Strategies for the improvement of secondary metabolite production in plant cell cultures

Heike DGmenburg and Dietrich Knorr

Department

of Food Technology,

Berlin University of Technology,

Berlin,

Germany

Plant cell and tissue cultures can be established routinely under sterile conditions from explants, such as plant leaves or stems. Strain improvement, methods for the selection of high-producing cell lines, and medium optimizations can lead to an enhancement in secondary metabolite production. However, most often trials with plant cell cultures fail to produce the desired products. In such cases, strategies to improve the production of secondary metabolites must be considered. One of the main problems encountered is the lack of basic knowledge of the biosynthetic routes, and mechanisms responsible for the production of plant metabolites. Where the productivity of the desired metabolites is limited by the lack of particular precursors, biotransformation using an exogenous supply of biosynthetic precursors may improve the accumulation of compounds. Feedback inhibition of metabolic enzymes as well as inhibition of membrane transport can be eliminated by the accumulation of synthesized products in a second phase introduced into the aqueous medium. Organ cultures often have sites of synthesis and storage of secondary metabolites in separate compartments. Elicitors, compounds triggering the formation of secondary metabolites, can be abiotic or biotic. Natural elicitors include polysaccharides such as pectin and chitosan. which are also used in the immobilization and permeabilization of plant cells. Immobilization provides several advantages, such as continuous process operation, but for the development of an immobilized plant cell culture process natural or artifcally induced secretion of the accumulated product into the surrounding medium is necessary
Keywords: Plant cell cultures; secondary metabolites; biotransformation; biosynthetic pathways; polysacchatides; chitosan: pectin; elicitation; penneabilization; immobilization: bioreactors

adsorption

culture;

Introduction
Recently, the production of secondary metabolites using plant cells has been the subject of extended research. It was expected that the biosynthetic capacity of plants could be exploited in vitro using plant cells and cell tissue systems analogous to microbial cells in fermentation processes. An important requirement for the improvement of secondary metabolite synthesis such as pigment and flavor production in plants is the understanding of the metabolic pathways and the enzymology of the biosynthesis of particular products. The knowledge of plant metabolic pathways is still very limited. In-depth studies of pathways in whole plants is often difficult because the biosynthetic activities may only

Address reprint requests to Professor Dietrich Knorr, Department of Food Technology, Berlin University of Technology, Konigin-Luise-Str. 22, D-14195 Berlin, Germany Received 7 November 1994; accepted 8 November 1994

be expressed in particular cell types within a specific plant organ or at a certain time of season. Cell cultures have a higher rate of metabolism than intact differentiated plants because the initiation of cell growth in culture leads to fast proliferation of cell mass and to a condensed biosynthetic cycle. This is the most important advantage of plant cell cultures as model systems for the study of biosynthetic pathways, as secondary metabolite formation can take place within a short cultivation time (about 2-4 weeks). Lack of progress in understanding of metabolic pathways had been shown to be the first barrier in developing commercial processes. A number of basic biologic and technical problems related to the characteristics of plant cells, such as the enormous sizes compared to microbial cells, growth as aggregates, slow growth rates, and high sensitivity to shear stress, are responsible for the low number of industrial processes and for the decrease in research activities regarding secondary metabolite production with cultured plant cells. As a result of the differences in growth and accumulation characteristics of plant cell cultures compared to microbial

Enzyme and Microbial Technology 17:674-684, 1995 0 1995 by Elsevier Science Inc. 655 Avenue of the Americas, New York, NY 10010

0141-0229/95/$10.00 SSDI 0141-0229(94)00108-4

Secondary growth and metabolite production, the well-established know-how in large-scale fermentation of microorganisms can rarely be transferred directly to biomass or metabolite production from plant cell cultures. Immobilization of shear-sensitive plant cell cultures has become a potential tool and could reduce or eliminate some of the problems arising with cultured plant cells. This article gives an overview, beginning with the cultivation of plant cells and evaluating tools, that may lead to improved secondary metabolite synthesis.

metabolite

production:

H. Ddrnenburg

and D. Knorr

market price of the product, market volume, culture growth rate, biomass yield, and product yield. The break-even point for plant cell culture processes has been quoted to $1,500 kg- of compound.2 Knowledge permitting the economic production of valuable compounds from plant cell culture systems has been a major goal in studying the biosynthesis of desired metabolites. Consequently, a considerable amount of applied research has been conducted to improve productivities of cultures (Table 1).

History and evolution

At the beginning of the century, Haberlandt attempted to cultivate isolated plant cells, but cell division was never observed in these cultures. In the 1930s the first in vitro cultures were established,2.3 and this was followed by a period of development of culture media and of cultivation methods.4 Twenty-five years ago, the prospect of the use of plant cells for chemicals production was not imaginable. The low yields of secondary metabolites in suspension cultures clearly were a bottleneck for commercialization. In these early efforts, plant cells in culture were treated in direct analogy to microbial systems, with little knowledge of plant cell physiology and biochemistry or the influence of bioreactor operation on the physiologic state of such systems. In 1982, at least 30 compounds were known to accumulate in plant culture systems in concentrations equal to or higher than that of the plant5 Concerning the production of natural plant metabolites, some in vitro systems exist that allow the large-scale production of economically important plant metabolites.6 A survey of the historical milestones in plant cell cultures is given by Schmauder and Doebel. Strategies to optimize growth and product formation began to develop separately during the period between 1975 and 1985. A combination of strategies for yield improvement resulted in the first commercial plant cell process.* The expectations of this two-stage process were high because shikonin production with cell cultures of Lithospermum erythrorhizon in 750-l bioreactors had been introduced to the market in Japan by Mitsui Petrochemical Industries in 1984. Shikonin can be used as a dye and a medicinal (antiinflammatory) compound, valued in 1983 at approximately $4,000 kg-.9 Three large-scale processes using plant cell cultures have been established in Japan. In the United States the development of two processes for commercialization was attempted-the production of vanilla flavor from plant cell cultures by Escagenetics*O and the production of sanguinarine by Papaver somniferum, but to our knowledge neither of these processes is currently in operation. However, Phyton Gesellschaft fur Biotechnik mbH began producing taxol, an anticancer drug, using several species of Taxus cultures, as well as other plant products in facilities with reactor capacities of up to 75,000 1. Cell cultures of Taxus may represent an alternative to extraction of stem bark as a source of taxol and related taxanes. Thus, largescale production with plant cell tissue culture for the generation of pharmaceuticals seems to be of continued commercial interest. Whether plant cell and tissue culture processes are economical for the production of secondary metabolites depends on a number of factors, including the

Strain improvement and medium optimization

Microbiologists have successfully used several approaches to accomplish overproduction in bacteria or fungi. Some of these approaches lead to an enhancement in secondary metabolite production within plant cells. Strain improvement begins with the choice of a parent plant with high contents of the desired products for callus induction to obtain highproducing cell lines. Statistically high-producing plants give rise to high-producing cell lines,15 but production levels in plant cells have also been shown to be variable. I6 The first step toward establishing highly productive cell lines is the selection of storage or production cells. Isolation and selection methods are often necessary during cultivation, because a major problem with plant cells is their inherent genetic and epigenetic instablity. Variability often leads to reduction in productivity with subculturing and has been attributed to genetic changes by mutation in the culture, or epigenetic changes, which are due to physiologic conditions. They can be reversed by changes in the culture environment as well as by screening for a desired cell population from the heterogenous population typically present in plant cell cultures. * Information concerning the factors regulating secondary metabolism is as important as the selection of highproducing cell lines in increasing the production of secondary metabolites. A number of physical and chemical factors that could influence secondary metabolism in plant cell cultures have been found. Optimization of the hormone concentration and combinations are often effective; high auxin levels, although good for cell growth, are often deleterious

Table 1 Factors influencing in plant cells3~4 Strain improvement Medium variation

secondary

metabolite

production

Culture conditions

Specialized

techniques

Selection Screening Nutrients Phytohormones Precursors Antimetabolites lnoculum size PH Temperature Light Agitation Elicitors immobilization Permeabilization Two-phase systems Two-stage systems

Enzyme Microb. Technol.,

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675

Review to secondary metabolite production. * Alterations in the environmental factors such as nutrient levels, light, and temperature may also be effective in increasing productivity (with the exception that reduced phosphate levels often stimulate product accumulation). I9 N-sources may also play an important role in product accumulation in plant cells. For example, in L. erythorhizon, shikonin synthesis was inhibited by ammonium ions, but these ions promoted cell growth. It was necessary to change to a medium containing nitrate ions at the end of the growth phase.8 Table 2 Glucosilation of monoterpenes
cuIturesz4 Glucosilation Monoterpenes Piperitone
L-( -)-Menthol

in Me&ha

suspension

(%) Mentha piperita 55.0 52.5 30.5 C5.0 C5.0

Mentha

canadensis 24.5 50.5 15.5 C5.0 C5.0

D-( + )-Neomenthol D-( + klsomenthol D-( + LNeoisomenthol

Genetic engineering
Genetic engineering includes isolation, characterization, and reordering of genetic material and its transfer to foreign organisms. Efforts to apply genetic engineering have at least two primary goals: the combination of properties otherwise found in different plants or different cells in a single organism, and incorporation of particularly active and specific regulation mechanisms. Initial trials in the context of secondary compound production were made with Lupinus polyphyllus, Peganum harmala, and Petunia hybrida cultures. I4 Calgenes Flavr Savr tomato is the first genetically modified whole food to be available to American consumers.20 Controlled ripening of fruit was achieved by inhibiting genes coding for polygalacturonase by antisense RNA. The tomato has been engineered for better ripening on the vine, which will permit improved flavor. special accumulation sites. Exogenous tetpenes have been shown to be rapidly metabolized by cell suspension cultures to form biotransformation and degradation products.25 Thus, biotechnologic plant cell culture processes for acceptable product yields are only conceivable if the desired product can be accumulated in a nonpolar organic phase or adsorbed by a resin.

Two-phase systems, adsorption cultures, and organ cultures

The sites of synthesis and storage of secondary compounds in plant cells often take place in separated compartments. The accumulation of monoterpenes in cultures such as Mentha species is most likely associated with the presence of highly specialized structures containing secretory and accumulatory elements, e.g., oil glands, glandular trichomes, or a glandular epidermis. Encapsulation of cytotoxic compounds also serves as a self-protection mechanism of intact plants. In undifferentiated callus or suspension cultures, these accumulation sites are missing. This is probably the reason for low yields of such compounds reached in these plant cell cultures. The addition of an artificial site for the accumulation of secondary metabolites had been shown to be an effective tool for increasing biosynthetic pathways in plant cell cultures. If the formation of a product is subject to feedback inhibition or intracellular degradation, the removal and sequestering of the product in an artificial compartment may increase total metabolite production. Table 3 summarizes several examples of two-phase or adsorption cultures. Such two-phase systems accumulate even traces of secondary metabolites from the culture medium, thus avoiding any type of feedback inhibition. Another effect may be the enhancement of secondary metabolite release from the cultures or the initiation of a release of compounds normally stored within the cells.36 Secreted secondary metabolites may be protected from degradation in the culture medium as a result of excreted catabolic enzymes and acids. Evaporation of the product into the gas phase can be reduced by trapping flavor compounds in artificial accumulation sites. Desired plant products can then be removed selectively from the culture systems.44 The product can be concentrated by in situ recovery, and downstream purification may be reduced if product removal from the culture medium and cells is selective. Consequently, recovery and purification are generally simplified, thus reducing production costs.

Biotransformation
The biochemical capability of cultivated plant cells to transform exogenously supplied compounds offers a broad potential and an interesting contribution toward modification of natural and synthetic chemicals.2 The enzymatic potential of cultured plant cells can basically be employed for bioconversion purposes. Plant enzymes are able to catalyze regio- and stereospecific reactions, and can therefore be applied for the production of desired substances. Plant cell cultures have also been used as model systems for the study of biosynthetic pathways. In Vanilla planifolia suspension cultures, for example, a linear biosynthetic pathway leading from phenylalanine directly to vanillic acid was postulated.22 In contrast to these results, Knorr et a1.23 demonstrated a nonlinear, more complex pathway in V. planifolia cluster cultures. The conversion of monoterpenes were studied with Mentha species in our laboratory. 24 Suspension cultures of Mentha canadensis and Mentha piperita were able to synthesize limonene as well as oxygenated, acetylated, or glucosilated monoterpenes. However, the yields of these compounds were low and monoterpene glucosides were accumulated in higher amounts than free monoterpenes. Mentha suspension cultures metabolized exogenous monoterpene ketones and monoterpene alcohols within 24 h and glucosilation occurred (Table 2). Glucosilation can be considered a detoxification mechanism of phytotoxic compounds by plant cells and results in accumulation of glucosilated and often water soluble products. Otherwise, the exogenously applied toxic monoterpenes are degraded and metabolized by cell cultures without Enzyme Microb. Technol., 1995, vol. 17, August

676

Secondary
Table 3 Examples of in situ adsorption with plant cell cultures

metabolite

production:

H. Ddrnenburg

and 0. Know

Adsorbents Miglyol k&ated charcoal

Cell cultures Matricaria chamomilla, Valeriana wallichii, Pimella anisum, Vitis vinifera, Mentha canadensis Vanilla fragrans, M. chamomilla, Nicotiana tabacum M. chamomilla, V. wallichii, P. anisum, Mentha piperita N. tabacum T. occidentalis, Galium vernum V. fragrans, T. occidentalis, Nicotiana rustica V. fragrans, Catharanthus roseus N. tabacum Mucuna pruriens, M. canadensis Eschscholtzia californica G. vernum neutral resin Thuja occidentalis,

Reference 26-29 30-32 26.33 34 35,36 32,37.39 32,39,46 41 29.42 43 36

Zeolith XAD-2 XAD-4 XADJ Polyethylenglycol p-Cyclodextrin Polydimethylsiloxan Wofatite RP-8: lipophyllic

carrier; XAD: ion exchanger,

Plants of Mentha species, for example, have epidermal oil glands on the leaves, which are the primary sites of monoterpene biosynthesis; these are the cells in which the cytotoxic substances were accumulated. Plant cell cultures, which do not have generally specialized accumulation sites, are not able to accumulate high levels of monoterpenes. Under the appropriate environmental conditions, mostly accomplished by variable combinations and concentrations of phytohotmones, different types of cultures can be induced. These may be differentiated shoots, somatic embryos, or root cultures. Shoot cultures of Mentha species grow in hormone-free medium; supplementation of phytohormones supported callus initiation by the cultures. Furthermore, the biosynthetic capacity of cultures declined with decreasing degree of differentiation.45 Metabolism of secondary products seems to correlate with organized cell structures. Shoot and root cultures were also induced by genetic transformation with Agrobacterium tumefaciens and Agrobacterium rhizogenes, respectively. Because of simultaneously transmitted hormone autotrophy, these shooty teratomas and hairy roots can also be cultivated without the addition of hormones. Such cultures are especially interesting because of their continued secondary metabolite production initiated by organ induction. Spencer et a1.46 examined the production of monoterpenes in shooty cultures of Mentha piperito citrota and Mentha piperito vulgaris transformed by A. tumefaciens T37 and observed oil glands on the leaves of the transformed cultures. The spectrum of terpenes produced in shooty teratoma cultures in general reflected that of the parent plant, but overall the yield of oil was lower than in leafy tissue. The composition of secondary metabolites produced by multiple-shoot cultures of Mentha canadensis resembled the distribution of monoterpenes found in plants.45 Addition of the triglycerid, Miglyol as a second phase to shoot cultures of M. canadensis during the last day of an incubation cycle of 4 weeks resulted in monoterpene production of more than twice ( 1. I mg g - fresh weight) that of the control cultures (0.5 mg g- fresh weight). The Miglyol phase dispersed into fine droplets in the aqueous medium and adhered to the shoots, forming an oily film on the surface of the plants. Higher portions of monoterpenes than in the aqueous me-

dium (4 pg gg fresh weight) were found in this phase (10 p.g g - fresh weight). 29 Flavor substances could also be accumulated in the medium by inclusion with B-cyclodextrins. These torus-like molecules with a nonpolar interior are able to generate inclusion complexes with hydrophobic compounds of the dimensions of monoterpenes.47 Figure 1 presents the effects of the addition of Miglyol and B-cyclodextrins to shoot cultures on monoterpene yields.

Elicitation
It is widely accepted that microbial invasion of intact plants will activate plant defense mechanisms including synthesis of antimicrobial metabolites. Molecules that stimulate secondary metabolism are called elicitors. Elicitors may form inside or outside plant cells, and are distinguished as endogenously or exogenously inducers. Depending on their origin, they are classified as biotic or abiotic.48.49 The primary reactions upon elicitation with a biotic elicitor are thought to be composed of recognition of the elicitor and its binding to a specific receptor protein on the plasma mem-

12 IO 8 6 4 2 0 7 --I10 14 lncubatlon 21 time IdI 28 :

Figure 1 Concentration of essential oils in multiple-shoot cultures of Mentha canadensis cultivated in different media.29 MS: Murashige and Skoog medium; P: phytohormones; CD: p-cyclodextrin; M: Miglyol (24 h of incubation)

Enzyme Microb. Technol.,

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677

Review brane. The next step in elicitation is thought to be inhibition of plasma membrane ATPase, which reduces the proton electrochemical gradient across this membrane.50 Gundlach et ~1.~~ demonstrated the integral role of jasmonic acid and its derivatives in the intracellular signal cascade that begins with the interaction of an elicitor molecule with the plant cell surface and results-ultimately, in the accumulation of secondary compounds. Such biotic elicitors include polysaccharides derived from plant cell walls (e.g., pectin or cellulose) and microorganisms (e.g., chitin, chitosan, or glucans), glycoproteins, and low-molecular-weight organic acids. Abiotic elicitors include ultraviolet irradiation, salts of heavy metals, and chemicals that disturb membrane integrity. Chitosan has been shown to be a very effective elicitor.52-54 Other polysaccharides produced and excreted by plant pathogenic microorganisms, such as xanthan and curdla@ or gellan, 56 have also been shown to be active elicitors in plant cell culture systems. Fukui et ~1.~ observed induction of shikonin formation by agar and found that polysaccharides containing acidic functional groups (agaropectin and pectic acid) were active for elicitation. However, these exogenous polysaccharides were less active than the endogenous polysaccharides of the cell wall.58 Several polysaccharides isolated from plants or microorganisms or produced extracellularly by microorganisms have been selected and screened for their elicitor effects (Table 4). Polysaccharides involved in interactions between plants and microorganisms (chitosan/chitin and pectin) gave the best results by activating secondary metabolism in Morinda citrifolia cultures and were selected for further investigations. A comparison of the elicitor effects of pectic acid and
Table 4 Elicitor effect of polysaccharides on anthraquinone synthesis in Morinda citrifolia cultures after 14 days of elicitation5 Polysaccharide Plants Guar gum CM-cellulose Pectic acid Pectin Konjac Cellulose 6-cyclodextrin Locust bean gum Alginate Microorganisms Alginate Rhamsan Xanthan Chitinb Chitosan Curdlan Levan Gellan Welan Elicitor Anthraquinones (%)

Chitin 50 showed an effect of Chitin 50 on anthraquinone biosynthesis at concentrations lower than that of pectic acid.59 Treatment with pectic acid predominantly induced synthesis of chitinase and, to a lesser extent, the synthesis of anthraquinones. In contrast, addition of Chitin 50 resulted in an increase in anthraquinone synthesis, whereas chitinase activities were relatively low. The plants response to different polysaccharides seems to be a result of progressive interaction between plants and microorganisms in specific plant defense strategies.60 The degree of acetylation of chitosan or chitin was found to be important in inducing defense metabolisms in the plant cell cultures. The origin of chitosans seemed to play a lesser role in signal recognition than their similarity to the chitin molecule, the key cell wall polysaccharide in fungi.59 Examinations of different galacturonic acid oligomers regarding their role in inducing anthraquinone synthesis in Morinda cihfolia showed a decrease in production when applying galacturonic acid with a short chain length of one to five units. Only oligomers with more than five units caused elicitor activities. Elicitation was also dependent on the source, the degree of esterification of pectin, or the age of M. citrifolia cultures from which cell wall pectins had been isolated. 59 Application o f p ectins to the cell culture caused a continued increase of anthraquinone synthesis with continuing incubation time. These phenomena indicated a degradation of the polymers by induced polygalacturonase activities to fragments serving as active elicitors.6 Application of cell wall pectins isolated from a 24-dayold suspension culture of M. citrifolia (late exponential growth phase) led to a maximum of anthraquinone production.59 The anthraquinone concentration within the cells decreased during the initial 4 davs of incubation nrimarilv as a result of cell enlargement (Figure 2). After this lag phase, the concentration of anthraquinones in M. citrifolia cultures increased. Pectin concentrations of 100 pg ml- medium
caused folk the a stimulated anthraquinone production in M. citri-

after 4 days of incubation. After an incubation time of 2 weeks anthraquinone concentrations 5.6 times higher than
control could be observed. Furthermore, a time-

Active Active Active Active (Inactive) Inactive Inactive Inactive Inactive Active Active Active Active Active (Inactive) (Inactive) Inactive Inactive

135 167 236 202 123 104 104 98 88

Anthraqu~nones 10

lug/g

FWI

Chltlnase

a~f~v~ty

hkatlg

FWI 100 i

Anfhraq~~nones

ChltlnaSe

8
(6)
r\ i

// I I

., \\/
:

//) 80 \I
60

132 131 152 234 130 120 122 96 90

(A)

\\
j, ,/

(c)

i/\

/ I
; / /I j, _
4 7

/x 5 / 1, I\ 40

1 2 i \/
0 0 2 4

\ i ,A

i; 1 \I
7 14 0 2 4

::---.

,J

\,
20

7 tme

14 [dl

,4O

Polysaccharide concentration were 250 Pg ml- medium with the exceotion of Chitin 50 = 100%. and control (from crab shells) Pronova Biopolymer a.s., Norway and chitosan (50 Fg ml-); compounds achieving anthraquinone concentrations >120% of the control were considered to be active elicitors

Incubation

Figure 2 Elicitation of stress metabolites by cell wall pectins isolated from Morinda citrifolia culturesso A: Control; B: 50 Fg ml-; C: 100 pg ml-

676

Enzyme Microb.

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1995, vol. 17, August

Secondary dependent induction of the synthesis of stress metabolites was observed. During the first few days of incubation after the addition of the elicitor, production of chitinase increased. After a decrease in enzyme activities biosynthesis of anthraquinones became the dominant factor (Figure 2).

metabolite
Table 6

production:

H. Dtjrnenburg

and D. Knorr

Polymers for plant cell immobilization6 Polymer Gelation mechanism Gel formation in the cold lonotropic gel formation lonotropic gel formation lonotropic gel formation lonotropic gel formation Gel formation in the cold, crosslinking lonotropic gel formation, gel formation in the cold Polymerization Crosslinking Crosslinking Gel formation in the cold, crosslinking lonotropic gel formation, crosslinking lonotropic-polyelectrolytic coacervate formation lonotropic-polyelectrolytic coacervate formation lonotropic-polyelectrolytic coacervate formation

Immobilization and permeabilization

The development of large-scale fermentation processes are complicated by several specific characteristics of plant cells. The transfer from flask or pilot plant-scale bioreactors is a problem because of the slow growth of plant cells, the low shear resistance, and the tendency for cell aggregation. Therefore, the immobilization of plant cell cultures has a number of advantages (Table 5). In general, cell immobilization provides continuous process operation, reuse of biocatalysts, separation of growth and production phases, and a simplified separation of biocatalysts from the culture medium,65 which allows productorientated optimization of the medium and reduction of cultivation periods. There are some additional advantages that become important for the immobilization of plant cells (Tuble 5). The choice of a suitable immobilization system is determined by several factors such as high cell density and a high arresting potential of the matrices; enzyme activity of biocatalysts should not be diminished by the immobilization process and the manipulation of the biocatalysts formed must be as simple as possible.i4 Since the immobilization of plant cells was introduced for the production of secondary metabolites,% gel entrapment has been the most widely used immobilization method because it is cheap, simple, and reproducible using mild conditions during the immobilization. A summary of polymers and gel-entrapment techniques used for plant cell immobilization is presented in Table 6. Currently, continuous operation is difficult to realize with immobilized plant cells because secondary metabolites are most often stored within the cell vacuole. Whether an immobilization and production process is economical depends on the capacity of the producing cells to secrete the desired metabolite into the surrounding medium. In a few

Agar6 Agarose6 Alginate66 k-CarrageenarF7 Chitosan6 Gelatine6 Gellan7 Polyacrylamide67 Polyacrylamide hydracide Polyphenylene oxide Agarose-gelatine6 Alginate-gelatine6

Table 5

Some

advantages of plant cell immobilization62-64

Reason/consequence Entrapment Elimination of nonproductive growth phase, reduced risk of declining productivity from a selected cell line Simplification of scale-up, protection from physical damage Continuous removal of metabolic inhibitors, improved process control and product recovery Increased cell densities, redifferentiation of plant cells, permeabilization of plant membranes, elimination of growth phase

Advantage Reuse of biocatalyst Maintainment of stable and active biocatalyst Protection of cells by matrix Employment of continuous process Increased productivity

cases species-specific secretion behavior could be observed.76 Secretion mechanisms are either natural or artificially induced. Natural secretion mechanisms can be passive diffusion processes or are based on active transport systems. 76-79 Artificially induced release of intracellularly retained products is perhaps the most obvious difficulty in process development. Permeabilization of plant membranes for the release of secondary metabolites was often connected with the loss of viability of the plant cells treated with permeabilizing agents and methods. Various methods have been used to initiate product release from cultured plant cells. These methods include treatment with solution of high ionic-strength,80 change of external PH,~ transfer to media lacking phosphate,82 permeabilization with dimethylsulfoxide (DMSO),83*84 chitosan,85.86 other chemicals,87 eletroporation, 88 ultrasonic89 or ultra-high-pressure treatments. It was also observed that usually intracellularly stored products were excreted by the cells in response to immobilization per se for unexplainable reasons. I3 Elicitors have been successful in dramatically increasing rates of secondary metabolite biosynthesis. 91 Interestingly, in suspension cultures elicitors also cause extracellular product accumulation. I3 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. Attempts have been made to permeabilize the plant cells only transiently, to maintain cell viability and have short time periods of increased mass transfer of substrate and metabolites to and from the cell.83.84 According to Brodelius and Nilssons cells treated intermittently can be permeabilized repeatedly. thus permitting the maximum use of the cells biosynthetic capacity. This mean a cyclic process in which periods of product release are followed by a short recovery period for the cells. The application of such a short time extraction procedure helps avoiding destrucEnzyme Microb. Technoi., 1995, vol. 17, August

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Review

tion of cells for product recovery. More recently, it was reported that long-term perrneabilization treatments have rarely been successful,92 and Brodelius93 concluded that chemical perrneabilization for the release of intracellularly stored products appear to be appropriate only in a very limited number if sustained cell viability is required. Chenopodium rubrum cells could be permeabilized by treatment with chitosan. This polycationic polysaccharide induces pore formation only in the plasmalemma of the plant cell cultures. The leakage caused by chitosan can be considered as leakage from a relatively small compartment, i.e., cytosol, similar to the action of poly-t_-lysin.94 Longterm permeabilization with chitosan showed a timedependent amaranthin release from C. rubrum cells into the culture medium at various chitosan concentrations.61 lncreased chitosan concentration caused an increase in pigment release during 5 h of treatment, which was correlated with cell death (Figure 3). The long-term treatment of C. rubrum cells with 100 )*g ml- of chitosan resulted in product release without disturbing cell viability, most likely due to the availability of some amaranthin in the cytoplasma.6 Because different chitosan concentrations resulted in different permeabilizing abilities, the effects of various degrees of deacetylation (positive charges) at given chitosan concentrations were tested. Figure 4 shows amaranthin release from C. rubrum cells in relation to the degree of deacetylation of chitosans. The amaranthin release at a given concentration of chitosan depended on the number of positive charges that could react with the negatively charged polygalacturonates of the plant cell walls.95 Consequently, pore formation in the plasmalemma can also be related to the degree of deacetylation of chitosans. Highly charged chitosan polymers induced a higher degree of pore formation in plasmalemma and caused faster amaranthin release than the less charged ones. This means that a critical charge density existed which led to loss of cell viability and initiated the diffusion of amaranthin from C. rubrum cells. Traditionally, most of the activities regarding perrneabilization of plant cell cultures are not membrane-specific. Anthraquinones in M. citrifolia are completely retained in

Amaranthm 60

release

I%1

50

40

30

20

47

50

56

74

82

92

Degree

of

deacetylation

I%1

Figure 4 Permeabilizing effects of chitosans with different degree of deacetylation on amaranthin release from Chenopodium

rubrum cellsz3

120

I%1

100

60

60

40

20

0 0 125 250 Chitosan 500 concentration 750 lug/mLl 1,000 2,000

Figure 3 Effect of chitosan concentration on cell viability and amaranthin release of Chenopodium rubrum celW

the vacuole of the cells; consequently, a prerequisite for the recovery of anthraquinones is the opening of the tonoplast. Domenburg and Knorr9* showed that the use of lipases specific to the plant membranes could serve as permeabilizing agents for M. citrifolia cultures while maintaining cell viability. We suggest that these enzymes caused only a brief perforation by interacting with some of the phospholipids in the double layer of the tonoplast. The polar-apolar characteristics of the phospholipids caused them to close the perforation quickly leading to restoration of an intact tonoplast9 The results of our permeabilization studies support the opinion that permeabilization of the tonoplast is a promising but not yet optimized method for inducing the release of natural products normally stored in the vacuoles of cultured plant cells. In addition to the perrneabilizing activities of chitosans, these polysaccharides have been used as effective elicitors (Table 4) and are known to be gel-producing systems suitable for cell immobilization.96 Viability studies with C. rubrum suspension cultures in chitosan solutions showed lethal effects at concentrations >400 pg chitosan ml - medium. For cell immobilization, chitosan concentrations >20 mg ml- were needed. Therefore, a polyanionic polymer interacting with the cationic charges of chitosan and protecting the cell of permeabilization9 and cell lysis was performed. This led to the development of coacervate capsules consisting of a liquid pectin core and a chitosan membrane that was stabilized by an interphasic membrane built via the ionic interactions between pectin and chitosan (Figure 5). The addition of pectin to chitosan-treated cell cultures had positive effects on cell growth and amaranthin accumulation in C. rubrum cells.6 Protective effects of pectin were also observed by addition to M. citrifolia cell cultures treated with chitosan (Figure 6). The additional interphasic membrane of pectin and chitosan in the coacervate capsules resulted in no additional barrier to mass transport of substances with molecular weights of 900 g/M.61 Cell cultures immobilized in pectin/ chitosan coacervate capsules were suppressed in cell growth, which reduced the problem of outgrowing cells

680

Enzyme Microb.

Technol.,

1995, vol. 17, August

Secondary

metaboiite

production:

H. Dtirnenburg

and D. Knorr

B C Figure 5 Electron microscopic photograph of a cross-section of a pectin-chitosan coacervate capsukbl A: Chitosan membrane; B: interphasic membrane; C: pectin core (magnification x 55,000)

during cultivation time. In contrast to pectin beads, which disintegrated after 2 weeks of cultivation, the coacervates were stable and were not dissolved by high-phosphate ions or polygalacturonases, which could be induced in the cell cultures.6

Bioreactors and scale-up

In 1959 the first report on the large-scale cultivation of plant cells appeared. 98 In the last few years, much success has been achieved in the field of plant cell fermentation and scaling-up. Plant cells can now be cultivated in volumes up to 75,ooo 1,99 specific bioreactor systems for plant cells have been developed, and productive plant cell culture systems have been established.m The different properties of plant cells compared with microbial cells have to be considered. Furthermore, only a few plant cell cultures are really single-cell cultures. Most often, plant suspension cultures consist of small aggregates, with sizes ranging from fine suspensions as small as lCKL500 pm (a few cells) to aggregates measuring several millimeters in diameter. OS

Mixing is affected, as the aggregates tend to settle sediment or adhere to the reactor wall. The objective in culturing plant cells is to provide the culture conditions to reproducibly optimize cultivation. Reproducing optimum conditions in bioreactors can be difficult with respect to the physical factors of mixing and shear. When the impeller speeds of mechanically agitated bioreactors are reduced to prevent shear damage to the plant cells, the ability of the impeller to disperse the gas bubbles is severely restricted. lo6 Mixing can be a serious problem in plant cell suspensions-especially when high cell concentrations and large volumes are used. For moderate cell concentrations (below 20 g l- dry weight), pneumatically agitated bioreactors are appropriate. 07 Plant cells require less oxygen than microorganisms because of their slow metabolism. In some cases a high oxygen concentration is even toxic to the metabolic activities of the cells08 and high sparge rates may strip nutrients such as carbon dioxide from the culture medium. lo9 Table 7 summarizes the advantages and disadvantages of each reactor type. Based on this, the airlift reactor seems to be the most desirable reactor for submerged cultivation of freely suspended plant cells. I0 Immobilized plant cell reactors have been developed as an alternative to suspension culture for the production of secondary metabolites. Immobilized cell operation usually implies continuous flow-through of the liquid medium with total retention of biocatalyst particles inside the reactor. Washout can be eliminated in a continuous cultivation system. Immobilized plant cell reactors have been classifiedbased on the mode of entrapment of the cells as well as on the agitation mode-as fluidized bed reactors, packed bed reactors, airlift reactors. stirred tank reactors, and membrane reactors. r3,ro4

Conclusions

and future directions

Chltosan/Pectm ImQ/mLl control 0.210 0.210 0 410 0 410.4 0 6/O 4 0.6/0.6 6 5 m 4 3 Fresh 2 Weight 1 1~1 0 m 20 40 60 80 100120140 [%I 0

1
2

Anthraquinones

Figure 6 Effect of pectin addition on cell viability of chitosantreated Morinda citrifolia culture@

Regarding the industrialization of plant cell culture processes, different strategies to increase biosynthesis of secondary metabolites and to alter the product spectrum have to be investigated. Knowledge of biosynthetic pathways of desired compounds in plants as well as in cultures is often still in its infancy, and consequently, strategies are needed to develop an information base on a cellular and molecular level. Because of the complex and incompletely understood nature of plant cells in in vitro cultures, case-by-case studies have been used to explain the problems occurring in the production of secondary metabolites from cultured plant cells. A key to the evaluation of strategies to improve productivity is the realization that all problems must be seen in a wholistic context. An optimum production process cannot be found by sequentially optimizing each step of the process individually, because strategies leading to improved product synthesis seem to be interactive. Nonetheless, substantial progress in improving secondary metabolite production from plant cell cultures has been made within the last few years. We believe that only continuation and increase of efforts in this field will lead to controllable and successful biotechnologic production of specific, valuable, and as yet unknown plant biochemicals. Enzyme Microb. Technol., 1995, vol. 17, August 681

Review
Table 7 Comparison of the performance of various standard bioreactor systems3,04 Reactor type Pneumatically Bioreactor performance Oxygen transfer Low shear Mixing Scale-up Limitations Mechanically agitated Bubble column ++ ++ + Easy Dead zones, settling of cells due to poor mixing agitated Airlift + +++ ++ Easy Dead zones at high cell densities

+++ + +++ Difficult Cell death, contaminations to moving parts

due

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