium containing 0.2 mg.L -1 2,4-D and 1 mg.L -1 kinetin and

developed liquid cultures in the presence of gibberellin, L-tryptophan and conditioned

medium. After 15 days in liquid suspension, cells yielded 0.0025% on a dry weight basis

camptothecin, i.e. 1/20 the amount produced by the intact plant. A.J. van Hengel et aL [84]

found 0.998 mg camptothecin per liter of medium; the compound was accumulated in C.

acumbzata cells cultivated in MS medium containing 4 mg-L -1 NAA. 10-

Hydroxycamptothecin, that is less toxic than camptothecin is a promising derivative and

is in clinical trials in the United States.

Homoharringtonine. Homoharringtonine, harringtonine and isoharringtonine, are derived

from Cephalotaxus harringonia [64]. These alkaloids are complex esters of the inactive

alcohol, cephalotaxine, and inhibit the growth of several neoplasms. Delfel and Smith [14]


found about 5-10 mg of cephalotaxine related compounds per kg dry wt. of callus tissues

grown for 3 to 6 months; these levels were approximately 1 to 3% of the amounts found

in the intact plant.

MS medium containing 1 mg.L - t kinetin and 3 mg.L -1 NAA can be used to

induce the callus from C. harringtonia [57,58]; radioimmunoassay showed that the levels

of cephalotaxine and its esters were approximately 1/300 of the intact plant.

Podophyllotoxin. Podophyllum peltatum, common to eastern North America contains the

antitumor lignan, podophyllotoxin. It is active against KB cells and certain viral diseases

and skin cancer [62]. A semi-synthetic derivative of podophyllotoxin, etoposide, is active

against brain tumor, lymphosarcoma and Hodgkins' disease and was approved by the US

Food and Drug Adminstration; Bristol-Myers Squibb is among the largest manufacturers

of the drug.

Podophyllotoxin accumulation by P. peltatum cell cultures was investigated initially

by Kadkade [44], who found that a combination of 2, 4-D and kinetin in the medium

supported the highest amount of its production. Red light stimulated production of the

compound in cell culture systems. Woerdenberg et al. [87] used 13-cyclodextin entrapped

coniferyl alcohol as a precursor for podophyllotoxin production in Podophyllum hexandrum

cell suspension cultures. Addition of 3 mM coniferyl alcohol yielded 0.013% on a dry

weight basis podophyllotoxin accumulation in cells; however, cells not provided the

precursor yielded only 0.003%. Confierin was a better precursor in stimulating the yield

(0.055%) of the compound. Cell suspension cultures of Callitris drummondii were also

reported to accumulate podophyllotoxin-l~-D-glucose. The cells produced approximately

0.02% (dry weight basis) podophyllotoxin in the dark, and 85-90% of the lignans extracted

were [3-D-glucosides; in the light, however, the yield of podophyllotoxin-l~-D-glucose

increased to 0.11%.

Smollny et al. [74] reported that callus tissues and suspension culture cells of Lilium

album produced podophyllotoxin. One of the cell lines produced 0.3% podophyllotoxin,

and lower amounts of 5-methylpodophyllotoxin, lariciresinol and pinoresinol after 3 weeks

of cultivation. Callus tissue of P. hexandrum was reported by Heyenga et al. [40] to

produce podophyllotoxin, 4'-demethyl-podophyllotoxin and podophyllotoxin-4-O-glucoside

when the callus was incubated in B5 medium containing 2, 4-D, gibberellic acid and 6-


benzylaminopurine. The levels of podophyllotoxin and its derivatives were similar to those

found in the intact plant from which the callus was induced.

Vinblastine and vincristine. The indolic alkaloids, vinblastine and vincristine are used in

cancer chemotherapy for treating various leukemias, Hodgkin's disease and solid tumors.

The drugs are produced commercially by extraction of large quantities of Catharanthus

roseus plant material. However, the intact plant, contains low concentrations of drugs

(0.0005% dry weight basis). As an alternative to whole plant extraction, plant cell cultures

have been employed in efforts to produce vinblastine and vincristine. The literature in this

area is voluminous. There are reports of vinblastine and vincristine being produced in cell

culture or organ culture, but the reports cite very low levels of compound, or the report

needs verification. There are no reports of vinblastine and vincristine accumulation in long

term plant cell suspension cultures, nor has the accumulation of vindoline been

demonstrated authoritatively.

Misawa and his colleagues at Allelix Inc. in Canada [56] studied the production of

vinblastine and established an economically viable process based on production of

catharanthine by plant cell systems and a simple chemical or an enzymatic coupling of

catharanthine with vindoline extracted from the intact plant.

Vinblastine is composed of catharanthine and vindoline; the plant produces much

more vindoline (0.2%) than catharanthine, and vindoline is less expensive than

catharanthine. Catharanthine production in high yield by selected C. roseus cell lines

derived from anther tissue has been demonstrated [31]. For example, cells are grown in

MS liquid medium supplemented with 3% sucrose, 1 mg.L -1 NAA and 0.1 mg.L -1

kinetin under continuous diffuse light. The results of the experiments showed that MS

medium was beneficial for catharanthine production; however, different amounts of

hormones were required for each cell line to achieve optimum results. Addition of various

compounds to the medium stimulated the production of alkaloids. Vanadyl sulphate,

abscisic acid and sodium chloride showed significant effects on the production of

catharanthine [73]. Subsequently, the Allelix group [24] tried to couple enzymatically, and

chemically, the catharanthine produced by the cell culture process with commercially

available vindoline. A crude preparation of 70% ammonium sulphate precipitated protein

from the cultured cells of C. roselts was used as an enzyme source. The reaction mixture

contained catharanthine, vindoline, Tris buffer, pH 7.0 and the crude enzyme; the mixture


was incubated at 30°C and for 3 hours. The products of the reaction were various dimeric

alkaloids including vinamidine, 3-(R)-hydroxyvinamidine and 3',4'-anhydrovinblastine. The

oxidized derivatives of anhydrovinblastine, leurosine and catharine were detected during

the early stages of the reaction. Manganese (II) chloride and either FAD or FMN

stimulated the coupling, but neither vinblastine nor vincristine was detected in the mixture.

However, a substantial amount of anhydrovinblastine was formed as the major reaction

product in the presence of excess sodium borohydride, added to the mixture after

incubation. The protein fraction with dimerizing activity was partially purified using gel

filtration and isoelectric focusing; five isozymes were resolved by Endo et al. [24]. All of

the fractions obtained had peroxidase activity. With the partially purified protein extracts

Endo et al. [24] showed that anhydrovinblastine was formed in approximately 50% yield.

It was found by the Allelix group that ferric ion catalyzes the dimerizing of catharanthine

and vindoline in the absence of the enzyme, and the products of the reaction were

anhydrovinblastine and vinblastine in 52.8% and 12.3% yield, respectively.

This relatively efficient process for producing vinblastine is likely to be commercially

viable. The technology was transferred from Allelix to Mitsui Petrochemicals Industry of

Japan for further development. Subsequently, the Mitsui group used high-cell density

cultures and improved further the yields of catharanthine to 230 mg.L -1 per week [31].

The yield of vinblastine via chemical coupling was also improved; the yield of vinblastine

from anhydrovinblastine was increased to 50% in the presence of ferric chloride, oxalate,

maleate and sodium borohydride.

Bede and DiCosmo [6,7] have also investigated the production of

anhydrovinblastine. A two-enzyme system, contained glucose oxidase and horseradish

peroxidase for sequential production of I-I20 2 and anhydrovinblastine, from glucose, and

catharanthine and vindoline, respectively. Peroxidase(s) require hydrogen peroxide for

dimerizing activity, but with excess H20 2 the reaction may be inhibited. Glucose oxidase

was used for the controlled, continuous production of H202 at relatively low but effective

levels, thus minimizing oxidative reactions. The yield of anhydrovinblastine in the presence

of various peroxidase enzymes is presented in Table 6. The pure enzymes were

immobilized on Eupergit C beads, and the system was shown to catalyze the synthesis of

anhydrovinblastine.


Table 6. Comparison of the ability of different peroxidase preparations to catalyze the formation of anhydrovinblastine [6].

Mean Concentration of Alkaloids Yield Peroxidase Activity (± standard deviation)

(U/assay) Catharanthine Vindoline AVLB AVLB [llmol] ll~moll [~tmol] (%)

HrP I 10.4 0.27 0.12 0.44 54.7 (± 0.03) (± 0.01) (± 0.07)

HrP VI 11.36 0.37 0.17 0.4 50.2 (-+ 0.01) (± 0.01) (-+ 0.01)

HrP VII 11.32 0.44 0.25 0.36 44.6 (± 0.01) (± 0.01) (_+ 0.02)

HrP IX 13.01 0.51 0.26 0.23 19.2 (_+ 0.03) (± 0.01) (± 0.03)

HrP XI 0 1.04 0.51 0 0 (± 0.20) (± 0.04)

HrP XII 11.04 0.68 0.36 0.14 17.8 (_ 0.03) (_+ 0.01) (± 0.02)

Lactoperoxidase 8.82 1.03 0.57 0 0 (± 0.12) (± 0.03)

Control 1.75 0.80 0 (_+ 0.07) (_+ 0.02)

The reaction mixture contained: Catharanthine (0.70 mM), vindoline (0.32 mM), GO (0.12 mg.mL -1 (1.0 U)), peroxidase preparation (approximately 11.0 U) and glucose (4.4 mM) were incubated in 2.5 mL of 0.1 M MES buffer, pH 6.8 for 45 minutes at 30°C. Peroxidase activity was calculated from spectrophotometric measurements of guaiacol oxidation. Final concentrations represent the mean of a triplicate experiment ___ the standard deviation. Control experiment was conducted in the absence of enzymes.

Paclitaxel. Under the intensive National Cancer Institute screening program of antitumor

compounds in the United States, Wall isolated a biologically active product from the bark

of Taxus brevifolia in 1965; the compound was active against KB cells and in 1969 pure

taxol was isolated and its chemical structure was elucidated in 1971 [85]. The diterpene

amide paclitaxel (Taxol ®) has shown activity against B16 mouse melanoma tumor, the

MX-1 human mammary xenograft and CX-1 colon xenografts. The mechanism action of

taxol is unique; it promotes microtubule polymerization. Taxol ® is now a trademark of

Bristol-Myers Squibb and is approved for the treatment of ovarian and breast cancer. The

chemistry and biology of paclitaxel have been reviewed recently [61].


The US Food and Drug Adminstration approved paclitaxel at the end of 1992.

Bristol-Myers Squibb produces the drug for use in the treatment of refractory ovarian

cancer; it is now being manufactured by extraction from the bark of wild-grown T.

brevifolia and needles of other Taxus spp. A semisynthetic method is also available for

paclitaxel production. The demand for paclitaxel is increasing and undoubtedly it will be

used for treating other neoplasms.

Plant tissue and cell culture technology, including biotransformation processes from

baccatin and related taxoids extracted from leaves of Taxus spp. [36] may lead to improved

production of paclitaxel. Cell culture methods for Taxus cuspidata and T. canadensis were

developed by DiCosmo's group [26-29] and others [78]. The cell cultures yielded taxol and

related taxanes. Subsequently, the group improved the growth of T. cuspidata cell cultures,

as well as improving the production of taxol by feeding phenylalanine to selected cell lines.

The kinetics of paclitaxel accumulation indicated that its appearance in the media of

suspension cultures had two peaks. The first, appeared in the early phase of the growth

cycle, and the second flush of paclitaxel appeared in the late stage of the growth cycle.

Further improvement to the production of paclitaxel was brought about by feeding cell

suspension cultures benzoic acid, N-benzoylglycine, serine and glycine. Indeed, there is

good evidence that paclitaxel and related taxoid production in Taxus cuspidata cell cultures

can be improved to industrially interesting levels.

Two US companies, ESCA genetics and Phyton Catalytic have suggested that

paclitaxel will be produced by commercially using proprietary cell culture technology.

However, details of the processes have not been disclosed; nonetheless, the production of

paclitaxel by cell culture methods on a commercial scale remains speculative. Japanese

industries, such as Nippon Steel Corp., Nippon Oil Company and Mitsui Petrochemical

Industry have investigated paclitaxel production using cell culture methods. For example,

Saito et al. [70a] of Nippon Steel reported a yield of 0.05% of paclitaxel (dry weight basis)

in T. brevifolia cultured cells. Tabata et aL [78a] of Mitsui Petrochemical has described the

production of 35 mg.L -1 paclitaxel, 18 mg.L -1 baccatin III, and 18 mg'L -1

cephalomannine simultaneously from suspension cultured T. baccata cells after 21 days of

cultivation.

Although the use of plant cell culture technology for the production of valuable

plant products has yet to be realized, we believe that success will occur as the


understanding of the metabolism of plant cells in culture improves, with concomitant

improvements to plant cell culture fermentation technology.

2.

3.

4.

5.

6.

7.

8.

8a.

9.

10.

11.

12.

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