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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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