Manipulating indole alkaloid production by Catharanthusroseus cell cultures in bioreactors: from biochemicalprocessing to metabolic engineering

Jian Zhao Æ Robert Verpoorte

Received: 5 July 2005 / Accepted: 29 November 2006 / Published online: 6 March 2007� Springer Science+Business Media B.V. 2007

Abstract Catharanthus roseus plants produce

many pharmaceutically important indole alka-

loids, of which the bisindole alkaloids vinblastine

and vincristine are antineoplastic medicines and

the monoindole alkaloids ajmalicine and serpen-

tine are antihypertension drugs. C. roseus cell

cultures have been studied for producing these

medicines or precursors catharanthine and vindo-

line for almost four decades but so far without a

commercially successful process due to biological

and technological limitations. The research thus

focused on the one hand on engineering the

bioreactor process on the other engineering the

cell factory itself. This review mainly summarizes

the progress made on biochemical engineering

aspects of C. roseus cell cultures in bioreactors in

the past decades and metabolic engineering of

indole alkaloid production in recent years. The

paper also attempts to highlight new strategies and

technologies to improve alkaloid production and

bioreactor performance. Perspectives of metabolic

engineering to create new cell lines for large-scale

production of indole alkaloids in bioreactors and

effective combination of these up- and down-

stream processing are presented.

Keywords Bioreactor process � Catharanthus

roseus � Cell factory � Gas regime � Indole

alkaloid � Large-scale cell culture � Metabolic

engineering � Monitoring and autocontroling

AbbreviationsABA Abscisic acid

ABC transporter ATP-binding cassette

transporter

ASa Anthranilate synthase a-

subunit

DCO2 Dissolved carbon dioxide in

liquid medium

DO2 Dissolved oxygen in liquid

medium

KLa Oxygen mass transfer

coefficient

MeJA Methyl jasmonate

SG Strictosidine glucosidase

STR Strictosidine synthase

TDC Tryptophan decarboxylase

Introduction

Plant secondary metabolites encompass a huge

number of natural compounds with a wide diver-

J. Zhao (&)Department of Pediatrics, Children’s NutritionResearch Center, Baylor College of Medicine, 1100Bates Street, Room 9016, Houston, TX 77030, USAe-mail: jzhao1@bcm.tmc.edu

R. VerpoorteSection of Metabolomics, Division of Pharmacognosy,Institute of Biology, Leiden University, 2300 RALeiden, The Netherlandse-mail: verpoort@chem.leidenuniv.nl

123

Phytochem Rev (2007) 6:435–457

DOI 10.1007/s11101-006-9050-0

sity in chemical structure. They provide human

beings with unique resources for medicines, food

additives, fragrances, and fine chemicals. The

daily life, health care, and other well being of

humans essentially depend on these plant prod-

ucts. Therefore, production of plant secondary

metabolites by cultivation of plants and chemical

synthesis are important agronomic and industrial

objectives. As a promising alternative to produce

plant secondary metabolites, plant cell culture

technology has many advantages over traditional

field cultivation and chemical synthesis, particu-

larly for many natural compounds that are either

derived from slow-growing plants or difficult to be

synthesized with chemical methods. Considering

the continuous decrease in arable lands and

increased considerations on environmental prob-

lems, production of plant secondary metabolites

by traditional plant cultivation and chemical

synthesis may be largely limited in the future.

Large-scale plant cell culture in bioreactors has

no such agronomic and environmental concerns

since the process is in factory independent of

seasons or climates, pathogens or other biofactors

that seriously affects field cultivation. Further-

more, plant cell culture is a renewable resource

and environmentally friendly. It is like an indus-

trialized biological factory for production of high-

quality natural products under strictly controlled

conditions. Although most plant cell culture

processes are not yet competitive for commercial

application due to the high-cost caused by low

productivity, to date there are already some

successful examples of commercial production of

valuable secondary metabolites by plant cell

cultures (Alfermann and Petersen 1995; Smith

1995). Shikonin by Lithospermum erythrorhizon

cell culture and berberine by Coptis japonica or

Thalictrum minus cell cultures are successfully

produced by Mitsui Petrochemical Industries

(Japan). Paclitaxel is commercially produced by

Taxus spp cell cultures in a two-stage process in

2500-l / 75000-l bioreactors by ESCA Genetics

(now Samyang Genex, South Korea) and Phyton

Catalytic company (USA). Also, ginseng saponin

production by Panax ginseng cell or root cultures

runs at a 20.000-l scale (Alfermann and Petersen

1995; Smith 1995). These successfully industrial-

ized processes largely depend on either a higher

productivity of secondary metabolites in cell

cultures like shikonin or extremely high market

values like paclitaxel, whereas most other plant

secondary metabolites have no such merits. Nev-

ertheless, these encouraging successes are driving

research of plant cell cultures towards further

breakthroughs, by overcoming biological limita-

tions such as low and unstable production of

interesting metabolites, and biotechnological lim-

itations including poor bioreactor performance or

uncontrolled processes. In other words, more

research efforts will be put in engineering of the

process and in the engineering of the cell factory.

Catharanthus roseus cell culture is one of such

an extremely interesting but unsuccessful exam-

ples that has been studied for more then three

decades (van der Heijden et al. 2004). The valu-

able secondary metabolites in C. roseus are terpe-

noid indole alkaloids, including the anticancer

medicines vinblastine and vincristine, as well as the

antihypertensive medicines ajmalicine and serpen-

tine. However, the highly valuable drugs vinblas-

tine and vincristine fail to accumulate in in vitro

cell cultures due to the absence of the biosynthesis

of one precursor vindoline. Ajmalicine and ser-

pentine can accumulate in the cell cultures to high

levels, yet their productivities are still too low to

compete with field cultivation. The rapid develop-

ment of semi-synthesis of vinblastine or vincristine

by coupling vindoline and catharanthine provides

another opportunity for C. roseus cell cultures as a

promising source of catharanthine. In C. roseus

plants, vindoline is abundant but catharanthine is

limited, but C. roseus cell culture could synthesize

much higher level of catharanthine than plants

(about 0.1% on dry weight basis) (Misawa and

Goodbody 1996). It would be possible to produce

catharanthine by plant cell cultures and to obtain

vindoline from field cultures, and then use chem-

ical or biochemical semisynthesis of vinblastine by

coupling catharanthine and vindoline (Misawa and

Goodbody 1996). Significant progress has been

made in the 1980s on coupling catharanthine and

vindoline into vinblastine or other bisindole alka-

loids in a much easier way and with an increased

efficiency (Misawa and Goodbody 1996). In addi-

tion, some novel bisindole alkaloid derivatives are

further developed as new anticancer medicines,

e.g., vindesine and vinorelbine (Souquet et al.

436 Phytochem Rev (2007) 6:435–457

123

2002). The advances in developing novel drugs

extend the potential applications of bisindole

alkaloids in pharmacotherapy and thus create an

increased demand for indole alkaloids (Rus-

zkowska et al. 2003; van der Heijden et al. 2004).

Production of indole alkaloids by C. roseus cell

cultures still is one of the greatest interests and

challenges that attract many researchers to explore

the technology. Therefore, C. roseus cell cultures

are now a well-developed model system for

biosynthesis and regulation of secondary metabo-

lites. Furthermore, it remains a potential alterna-

tive for production of indole alkaloids with the

expectation of breakthroughs in bottlenecks of the

biotechnology such as creating high-alkaloid-yield

cell lines by genetically engineering the metabolic

flux and improving large-scale performance of

bioreactor processing.

Most aspects of C. roseus cell cultures affecting

production of indole alkaloids have been exten-

sively investigated with respects to optimizing

medium components, culture conditions, and

bioreactor processing. Van der Heijden and

Verpoorte (1989) and Moreno et al. (1995)

reviewed more details about the progresses made

in the period before their reviews. Obviously,

most progress about bioreactor processing was

achieved in the 1980s–1990s, which reflects the

large research effort focused on C. roseus cell

cultures and indole alkaloid production in this

period. But after finding that C. roseus cell

cultures were unable to produce bisindole alka-

loids and the failure in upscaling the cell culture

process for commercial application, research

turned away from biochemical engineering of

the process and focused more on studies of the

regulation of the biosynthetic pathways, i.e.,

looking for strategies to engineer the cell factory

itself. The biosynthetic routes, the enzymes

involved and their encoding genes, transcription

factors and regulatory signaling compounds for

production of indole alkaloids became the focus

of the research (for review, see Memelink et al.

2001; van der Heijden et al. 2004; Verpoorte et al.

2002; Zhao et al. 2005a). At present engineering

of metabolic fluxes is regarded as the key to

achieve a commercially viable indole alkaloid

production (Verpoorte et al. 2002). Once meta-

bolic engineering of indole alkaloid production is

successful based on the understanding of biosyn-

thetic genes and regulatory mechanisms, all the

previously developed knowledge on bioreactor

processing will be useful to engineer a final

industrial process, which may compete success-

fully with field cultivation.

This review summarizes the progress made in

engineering the bioreactor process of C. roseus

cultures for production of indole alkaloids, and

highlights recent advances in engineering alkaloid

production in the cell factory itself. New technol-

ogies in bioreactor processing of other plant cell

cultures that might also be useful for transgenic

C. roseus cell cultures will be discussed. All

aspects from the cell factory, plant cell culture,

bioreactor, to downstream bioreactor processing

and product recovery will be discussed.

Optimization of growth conditions

The medium components, growth regulators, pH

value, as well as culture conditions including

temperature, light, aeration, and agitation are

important factors affecting biomass accumulation

and indole alkaloid production. The medium is the

basic environmental and nutrimental condition for

plant cell cultures. Medium optimization and

manipulation of culture conditions thus is the most

fundamental approach in plant cell culture tech-

nology. Such optimization in combination with

selection of high-yielding cell lines may lead to a

20–30-fold increase of alkaloid production (Ver-

poorte et al. 1997, 2002). C. roseus suspension cells

or hairy root cultures in bioreactors behave gen-

erally almost similar as in shake-flasks in terms of

nitrogen and phosphorus consumption and growth

(Bhadra and Shanks 1997). In a two-stage turbine

stirred bioreactor process, nitrate depletion in the

medium is synchronically correlated with the start

of indole alkaloid accumulation (Schlatmann et al.

1995b). Schlatmann et al. (1995a, b) showed the

importance of an optimized glucose concentration

for ajmalicine production in a 3-l turbine stirred

bioreactor (turbine impeller speed at 250 rpm).

Growth regulators

Growth regulators have significant effects on

indole alkaloid production. Auxins and cytokinins

Phytochem Rev (2007) 6:435–457 437

123

are basic requirements for proliferation and

growth of in vitro plant cell cultures. However,

2, 4-dichlorophenoxyacetic acid (2, 4-D) signifi-

cantly inhibits indole alkaloid biosynthesis

whereas cytokinins such as benzyladenine pro-

motes cell differentiation and stimulate indole

alkaloid production. Auxins not only suppress

expression of biosynthetic genes, but affect pre-

cursor supply, e.g., by inhibiting upstream bio-

synthestic pathways or metabolite trafficking

(Whitmer et al 1998a, b; El-Sayed and Verpoorte

2000). Abscisic acid (ABA) is not a necessary

growth regulator for plant cell cultures, but it is

an important phytohormone that mediates vari-

ous abiotic stress responses in plants. It was

already shown that both ABA, salts or osmotic

stress induce an increase in ajmalicine and cath-

aranthine production (for reviews, Van der Heij-

den and Verpoorte 1989; Moreno et al. 1995).

Recently, indole alkaloids and biosynthetic en-

zymes in salicylic acid-, ethylene-, ABA-, methyl

jasmonate (MeJA)-, and gibberellic acid-treated

seedling cultures were profiled by El-Sayed and

Verpoorte (2004). MeJA generally induces pro-

duction of all indole alkaloids. SA induces

serpentine and tabersonine production at lower

concentrations and induces vindoline accumula-

tion at higher concentrations. ABA and ethylene

promoted metabolic fluxes towards ajmalicine,

serpentine, tabersonine, and vindoline biosynthe-

sis whereas gibberellic acid has little effect on

alkaloid production. Consistently, a recent report

showed cytokinin and ethylene or combination of

these two hormones to promote expression of the

secologanin biosynthetic branch of indole alka-

loid production (Papon et al. 2005).

Light and temperature

Light positively affect indole alkaloid production

in C. roseus cell and tissue culture (Zhao et al.

2001e), but effects of temperature on C. roseus

cell culture are not significant (see review, van der

Heijden and Verpoorte 1989; Moreno et al.

1995). A recent investigation in a two-stage

bioreactor process showed that the growth of C.

roseus cells and ajmalicine production was found

to be optimal at 27.5�C (ten Hoopen et al. 2002).

pH and alkaloid storage

Effect of pH value on plant cell growth and

secondary metabolite production is mainly

through influencing membrane properties and

transport activity. The capability of a cell line to

produce secondary metabolites depends not only

on biosynthesis activity, but also on transport and

storage systems. It was shown that ajmalicine and

serpentine are stored in the vacuole (Blom et al.

1991). The transport and storage processes for

indole alkaloids are affected by the pH. Renaudin

and his colleagues and Blom and colleagues have

developed a hypothesis named as the ion-trap-

ping-model, which proposed that ajmalicine and

serpentine are taken up into the vacuole in

unprotonated forms and trapped by protonation

(Renaudin 1989; Blom et al. 1991). These studies

show that the pH gradient is very important for

storage and release of indole alkaloids in C. roseus

cell cultures. The biochemical mechanism about

transport of indole alkaloids in C. roseus cell

cultures recently have been explored at protein

and gene levels, indicating that besides ion-

trapping and diffusion through membranes also

active and selective transport by multiple types of

ATP-binding cassette (ABC) transporters are

involved in alkaloid accumulation in vacuoles

(Roytrakul 2004, see in this issue).

Bioreactor process

The bioreactor process is the most important and

difficult step in the scale-up of plant cell cultures

for production of valuable secondary metabolites.

The bioreactors used for plant cell cultures are

modified from bacteria fermenters. Because of

the different properties of plant cell cultures from

microorganisms, such as slower growth rate,

adhesive and larger cell walls, sensitive to shear

force, and tendency to form cell aggregates,

bioreactor behavior of plant cell cultures is very

different from that of microorganisms. It is often

observed that scale-up of plant cell cultures in

bioreactors yields much lower biomass and, par-

ticularly, secondary metabolite production com-

pared with that in shake flasks (Moreno et al.

1995). Shear stress, gas regime (O2 and CO2,

438 Phytochem Rev (2007) 6:435–457

123

ethylene, as well as other unknown gaseous

compounds), liquid–gas mass transfer efficiency,

and other process parameters all contribute to

these problems. Since these parameters are

dependent on the bioreactor process design and

operation, optimizing bioreactor processing is a

great challenge for researchers (Leckie et al.

1991b, c). Reducing shear force to a reasonable

level but at the same time increasing mixing

efficiency is one of main goals during bioreactor

design. Regardless the types of bioreactor, also

different cultivation modes have been explored

for plant cultures. Plant cell cultures may be

grown at high-density, as immobilized cells, and

in a continuous two-phase cultivation system.

Bioreactor design and optimization

Several types of bioreactors have been used for

plant cell cultures. In terms of mixing forces,

mechanical stirring (like impeller stirring bioreac-

tor), air sparging (such as airlift bioreactor), and

combinations (mechanical stirring together with

aeration, like stirred-jar reactor) and variations of

mechanical stirring and airlift bioreactors have

been used as well as a membrane reactor with two

permeable membranes to deliver nutrients and to

export waste and products from the plant cells into

the center tube. Based on the experience with

bioreactor operation and the characteristics of

plant cultures, various modified bioreactor con-

figurations were developed, such as loop-fluidized

bed, spin filter, continuous stirred turbine, hollow

fiber, membrane stirrer for bubble-free aeration,

hybrid reactor with a cell-lift impeller and a

sintered steel sparger, as well as a centrifugal

impeller bioreactor (for review, see Zhong 2001).

A comparison of a variety of bioreactors with

different agitation and aeration systems, as to

their performance on biomass and secondary

metabolite production of C. roseus cells showed

the airlift bioreactor as the most suitable system

(Misawa 1994). A double helical-ribbon impeller

bioreactor with working volume of 11-l was

designed for high-density C. roseus cell culture

(Jolicoeur et al. 1992), a Maxblend fermentor for

high-density culture was developed and tested for

rice and C. roseus cell cultures (Yokoi et al. 1993).

A biofilm reactor that contains a horizontal

biofilm as a matrix to support cell cultures and

circulated production medium to support living of

cells was developed, showing less growth rate but

being more effective in maximizing indole alka-

loid titers than suspension cultures (Kargi et al.

1990; Kargi and Ganapath 1991). A surface-

immobilized bioreactor for C. roseus cell cultures

had also been tested (Archambault et al. 1990;

Archambault 1991). Recently Ramakrishnan and

Curtis (2004) developed a trickle-bed bioreactor

for root cultures. In terms of operating C. roseus

cell cultures in bioreactors, several modes such as

batch, semi-batch, fed-batch, immobilized culture,

and continuous cultures have been used. But

the most common one in the above-mentioned

bioreactor processes is the batch culture.

Agitation and shear stress

Mechanical agitation and sparging aeration are

very important parameters for the culture of plant

cell suspensions. They are responsible for mixing

the plant cells with the medium and thus to

facilitate homogenous nutrient uptake, and also

for providing a good O2 and CO2 supply. How-

ever, mechanical agitation and sparging aeration

cause hydrodynamic forces on the cells. The cells

subjected to these shear forces show many phys-

iological and morphological changes, such as

aggregate size and shape, cell wall composition,

oxygen uptake rate, cell integrity and viability,

and eventually biomass accumulation and sec-

ondary metabolism. The effect of shear force on

C. roseus cell cultures has been investigated in

various bioreactors (Meijer et al. 1987; Leckie

et al. 1991b, c; Smith et al. 1990; Kargi et al. 1990;

ten Hoopen et al. 1994). The major conclusions of

these studies were different from each other for

many years and this was the paradigm for

developing a plant cell culture system. Plant cells

are not extremely sensitive for shear forces,

depending on the cell lines; some are moderately

or even almost not sensitive for shear. (Fig. 1)

Because of the close relationship between

agitation and oxygen supply, many studies have

been conducted on the effect of these parameters

on C. roseus cell cultures in bioreactors. DO2 in

cell culture can be controlled by both agitation

speed and aeration rate, which further will affect

Phytochem Rev (2007) 6:435–457 439

123

hydrodynamical conditions and gas regime in the

cell culture. C. roseus cell cultures are more

sensitive to hydrodynamic forces than bacteria.

However, due to the much slower growth of plant

cells, there is no need for high stirrer speeds and

high aeration levels. Most bioreactors for plant

cell cultures are from origin microbial fermenters.

In such fermenters C. roseus cells showed a lower

growth rate and viability under higher agitation

speed and aeration rate due to a high shear force

and DO2 supply. On the other hand, C. roseus

cells tend to form even larger aggregates at

lower agitation speeds, and the cell morphology

and aggregate size also largely depend on the

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Fig. 1 Schematic illustration of engineering the bioreactorprocess and cell factory of Catharanthus roseus cellcultures for indole alkaloid production. Upstream engi-neering is mainly to generate high-alkaloid-yield cell linesfor large-scale production of indole alkaloids in bioreactor.A simple bioreactor model shows an airlift bioreactor withvarious probes for plant cell culture process. Severalimportant steps were shown in frames with or withoutrecycle arrows, which show these steps are developingtechnologies and processes. A chromatography column isused to show recovery and purification at downstreamprocess. A zoom-in C. roseus cell shows the cellularprocess of indole alkaloid biosynthesis. Hormonal andenvironmental stimulations (elicitation) initiate signaltransduction leading to jasmonate (JA) biosynthesis inplastids, which signaling further activates activation oftranscription factors (TFs). Using STR gene as ansimplified example to show transcription factors, likeORCA3 binding to GCC box, activate STR gene expres-sion, STR further is targeted into vacuole, where itcatalyzes strictosidine biosynthesis from cytosolic trypt-amine (Trp) and precursor of from plastid-derived gera-

niol, which converted into geraniol-10-hydroxylate bytonoplast-localized P450: geraniol 10-hydroxylase (GH).Strictosidine is transported to ER and converted by ER-localized strictosidine glucosidase (SG) into cathenamine,which is translocated to the cytosol, where it is furthermodified to synthesize ajmalicine and catharanthine.Strictosidine-derived tabersonine was further modifiedinto intermediates that could be taken up into plastids,where it modified into precursors for vindoline biosynthe-sis. The final two steps of vindoline biosynthesis werecarried out in the cytoplasm. These monoindole alkaloidsare taken up into vacuoles, most probably by the action ofMRP-type ABC transporters or ABC transporter-likeproteins. Inside the vacuole, vindoline and catharanthineare coupled by peroxidases into bisindole alkaloidswhereas ajmalicine is converted into serpentine. Undersome circumstances (such as elicitation), these indolealkaloids were exported into the cytosol and then furthersecreted into the medium, probably by the action of MDR-type ABC transporters. The bioreactor processing of C.roseus cell cultures consists of numerous cell factories forproduction of indole alkaloids

440 Phytochem Rev (2007) 6:435–457

123

hydrodynamic shear forces in the surrounding

fluid (Leckie et al. 1991a; Schlatmann et al.

1995a, b). However, various experiments suggest

that shear force effects on C. roseus cell cultures

are relatively not such a serious problem in

normal operation (Leckie et al. 1991b, c; Schlat-

mann et al. 1994, 1995b). C. roseus suspension

cell cultures tend to adhere to the walls of the

culture vessels and accumulate at the headspace,

particularly at high cell densities. A series of

studies on processing C. roseus cell cultures in 3-l

and 15-l turbine stirred tanks and other bioreac-

tors have provided many useful data for under-

standing of the issues mentioned above (Leckie

et al. 1991b, c; Kargi et al. 1990; Kargi and Potts

1991; Schlatmann et al. 1993, 1994, 1995a, b).

Gas regime

The importance of gas components in the head-

space of culture vessels has long been recognized.

Early studies showed that a limited oxygen supply

to C. roseus cell culture incubated in the 4-l

stirred tank bioreactor caused a reduced biomass

accumulation, but also a high gassing rate in the

bioreactor reduced biomass production (Pareil-

leux and Vinas 1983). Gases like oxygen, carbon

dioxide, and ethylene accumulate in plant cell

culture systems. Additional oxygen supply is

essential for energy generation and various met-

abolic pathways in the bioreactor process. Carbon

dioxide is the main metabolic gas component

produced by plant cells. Ethylene is a gas borne

phytohormone that is necessary for development

and growth, as well as defense response. There

are other unknown gaseous factors also affecting

cell growth, biomass and secondary metabolite

production in plant cell cultures. Recently

about 76 volatile components were identified

from C. roseus plants, including alkanes, alcohol,

aldehydes, ketones, fatty acids, fatty acid esters,

terpenoids and phenylpropanoids (Brun et al.

2001). It is very likely that in vitro C. roseus cell

cultures also generate some of these volatiles,

which accumulate in the headspace and affect the

cell culture process and production of biomass

and indole alkaloids. Particularly, some alde-

hydes, ketones, fatty acids, and fatty acid esters

derived from oxylipin biosynthesis pathways may

have interesting effects that researchers have not

investigated yet (Zhao et al. 2005a).

Catharanthus roseus cell cultures scaled up in

simple stirred bioreactors often have much lower

biomass accumulation and indole alkaloid produc-

tion than in shake flasks. It was shown that gas

composition and shear forces are the main reasons

for this difference (Schlatmann et al. 1993).

Recirculation of exhaust gases in the stirred

bioreactor partly restored the biomass and indole

alkaloid production in a stirred tank reactor or

bubble column, suggesting that exhaust gases play

an essential role in biomass and indole alkaloid

production (Schlatmann et al. 1993). Therefore,

effects of gas regime on the culture process were

extensively studied in C. roseus cell cultures in

various bioreactors. In a 15-l turbine stirred

bioreactor, ajmalicine production in high dissolved

oxygen (DO2) conditions (80% of air saturation)

was 5-fold higher than that in low DO2 (15% of air

saturation) (Schlatmann et al. 1994). A linear

relationship between DO2 and ajmalicine produc-

tion was observed in DO2 between 29% of air

saturation and 43% of air saturation (Schlatmann

et al. 1993). A high-density cell culture produced

much lower ajmalicine than a low- density cell

culture (with high DO2), but an increase in DO2 to

high-density cell culture could not restore the

ajmalicine production (Schlatmann et al. 1994).

During two-stage culture for optimal growth and

alkaloid production, aeration rate for the growth

stage was usually set as 228–300 l/h, stirrer speed at

minimum of 200 rpm; in that case, DO2 was about

40% (Schlatmann et al. 1994).

According to Pareilleux and Vinas (1983), the

critical dissolved oxygen concentration for C.

roseus cell suspension is 0.05 mmol/l (20 % of air

saturation). The respiration rate was measured to

be around 0.15–0.3 mmol/g cells/h (without oxy-

gen limitation for cell cultures) (Pareilleux and

Vinas, 1983). The optimal value for oxygen mass

transfer coefficient KLa for C. roseus cell cultures

in bioreactor ranges between 15 and 20 h–1.

However, different optimal KLa values were

found for growth and alkaloid production in a

12.5-l stirred tank bioreactor: KLa for serpentine

production is 16.0 h–1 and for ajmalicine are

4.5 h–1 (Leckie et al. 1991a). High KLa values

caused increased aggregation of the cultures,

Phytochem Rev (2007) 6:435–457 441

123

depressed biomass yields, and altered patterns of

alkaloid accumulation (Leckie et al. 1991a). The

KLa for optimum biomass production were

among 4.5 h–1 and 12.5 h–1 (Leckie et al. 1991a).

Smith et al. (1990) developed a bioreactor system

to determine and control dissolved concentrations

of oxygen and carbon dioxide (DCO2) at constant

shear force. It was shown that DO2 set at 50% of

air saturation and DCO2 set as 20 mbar at starting

point could keep the culture system in long-term

balance (Smith et al. 1990). Diaz et al. (1996)

studied the partial pressures of dissolved oxygen

and dissolved carbon dioxide in a bioreactor.

Doran (1998) reported a new technology to

improve oxygen delivery to hairy root cultures

by using membrane tubing aeration and perflu-

orocarbons.

For catharanthine production in a 2-l jar

bioreactor with 300 g cells as inoculums, an

increase in DO2 (8 ppm) significantly enhanced

catharanthine production from 180 mg/l/week to

230 mg/l/week. In the same bioreactor, Schlat-

mann et al. (1994) showed that with certain levels

of DO2, more CO2 promoted biomass accumula-

tion and ajmalicine production. Aeration affects

CO2 supply in cell cultures. Both higher dissolved

CO2 caused by too high airflow rate and lower

DCO2 caused by too low airflow rate are not good

for C. roseus cell growth (Ducos and Pareilleux

1986; Hegarty et al. 1986).

Rheology in cell culture

Rheological studies on plant cell suspension

cultures provided important information for

improvement of the cell culture process. Studies

including viscosity, aeration, mass transfer, shear

stress and cell growth, as well as metabolite

production in plant cell cultures can provide

insights into problems of the bioreactor process.

Rheological effects of treatments with e.g., algi-

nate, and sugar or with osmotic regulators such as

sorbitol and mannitol on biomass and indole

alkaloid production are very clear (Zhao et al.

2000c; Zhao et al. 2001a). Even with rigorously

mixing, DO2 and DCO2 are significantly reduced,

yet accumulation of secondary metabolites was

not changed much, suggesting specific rheological

effects on alkaloid biosynthesis. Rheological

effects also dramatically influence the bioreactor

process when C. roseus cell cultures are cultured

at high density. High-density cell culture can

improve volumetric productivity of plant second-

ary metabolites. High-density cell cultures to-

gether with other treatments to initiate

biosynthesis of target secondary metabolites

could considerably improve the productivity

(Zhao et al. 2001a; Zhong 2001). However, a

bioreactor process of high-density cell cultures

may result in lower indole alkaloid productivity in

part due to the decreased DO2 and nutrient

limitation because of the decreased mass transfer

(Schlatmann et al. 1994, 1995c). The significantly

reduced mass transfer is mainly due to the low

dynamics in the high-density cell culture. Increas-

ing DO2 indeed can partly recover ajmalicine

productivity (Schlatmann et al. 1994, 1995c).

Bioreactor process monitoring

An important part of cell culture process is to

monitor the biomass concentration of the plant

cells or even secondary metabolites over the

growth cycle, since it is essential to know how

culture cells are growing and how target metab-

olites are accumulated. Bioreactor processing of

plant cell cultures represents a physically, chem-

ically, and biologically dynamic system, in which

different levels of interactions are ongoing: cells

with their environment, cells with cells, between

subcellular organelles, and cells with endogenous

molecules in the culture. To obtain as many

details as possible about changes in these vari-

ables is a prerequisite for optimizing and control-

ling the bioreactor process.

Shear forces often exert negative effects on

plant cell growth and secondary metabolite accu-

mulation, quantitatively determining shear forces

and their effects on plant cell cultures can be done

by using special flow and shearing devices such as

corvette-type apparatus, recirculating flow capil-

lary, and submerged jet (Zhong, 2001). The shear

damage effects can also be detected by measuring

O2 uptake rate, cell growth rate, conductometry,

osmotic pressure, and O2/CO2 concentrations in

the bioreactor inlet and outlet gases. Many

important parameters, such as KLa for O2 and

CO2, mixing conditions, DO2, DCO2, and viscosity

442 Phytochem Rev (2007) 6:435–457

123

of cell culture always change during a bioreactor

process of plant cell cultures due to cell growth

and death. For example, KLa values were found to

increase by about 10–20% compared to their

corresponding initial KLa, but as the cell density

increased, KLa ultimately decreased (Ho et al.

1995). Biomass also decreased with KLa, probably

due to the hydrodynamic forces at impeller speeds

of 100–325 rpm as at the aeration rate of 0.43 vvm

no oxygen starvation was observed (Ho et al.

1995). Kinetic monitors for above parameters are

usually present in various commercial bioreactors

and are important to evaluate the culture system

and establishing a mathematical model for auto-

controling the culture system. On-line monitoring

of an established bioreactor process based on a

computer-aided monitoring system was applied to

a plant cell process (Zhong 2001). Komaraiah

et al. (2004) developed multisensor array gas

sensors to continuously monitor changes of the

gas regime in plant cell cultures. Analyzing the

multiarray responses using two pattern recogni-

tion methods, principal component analysis and

artificial neural networks showed that plant cell

suspension cultures can generate volatile emis-

sions and these emissions may be detected using

electronic noses sensor arrays. Analysis of these

process variables in turn can predict the biomass

concentration and the secondary metabolite pro-

duction (Komaraiah et al. 2004). Recently, a

multiwavelength fluorescence probe was tested

for in situ monitoring of Eschscholtzia californica

and C. roseus cell cultures (Hisiger and Jolicoeur

2005). Using endogenous fluorophores with dif-

ferent excitation and emission peaks of plant

(secondary) metabolites, this probe can be used to

monitor NAD(P)H as marker of cell activity and

riboflavins for cell concentration and growth. The

real-time production of tryptophan, tryptamine,

ajmalicine and serpentine could also be monitored

with this probe (Hisiger and Jolicoeur 2005). This

provides a very useful tool for the control and

optimization of plant cell processes. Except for

monitoring the whole culture system, assaying

enzyme activity and detecting gene expression of

C. roseus cell cultures in bioreactors also are

important to understand the kinetic process.

Previous studies have already provided insight in

the enzyme activities in C. roseus cell cultures

grown in a bioreactor under different cell-densi-

ties and sugar concentrations (Schlatmann et al.

1995a, b). It was reasoned that shear stress, special

gas regime, high pressure, and other unknown

factors in bioreactor-processed C. roseus cell

cultures may change expression of some genes

critical for indole alkaloid production.

Mathematical model and process control

Mathematical models describing the bioreactor

process are essential tools for designing, optimiz-

ing, scaling up, and auto-controlling the bioreac-

tor operation and cell culture process. Several

mathematical models for plant cell cultures were

developed (Bailey and Nicholson 1989; Bailey

and Nicholson 1990; De Gunst et al. 1990).

A basic structured kinetic model was established

and used for batch tobacco suspension cultures,

regarding structural component production, sec-

ondary metabolite synthesis and cellular respira-

tion (Hooker and Lee 1992). Models for

utilization of nutrients by C. roseus cell cultures

were also established, e.g., a bioreactor system for

controlling dissolved concentrations of both DO2

and DCO2 simultaneously (Smith et al. 1990); an

unstructured mathematical model in glucose lim-

ited chemostats showed a linear relation between

specific glucose uptake, oxygen consumption, and

carbon dioxide production as a function of the

growth rate (van Gulik et al. 1992). Furthermore,

they developed a structured mathematical model

for the description of the kinetics of growth and

intracellular accumulation of glucose and phos-

phate, as a function of glucose and phosphate

supply; this structured model well described the

growth of C. roseus cell suspensions (van Gulik

et al. 1993). More recently, based on the descrip-

tion of metabolic events during the production

stage, a simple structured model for maintenance,

biomass formation, and ajmalicine production by

non-dividing C. roseus cells was established (Sch-

latmann et al. 1999). This model describes stoi-

chiometry of biomass (including two parts, active

biomass and storage carbohydrates) and ajmali-

cine production kinetics of non-dividing C. roseus

cells in the second stage of a two-stage batch

process. It provides a satisfactory description of

the results even though ajmalicine production did

Phytochem Rev (2007) 6:435–457 443

123

not fit well due to accumulation of inhibiting

gaseous metabolites (Schlatmann et al. 1999).

Continuous culture is preferred for the develop-

ment of mathematical models, because of the

potential of steady state conditions. However, no

good mathematical model is established yet to

describe the important model of the bioreactor

process. Establishing a well-fitting mathematical

model for a plant cell culture process is difficult

due to the uncertainty and the nonlinear nature of

the bioprocess. Recently artificial intelligence

methods were used for the design and control of

microbial processes, this knowledge-based meth-

odology can also be used for plant cell culture

(Zhong 2001). Recently Leduc et al. (2006)

developed a kinetic metabolic model describing

C. roseus hairy root growth and nutrition condi-

tions. The model used intracellular nutrients and

energy shuttles to describe metabolic regulation,

providing an efficient tool for estimating the

growth rate. A suitable metabolic model should

be made on the basis of measurements of many

metabolic pathways.

High-density culture

Calculation of cost-effectiveness of a plant cell

culture process for secondary metabolite produc-

tion has shown that only with a high biomass it is

possible to reach a low price, which means that

one has to reach maximal biomass by high-density

culture to obtain a cost-effective production of

secondary metabolites (Verpoorte et al. 2002).

But high-density cell culture of C. roseus was

shown to have 5-fold lower productivity than the

low-density culture, due to low DO2 and other

factors (Moreno et al. 1993a; Schlatmann et al.

1995c), optimizing culture conditions such as DO2

may increase the volumetric productivity of

indole alkaloids. At a density of about 20–30 g/l

cell cultures have only 5–10% free medium,

which makes it difficult to recover anything from

the medium, consequently, the products must be

extracted from the biomass (Verpoorte 2002).

Other plant cell cultures have been grown in high-

density for producing useful secondary metabo-

lites (Zhong 2001). It was shown that high-density

cell cultures gave much higher productivity of

target secondary metabolites, particularly in com-

bination with other strategies, thus corresponding

bioreactor configurations have been developed

for high density cultures, such as a double helical-

ribbon impeller reactor and maxblend fermentor

(Jolicoeur et al. 1992; Yokoi et al. 1993; Zhong

2001; Zhao et al. 2000a, 2001a). It thus seems that

high-density culture could be best for production

of intracellular-accumulated secondary metabo-

lites in combination with stimulation strategies. In

a two-stage culture strategy, high-density cultures

can be treated at the second stage in many ways

to obtain the target secondary metabolites, such

as elicitation, immobilization, two-phase and

continuous cultivation (including semi-continuous

and fed-batch cultures).

Continuous culture

It was suggested that continuous culture of plant

cells might be an economical process for commer-

cial production of secondary metabolites by plant

cell cultures. The continuous culture technique

would enable cell cultures in suitable bioreactors

to continuously synthesize and release secondary

metabolites for long time, upon feeding nutrients,

precursors, or stimulation substances. However,

due to the technical and practical limitations on

keeping cell viability and stability, sterile opera-

tion of bioreactors, and some other factors, such

processes have not been realized and cost calcu-

lations show that the costs will be higher than in

case of a fed-batch culture (Verpoorte et al. 2002;

Zhong 2001). However, for modeling the second-

ary metabolite production the continuous cultures

are an important research model. Pareilleux and

Vinas (1984) first tried continuous culture of C.

roseus cells for indole alkaloid production. Van

Gulik et al. (1992, 1993) studied C. roseus cell

cultures in stirred tank bioreactors operated in

batch and continuous modes. Comparison of

stoichiometry of C. roseus growth in steady-state

glucose limited chemostats and dynamic condi-

tions showed that they are very different (van

Gulik et al. 1992). The chemostat culture tech-

nique is useful to obtain reliable data on the

stoichiometry of the growth of plant cells in a

stirred bioreactor. Several other groups have also

studied growth kinetics, stoichiometry, and mod-

eling of the growth of suspension-cultured plant

444 Phytochem Rev (2007) 6:435–457

123

cells by using semi continuous or fed-batch cul-

tures to achieve steady-state growth (for a review,

see Zhong 2001). The results suggest that removal

of indole alkaloids away from cells facilitates the

metabolic flux to production of more alkaloids due

to deletion of feedback inhibition. This strategy

could be important for efficient up scaling of plant

cell cultures as both productivity and recovery of

secondary metabolites are improved. A continu-

ous culture process that adapts this strategy might

be an economical way for production of plant

secondary metabolites although in such a system

high-density biomass is not possible as free

medium is required for the extraction of these

compounds. The improved productivity per unit of

biomass has a tradeoff in terms of a decrease in

volumetric productivity. The optimum balance

between the two must be determined to come to

the true commercial production system.

Two-phase culture

Two-phase cultivation systems of plant cells have

been developed to improve production of sec-

ondary metabolites. Both liquid–liquid and li-

quid–solid systems have been used to concentrate

secondary metabolites from the cells and the

medium into the second phase. Introduction of

such an additional phase is not only of interest for

in-situ extraction and prevention of degradation,

but also may enhance the metabolic flux toward

desired products by reducing the feedback inhi-

bition by removal of the products from their

biosynthesis site (intracellular compartments).

Based on this theory, different two-phase culture

systems were developed for plant cell cultures.

Byun and Pedersen (1994) used a two-phase

airlift bioreactor in combination with elicitation

for a significantly enhanced production of benz-

ophenanthridine alkaloids in cell suspensions of

E. californica. The use of Amberlite XAD-7 resin

in C. roseus cell cultures can dramatically

improve both volumetric productivity and recov-

ery of indole alkaloids (Brodelius and Pedersen

1993; Payne et al. 1998; Lee-Parsons and Shuler

2002). The alkaloids are absorbed on the resin,

which dramatically improves the production of

alkaloids. Tikhomiroff et al. (2002) tried C. roseus

hairy root cultures in a two-liquid-phase bioreac-

tor, which was designed to extract indole alka-

loids with silicon oil. This two-phase culture

system could efficiently absorb tabersonine and

lochnericine and thereby increase the production

of the two indole alkaloids by 100–400% and

14–200%, respectively, without significantly

affecting the availability of nutrients and hairy

root growth. In combination with elicitation by

jasmonic acid, specific production of all indole

alkaloids including non-silicon oil-absorbed ser-

pentine further increased.

A polyurethane foam draft tube as the immo-

bilizing matrix was applied to an airlift bioreactor

to carry out a two-phase C. roseus culture. The

bioreactor was connected to a neutral polymeric

resin column to absorb indole alkaloids. The total

secreted indole alkaloids reached 380 mg/l, most

of the intracellular alkaloid produced by C. roseus

cells was secreted into the medium (Yuan et al.

1999). The volumetric oxygen transfer coefficient

KLa in cell cultures processed in an organic solvent

two-phase culture system, as well as rheological

properties of the system, was studied regarding the

effects of organic solvent, agitation speed, and

aeration rate (Wu et al. 2000). Such data are of

interest for developing optimal conditions for

growth and secondary metabolite production.

Strategies to improve the productivity of the cell

factory

Feasibility of the commercial production of a

valuable secondary metabolite by plant cell cul-

tures largely depends on the economics of the

production process, which in turn depends on

productivity. Economics for a bioreactor process

for a plant cell culture producing indole alkaloids

was calculated (Verpoorte et al. 1999), showing

that current productivity of ajmalicine (maximum

0.3g/l) (Verpoorte et al. 2002) is not enough for

an economical feasible large-scale production.

Since the low productivity of indole alkaloids in

C. roseus cell cultures is one of the obstacles

towards commercial production, extensive efforts

are made to overcome the biological limitation.

Selection or creation of new high-alkaloid-yield

cell lines, eliciting C. roseus cell cultures, precur-

sor-feeding, or metabolic engineering of biosyn-

Phytochem Rev (2007) 6:435–457 445

123

thetic pathways are potential strategies to im-

prove indole alkaloid productivity. The whole

idea is to create high-indole alkaloid-yield cell

lines by stable genetic over-expression of the

expected biosynthetic pathways or inhibiting

competitive pathways, to improve bioreactor

performance by to-the target eliciting, precursor

feeding, optimizing growth and production of

engineered cell cultures, and to release indole

alkaloids into medium and recover them effi-

ciently by processing technologies.

Selection and creation of high-alkaloid-yield

cell lines

Catharanthus roseus cell cultures often are heter-

ogeneous and are composed of low-alkaloid-

yielding, high-alkaloid-yielding, as well as non-

alkaloid-producing cells. Like bacteria colony

isolation, i.e., selection of cell lines with suitable

and uniform genetic, biochemical, and physiolog-

ical characteristics, is an important approach to

improve productivity of target secondary metab-

olites. Researchers used radioimmunoassay and

fluorescence assay methods to screen cell lines to

obtain high-yield cell lines. UV- and radioactive-

irradiation, as well as other mutagenic treatments

have been applied to further widen the genetic

resources (for review, see Moreno et al. 1995).

The random mutagenesis and the laborious

and time-consuming screening are rather like a

lottery with an unpredictable outcome, therefore

now the preferred strategy is to genetically

engineer cells in a clearly targeted approach.

Advances in understanding the biosynthesis and

its regulation resulting in the cloning of a number

of genes involved in the pathway are the basis of

metabolic engineering. Developments in the field

of molecular biology have greatly facilitated the

unraveling of plant metabolism. To date many

successful examples of improved secondary

metabolite production by genetic modifications

have been reported (for a review, see Verpoorte

and Memelink 2002).

Elicitation of indole alkaloid biosynthesis

Elicitation of cell cultures with various abiotic

and biotic elicitors or signal molecules often

results in a dramatic increase in yield of certain

secondary metabolites, probably due to the

defense role of these secondary metabolites.

Production of indole alkaloids is also induced by

biotic or abiotic stresses. Examples are many: salt

stress using NaCl and KCl, osmotic stress using

sorbitol, mannitol (Moreno et al. 1995; Zhao

et al. 2000c), polyethylene glycol, polyvinyl pyrr-

olidone, and sodium alginate (Aoyagi et al. 1998;

Zhao et al. 2000c), metal stress with sodium

orthovanadate, vanadyl sulphate and some rare

earth elements (Zhao et al. 2000b), stimulation

with various chemicals (Zhao et al. 2000d; 2001c),

fungal elicitors and hormones (Namdeo et al.

2000; Zhao et al. 2001d; El-Sayed and Verpoorte

2004). Application of the elicitation to C. roseus

cell cultures not only improves indole alkaloid

biosynthesis in short time, but causes also excre-

tion of the products into the medium. Combina-

tion of two or more elicitors that can

synergistically induce metabolic fluxes towards

indole alkaloids even further improves the pro-

ductivity of target compounds and performance

of bioreactor processing. A C. roseus cell line was

cultured in a 14-l bioreactor with 80% decrease in

total alkaloid production compared to the shake

flask culture, but combined osmotic stress with

1 mM trans-cinnamic acid treatment restored the

original alkaloid amounts (Godoy-Hernandez

et al. 2000). A combined elicitor treatment with

an Aspergillus niger mycelium and tetramethy-

lammonium bromide resulted in much higher

ajmalicine production in a 20-l airlift bioreactor

(Zhao et al. 2000a). In addition, a synergistic

effect on indole alkaloid accumulation was

observed in C. roseus cell cultures when treated

with combined elicitation with malate and sodium

alginate, resulting in a higher catharanthine yield

in flasks and a 20-l airlift bioreactor compared

with control (Zhao et al. 2001a). Treatment of

C. roseus cells with ABA induced catharanthine

and ajmalicine accumulation. In a 30-l airlift

bioreactor, 8.3 mg/l ABA was added to 7-day-old

C. roseus cell culture and 82.25 mg/l of catharan-

thine can be obtained after three days of further

culture (Smith et al., 1987). The effect of a

combination of treatments was nicely illustrated

for taxane diterpenes production that was dra-

matically increased when suspension cultures of

446 Phytochem Rev (2007) 6:435–457

123

Taxus chinensis were treated with MeJA, sucrose

feeding, and ethylene exposure (Dong and Zhong

2002).

Precursor feeding and metabolic flux

distribution

The indole alkaloids derive from precursors from

two biosynthetic pathways, the terpenoid pathway

and shikimate pathway. By feeding precursors an

improved production of indole alkaloids can be

achieved. Commonly, precursors from the shiki-

mate pathway such as tryptophan or tryptamine

and precursors from the terpenoid pathway such

as loganin, loganic acid, and secologanin are used

to effectively promote indole alkaloid production.

Of the iridoid precursors, loganin is most effi-

ciently incorporated into indole alkaloids (Mo-

reno et al. 1993b; Whitmer et al. 1998a, 2002a, b).

Precursor feeding time usually significantly af-

fects feeding outcomes. It is concluded that

feeding cells in exponential growth phase (Mo-

reno et al. 1993b; Silvestrini et al. 2002) with

precursors gives the maximum outcome, probably

due to active uptake and biosynthesis capability

of cell cultures at this stage. There are many other

branches in the terpenoid and the shikimate

pathway leading to the production of other

compounds, which compete for the precursor

pools with indole alkaloid production. Inhibiting

production of these undesirable compounds may

enhance precursor flow to indole alkaloids. For

example, trans-cinnamic acid (inhibitor of pheno-

lic compound biosynthesis), phenobarbital, an

inducer of Cytochrome P450 enzymes like gera-

niol-10-hydroxylase, or an inhibitor of Cyto-

chrome P450s, all affect production of indole

alkaloids with predicted results (Contin et al.

1999). Fed with tryptophan in a 20-l airlift

bioreactor, C. roseus cell suspension produced

0.31 mg/g dry mass ajmalicine after 14 d of

cultivation (Fulzele and Heble 1994). Fed with

stemmadenine C. roseus cell cultures accumu-

lated more catharanthine, tabersonine and cond-

ylocarpine, suggesting that stemmadenine is an

intermediate in the pathway to catharanthine and

tabersonine (El-Sayed et al. 2004). Recent feed-

ing studies on elicited cells or transgenic cell lines

clearly showed that the terpenoid pathway is

limiting the production of indole alkaloids, feed-

ing both tryptamine and loganin the cell factory

can even produce much higher levels of alkaloids.

This proves that the capacity of the cell factory

for producing alkaloids in fact is much higher

than the actual production (Whitmer et al. 1998a;

2002a, b).

Release and recovery of indole alkaloids

Excretion of the secondary metabolites into the

medium makes recovery of these chemicals much

easier. However, there is only a minor portion of

indole alkaloids released into the culture medium,

with improved production of indole alkaloids

most of the synthesized indole alkaloids are

intracellularly stored. The lack of appropriate

methods to release secondary metabolites into the

medium is a problem for developing an industrial

process in which extraction from the medium is

desired. Techniques that cause continuous pro-

duction and release of secondary metabolites

from plant cells without decreasing their viability

and biosynthesis capability could be of interest.

From studies on immobilized and two-phase C.

roseus cell cultures researchers have established

some methods for releasing indole alkaloids

(Brodelius and Pedersen 1993; Payne et al.

1998). These include chemical permeabilization,

elicitation, oxygen or phosphate limitation, pH

gradient variation, pressure or heat shock, ultr-

asonication, and electropermeabilization. Brode-

lius and Pedersen (1993) tested several

permeabilizing agents such as DMSO and Tri-

ton-X-100 on C. roseus cell cultures and found

that they effectively released indole alkaloids but

also reduced cell viability. In situ extraction of

indole alkaloids from cell cultures with neutral

resins, on the other hand, was shown to stimulate

production of indole alkaloids (see above)

(Brodelius and Pedersen 1993; Payne et al. 1998;

Lee-Parsons and Shuler 2002). Also some other

release methods stimulate the production of plant

secondary metabolites, for example, various elic-

itors, ultrasonication (Wu and Ge 2004), and

electropermeabilization (Yang et al. 2003).

Therefore release of indole alkaloids from C.

roseus cell cultures is an important strategy to

improve overall productivity. Based on the ionic

Phytochem Rev (2007) 6:435–457 447

123

properties of the indole alkaloids, electropermea-

bilization of C. roseus cell membranes and simul-

taneous electrophoric transport and collection of

indole alkaloids was explored (Yang et al. 2003).

Both batch and continuous electrophoretic tubu-

lar membrane reactors were developed for simul-

taneous release, transport, and collection of ionic

metabolic products, and validated by using

C. roseus and Beta vulgaris cell cultures for

positively and negatively charged plant secondary

metabolites (Yang et al. 2003). Promising results

were obtained with the application of an oscilla-

tory electrical field, it appeared to improve

production of secondary metabolites while retain-

ing high cell viability (Yang et al. 2003).

Metabolic engineering of the cell factory

for indole alkaloid production

Metabolic engineering requires detailed knowl-

edge of the various metabolic pathways in an

organism. This knowledge comes from studies on

the enzymes involved in the pathway, their

characterization and measuring their activity

and determining its regulation. Based on this

enzymes can be selected as targets for cloning of

the gene and subsequent engineering of the

cellular biosynthetic machinery, e.g., to increase

the metabolic flux to desired secondary products

and thus improve the productivity of the plant or

plant cell culture for the target metabolite. This

goal can be achieved either by redirecting meta-

bolic fluxes by overexpressing the target pathway;

suppressing other pathways that compete with the

target pathway for precursor pools; suppressing

catabolic pathways of the product of interest; or

any combination of these. In other words, meta-

bolic engineering is expected to control as many

closely related metabolic pathways as possible to

achieve maximal effects. Metabolic engineering

provides completely new perspectives with great

potential for production of important plant sec-

ondary metabolites. Despite the limited knowl-

edge on the metabolic pathways and genes

involved, the available information and materials

obtained from studies on C. roseus have already

provided the tools for starting the metabolic

engineering of the alkaloid biosynthesis.

Signal transduction to indole alkaloid

biosynthesis

Since various biotic and abiotic elicitation meth-

ods have been found that successfully stimulated

net indole alkaloid production in various C. roseus

cultures, investigation of the signal transduction

pathway(s) involved in the response to elicitation

will be of great importance (for a review, see

Zhao et al. 2005a). Studies of signal transduction

and regulatory mechanisms underlying the induc-

tion of the biosynthesis of indole alkaloids by

elicitation with yeast extract or MeJA lead to

identification of several transcription factors that

control the alkaloid biosynthetic genes (Meme-

link et al. 2001). Overexpression of such tran-

scription factors might be used to turn on a

complete pathway. Studies on elicitation revealed

various signaling components that mediate elici-

tor-or other stress-induced indole alkaloid pro-

duction (Zhao et al. 2005a). Ca2+, reactive oxygen

species, and nitric oxide are found to be compo-

nents in the elicitor signaling pathway leading to

indole alkaloid production (Zhao et al. 2001b; Xu

et al. 2005). Elicitor induction of endogenous

jasmonate biosynthesis was proved to be an

important step in mediating the elicitor-induced

strictosidine synthase (STR) and tryptophan

decarboxylase (TDC) gene expression and indole

alkaloid production. Different protein kinases

may be activated upstream and downstream of

jasmonate biosynthesis (Menke et al. 1999). Cal-

cium ions influx is identified as a prerequisite for

fungal elicitor-induced oxidative stress, jasmonate

biosynthesis, and indole alkaloid production

(Zhao et al. 2001b; Pauw et al. 2004; Lee-Parsons

and Erturk 2005). Even though H2O2 was shown

to stimulate biosynthesis of secondary metabo-

lites, it is still a question whether H2O2 is a

signaling compound for indole alkaloid biosyn-

thesis (Zhao et al. 2001b; Pauw et al. 2004). In

addition, nitric oxide burst was also observed in

elicitor-induced C. roseus cell cultures and medi-

ates catharanthine production (Xu et al. 2005).

Plant cell cultures in bioreactors often suffer from

oxidative stress due to improper culture condi-

tions such as oxygen supply, mechanical damages,

and nutrition imbalance even without elicitation.

Such oxidative stress can exert multiple effects on

448 Phytochem Rev (2007) 6:435–457

123

plant cell growth and secondary metabolism

(Zhao et al. 2000a; Zhao et al. 2001a; Zhao et al.

2005b). Reactive oxygen species generated during

oxidative stress may be the main factors for these

effects. Recently a new group of jasmonate-like

oxylipins generated in tobacco under H2O2 stress

was identified as inducers of plant secondary

metabolite accumulation (Thoma et al. 2003).

Almost all responses to elicitation and physi-

ological conditions inducing indole alkaloid bio-

synthesis may be mediated by the jasmonate

pathway. Cytosolic Ca2+ spiking is required for

fungal elicitor-induced jasmonate biosynthesis

(Menke et al. 1999; Pauw et al. 2004). Auxin

suppression of STR and TDC expression and

indole alkaloid production is also due to suppres-

sion of endogenous jasmonate biosynthesis. Re-

moval of auxins from C. roseus cell cultures and

exposure of the cell cultures to kinetins may

require intracellular Ca2+ spiking to resume

indole alkaloid biosynthesis, and most probably

recovery of jasmonate biosynthesis (Gantet et al.

1998). The cytokinin-induced alkaloid biosynthe-

sis is mediated by a two-component system, like

the cytokinin signaling pathway in Arabidopsis

(Papon et al. 2003). How jasmonate signaling

mediate elicitation as an integral signal to induce

indole alkaloid biosynthesis is not well known yet.

However, several transcriptional factors have

been found that are specifically induced by

elicitors or MeJA. These transcription factors

bind to promoter regions of STR and TDC

(Memelink et al. 2001; van der Fits and Memelink

2000).

Biosynthetic and metabolic pathway, gene

and enzyme characterization

The biosynthesis of indole alkaloids has been

extensively studied and many details about

enzymes and genes involved have been reported,

yet many genes involved in the secologanin and

catharanthine biosynthesis remain unknown

(Verpporte et al. 1997; van der Heijden et al.

2004). Except for several transcription factors for

the elicitor and jasmonate signaling, other regu-

latory mechanisms are not known, e.g., intra- and

intercellular metabolite transport, enzyme or

regulatory factor trafficking, compartmentation,

and degradation of indole alkaloids (catabolic

pathways) (St-Pierre et al. 1999; Memelink et al.

2001; van der Heijden et al. 2004). Despite that so

far only a limited number of biosynthetic genes

are available, metabolic engineering of the indole

alkaloid biosynthesis has generated already some

interesting results (van der Heijden et al. 2004).

Inter- and intra-cellular transport of indole

alkaloids and trafficking of proteins

Numerous studies have shown that biosynthesis

and storage of plant secondary metabolites take

place in different subcellular organelles of the

cells. Indole alkaloid biosynthesis and storage

involve multiple machineries in different cellular

and subcellular compartments. Firstly it was

shown that many different cell types are impli-

cated in vindoline formation and storage: earlier

steps for tryptamine and secologanin biosynthesis

occur at epidermis cells, and later steps for

vindoline formation take place at mesophyll,

idioblast, or laticifer cells (Murata and De Luca

2005; Mahroug et al. 2006). Secondly it was found

that, different subcellular compartments are

involved in indole alkaloid biosynthesis, these

include at least the plastids, endoplasmic reticu-

lum, and vacuole. Therefore, the assembly of the

indole alkaloid biosynthesis pathway requires not

only appropriate enzyme trafficking but also

efficient transport of substrates and metabolites.

Transport of biosynthetic intermediates and

metabolic products in and out of the chloroplasts

and vacuoles through endomembranes inside the

cells or from cell to cell through the plasma

membrane are essential parts of the biosynthesis

(St-Pierre et al. 1999; Yazaki 2005). Recent stud-

ies have revealed that different ABC transporter

and H+-antiporters are involved in these func-

tions. For example, efflux of berberine from C.

japonica cells is finished by one type of ABC

transporter, a multidrug resistance protein

(MDR), CjMDR1 (Yazaki 2005). The barley

secondary metabolite saponarin is imported into

barley vacuoles by a proton-motive forced H+-

transporter but into Arabidopsis vacuoles by an

ABC transporter (Frangne et al. 2002). These

studies provide insights and tools for production

of plant secondary metabolites. Most recent

Phytochem Rev (2007) 6:435–457 449

123

studies using an in vitro vacuolar uptake assay

and pharmacological methods have shown that

influx and efflux of alkaloids and its precursors

through the vacuolar membrane or C. roseus cell

plasma membrane involves different types of

ABC transporters (Roytrakul 2004). Roytrakul’s

study indicated that ajmalicine, catharanthine,

and vindoline could be taken up into the vacuole

by a subfamily of ABC transporters, the multi-

drug resistance associated proteins (MRP); while

strictosidine and secologanin could be taken up

into the vacuoles by ABC transporter-like pro-

teins; and tryptamine uptake into the vacuole may

involve H+-antiporters (Roytrakul 2004). All of

these alkaloids could be secreted from the vacu-

ole by MDR-type transporters (Roytrakul 2004).

Since these ABC transporters and H+-antiporters

are less specific in substrate preference and highly

regulated (Yazaki 2005); their activity definitely

affects production of indole alkaloids in C. roseus

cell cultures. Overexpression of indole alkaloid-

biosynthesis genes in tobacco cells showed that

strictosidine generated by transgenic tobacco cell

cultures fed with secologanin and tryptamine are

exported into the medium and not stored in the

vacuole (Hallard et al. 1997; Verpoorte, unpub-

lished results). Apparently every plant species has

different selective transport systems. Further

study on the transport of precursors, intermedi-

ates and indole alkaloids may lead to discovery of

new strategies that could facilitate the production

of certain indole alkaloids. Metabolic engineering

of transport of related metabolites can improve

overall production of indole alkaloids in C. roseus

cell cultures.

Protein trafficking involved in plant secondary

metabolism is not well understood until recent

years, but is thought to play an important regu-

latory role. Characterization of CaaX-pren-

yltransferases from C. roseus cell cultures

supported this idea (Courdavault et al. 2005).

Prenyltransferases are a group of heterodimeric

enzymes that can modify and re-localize many

regulatory proteins such as transcription factors

and signal components, and membrane-associated

enzymes or membrane trafficking proteins. RNAi

suppression of subunits of the protein dramati-

cally inhibits expression of early steps of indole

alkaloids and blocks indole alkaloid production,

suggesting that proper targeting of some regula-

tory proteins is essential for regulation and

expression of indole alkaloid biosynthesis genes

(Courdavault et al. 2005).

Manipulating biosynthetic genes

Genetic modification results showed that the

production of indole alkaloids in C. roseus cell

cultures was not affected by the overexpression of

TDC (Canel et al. 1998; Whitmer et al. 1998a, b;

Goddijn et al. 1995; Whitmer et al. 2002b). How-

ever, feeding with tryptophan and more loganin

(6.4 mM) resulted in about 400 mg/l of total indole

alkaloids including strictosidine, ajmalicine, ser-

pentine, catharanthine and tabersonine as major

components. On the other hand, overexpression of

STR resulted in a higher production of total indole

alkaloids at a level of almost 300 mg/l (Whitmer

et al. 2002a). Like other high-yield cell lines,

genetic or epigenetic instability of secondary

metabolite biosynthesis also occurs in transgenic

lines. The levels of indole alkaloids in STR-

transgenic cell lines decreased gradually after

years of subculture maintenance, but the high

capacity of indole alkaloid production can be

restored by precursor (like loganin) feeding (Whit-

mer et al. 2003). These results not only suggest the

feasibility of metabolic engineering for indole

alkaloid production, but also indicate that other

unknown regulatory mechanisms exist to control

the metabolic fluxes. Results also suggests that

there seems to be a physiological barrier for the

production of more than about 400 mg/l, as feed-

ing larger amounts of precursors does not lead to

further increase of alkaloids. The regulation

includes probably substrate and product traffick-

ing, compartmentation and metabolism, since

transgenic cells still kept high enzyme activity.

The results from transgenic tobacco and Cinchona

officinalis hairy roots expressing the TDC and STR

genes also point to the importance of subcellular

trafficking and storage for production of secondary

metabolites (Hallard et al. 1997; Verpoorte et al.

2002).

Metabolic engineering was also tried on C.

roseus hairy root cultures. Overexpression of a

truncated hamster 3-hydroxy-3-methylglutaryl-

CoA reductase, a key enzyme in mevalonate/

450 Phytochem Rev (2007) 6:435–457

123

acetate pathway for terpenoid biosynthesis, in C.

roseus hairy roots was reported to result in an

increased production of ajmalicine and catharan-

thine or serpentine compared with control (Ay-

ora-Talavera et al. 2002). Transgenic hairy root

cultures of C. roseus were reported with a

glucocorticoid-inducible promoter controlling an

Arabidopsis feedback-resistant anthranilate syn-

thase a-subunit (ASa) or b-subunit (Asb) (Hughes

et al. 2004a). The transgenic hairy roots produced

more than 20-fold higher levels of tryptophan or

tryptamine under certain induction conditions

compared with control whereas most indole

alkaloids were not significantly altered with the

exception of lochnericine, which increased 81%

after a 3-day induction period (Hughes et al.

2004a). Furthermore, glucocorticoid inducible

expression of TDC alone or in combination with

ASa in transgenic C. roseus hairy root cultures

showed an increased TDC activity after induction

(Hughes et al. 2004b). The induced TDC line

showed no significant increase in tryptamine level

but 129% increase in serpentine production,

whereas TDC-ASa line showed 6-fold increase

in tryptamine level but no increase in serpentine

production (Hughes et al. 2004b). Secologanin

biosynthesis limitation in such transgenic hairy

roots was confirmed (Peebles et al. 2006). In

feeding 1-deoxy-D-xylulose to ASa-overexpress-

ing hairy root line, an increase of 125% in

hoerhammericine was observed, while loganin

feeding increased catharanthine by 45%. In

feeding loganin to the ASa- and ASb-overex-

pressing hairy root line, increases of 26% in

catharanthine, 84% in ajmalicine, 119% in loch-

nericine, and 225% in tabersonine were observed

(Peebles et al. 2006).

Manipulating transcription factors

It is generally recognized that signal transduction

pathways, controlling the biosynthetic genes of

secondary metabolites, do so via transcription

factors, which activate or suppress biosynthetic

pathways (for review, see Zhao et al. 2005a).

Identification and subsequent manipulation of

these transcription factors becomes very attrac-

tive for metabolic engineering of plant secondary

metabolites (Gantet and Memelink 2002). The

idea has been validated for various plant second-

ary metabolite pathways (for review, see Gantet

and Memelink 2002). Searching transcription

factors that bind to the promoter region of the

STR gene led to the identification of two impor-

tant AP2/ERF family transcription factors

ORCA2 and ORCA3, which are specifically

induced by jasmonate and thought to mediate

jasmonate-induced STR and TDC expression and

indole alkaloid production (van der Fits and

Memelink 2000). Ectopic expression of ORCA3

in C. roseus cultured cells resulted in an increased

expression of several indole alkaloid-biosynthetic

genes, and an almost 3 fold increased indole

alkaloid production upon feeding of loganin if

compared with control (van der Fits and Meme-

link 2000). Because of the difficulty to overex-

press multiple biosynthetic genes at the same time

and the facts that one transcription factor may

regulate many functional-related secondary

metabolism genes, metabolic engineering of sec-

ondary metabolism by manipulating transcription

factors can be very efficient and successful.

However, multiple targets and low specificity of

transcription factors may also bring problems,

e.g., if competitive pathways are induced as well.

Heterologous construction of metabolic

pathway

Since microorganism cultures have been success-

fully scaled up for production of various pharma-

ceuticals, the overexpression of plant or mammalian

pathways was attempted in microorganisms such

as yeasts and bacteria. Geerlings et al. (1999,

2001) have made a pioneering effort in yeast by

using C. roseus genes STR and SG (strictosidine

glucosidase). Functional enzymes were expressed

and found in the culture medium and the cells,

respectively. Upon feeding of secologanin and

tryptamine, yeast produced 2g/l of strictosidine in

the medium, and after releasing SG enzyme by

breaking the yeast cells strictosidine was con-

verted into cathenamine by the action of SG. By

feeding the juice of Symphoricarpus albus berries,

which are rich in sugar and secologanin, strictos-

idine and cathenamine can be produced. Bacteri-

ally expressed STR from Rauvolfia serpentina

can functionally synthesize strictosidine after

Phytochem Rev (2007) 6:435–457 451

123

immobilization to a matrix and adding precursors

(Shen et al. 1998). Heterologous construction of a

plant metabolic pathway in well-engineered

microorganisms such as bacteria or yeasts cer-

tainly is a great idea, yet it can be a long-term

project to clone and functionally express all

metabolic genes and overcome metabolic traffick-

ing problems. Recently a preliminary trial was

carried out for reconstructing biosynthesis path-

way of the highly valuable anticancer diterpenoid

taxol in yeast cells by over-expression of 5

sequential pathway genes (Dejong et al. 2006).

Eight single genes could be functionally expressed

in yeast (Jennewein et al. 2005), however, when 5

of these genes were expressed in a single yeast

cells, only metabolites from the first two steps

were detected (Dejong et al. 2006), suggesting

enzyme or protein trafficking problems. These

studies are important to identify such problems

and eventually find solutions to overcome them. A

success was made recently on production of

antimalarial sesquiterpene precursor artemisinic

acid in yeast. The heterologously constructed

metabolic pathway by expressing amorphadiene

synthase and a cytochrome P450 monooxygenase

from Artemisia annua in yeast generated up to

100 mg/l of artemisinic acid (Ro et al. 2006). This

engineered yeast shows a potential for further

yield optimization and industrial scale-up.

Upscale of transgenic cell cultures

Obviously volumetric productivity of some of

these transgenic C. roseus cell lines overexpressing

TDC or STR is very high and they may have great

potential for being used in a larger scale bioreac-

tor process to produce indole alkaloids. But they

all need to be fed with high concentrations of

tryptophan and loganin, which are also costly.

Ratio of cost and effect for the production

processes using these transgenic cell lines may be

high in a fed-batch bioreactor. Actually the

productivity of transgenic lines with feeding is

still too low for a commercial process. Co-over-

expression of TDC and STR in a C. roseus cell line

may further improve the productivity of indole

alkaloids. However, since insufficient precursor

supply is the major problem in these transgenic

cell lines, a strategy may be necessary to increase

these precursor pools by activating upstream

genes or directly manipulating a key upstream

gene in the biosynthesis pathway for terpenoid

precursors. On the other hand, of the many

biosynthetic genes involved in indole alkaloid

biosynthesis, overexpression of only two or three

genes may not be effective to promote overall

productivity. From metabolomics point of view,

there are many trade-offs at different levels

among metabolic pathways such as direction and

rate of metabolic fluxes or size, location, and

distribution of precursor pools to ensure normal

cellular processes and physiological functions

(Stephanopoulos 1999). Such metabolic balances

are essential for plant cells; any imbalance in

metabolic fluxes could hamper growth or even be

lethal (Stephanopoulos 1999; Manzano et al.

2004). The instability of high-alkaloid-yield cell

lines obtained from selection or genetic manipu-

lation may arise from an unbalanced metabolic

fluxes. High levels of indole alkaloids and/or

feedback inhibition could initiate regulatory

mechanisms minimizing the overuse of precursors

by the alkaloid biosynthetic pathway and slowly

come back to the balance of the whole metabolic

network. However, here the biochemical engi-

neering of the process may solve these problems,

as for example feedback inhibition can be over-

come in two-phase systems (see above). The

limited achieves in metabolic engineering of

indole alkaloid production reflect our limited

knowledge about metabolic pathways and their

regulations. New technologies in genomics, pro-

teomics, and metabolomics will lead to an in-

depth understanding of the biosynthesis and

metabolic pathways for the indole alkaloid and

the regulatory mechanisms (Verpoorte and

Memelink 2002; Choi et al. 2004; Jacobs et al.

2005; Zhao et al. 2006; Rischer et al. 2006).

Networks drawn on the basis of these gene-to-

gene, protein-to-protein, protein-to-metabolite,

and metabolite-to-metabolite collections will help

to gain a whole view of indole alkaloid biosynthe-

sis and metabolism, as well as their regulations.

Perspectives

In theory, the productivity of the plant cell factory

for natural compounds should be unlimited.

452 Phytochem Rev (2007) 6:435–457

123

Actually, the main limitation for commercial

production of indole alkaloids by C. roseus cell

cultures remains the low level of alkaloid pro-

duction of the cells in the bioreactor. In the past

years biochemical engineering approaches has

resulted in optimized conditions for growth and

production in the bioreactor. These experiences

with the increased knowledge about the cell

factory itself and perspectives of metabolic engi-

neering eventually will lead to considerably high-

er productivity, but it will involve a combination

of methods. Close collaboration between the

biochemical engineers, metabolic engineers and

cell biologists is required to reach this goal.

Acknowledgements We thank guest editor to give us thechance to express and exchange our ideas with thisresearch community. Although there are many otherexcellent publications on bioreactor processingCatharanthus roseus cell culture and related aspects, weregret not be able to cite them due to space limitation.

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