181
Engineering secondary metabolite production in plants
R Verpoorte* and J Memelink†
Recent achievements have been made in the metabolic
engineering of plant secondary metabolism. Various pathways
have been altered using genes encoding biosynthetic enzymes
or genes encoding regulatory proteins. In addition, antisense
genes have been used to block competitive pathways, thereby
increasing the flux towards the desired secondary metabolites.
Addresses
*Division of Pharmacognosy, Leiden/Amsterdam Center for Drug
Research, Leiden University, Leiden, The Netherlands;
e-mail: verpoort@lacdr.leidenuniv.nl
† Institute of Molecular Plant Sciences, Leiden University, Leiden,
The Netherlands; e-mail: memelink@rulbim.leidenuniv.nl
Current Opinion in Biotechnology 2002, 13:181–187
0958-1669/02/$ — see front matter
© 2002 Elsevier Science Ltd. All rights reserved.
Abbreviations
4HB 4-hydroxybenzoic acid
ICS isochorismate synthase
IPL isochorismate pyruvate lyase
PMT putrescine N-methyltransferase
SA salicylic acid
STR strictosidine synthase
TDC tryptophan decarboxylase
Introduction
Plant secondary metabolism has multiple functions
throughout the plant’s life cycle. These functions can be
classified as mediators in the interaction of the plant with
its environment, such as plant–insect, plant–microorganism
and plant–plant interactions [1,2]. The production of
secondary metabolites forms part of the plant’s defence
system, for example, the constitutive production of
antifeedants and phytoanticipins, and the inducible
production of phytoalexins [2]. Secondary metabolism also
plays a role in plant reproduction, for example, in attracting
pollinators and in male fertility. Secondary metabolites
determine important aspects of human food quality (taste,
colour and smell), and plant pigments are important for
the diversity of ornamental plants and flowers. Moreover,
several plant secondary metabolites are used for the
production of medicines, dyes, insecticides, flavours and
fragrances. Secondary metabolism is thus an interesting
target for plant breeding. Molecular breeding, by applying
genetic engineering, is in this respect a promising
approach. Although in the past 10 years quite some
research has been done in this field [3•], a major constraint
has been the poor characterization of plant secondary
metabolic pathways at the level of biosynthetic inter-
mediates and enzymes. Consequently very few genes
from plant secondary metabolism are available. The best-
studied pathway at the genetic level is the one leading to
the formation of flavonoids and anthocyanins [4•,5]. Most
of the genes in the anthocyanin pathway have been cloned
by a combination of biochemistry, molecular biology and
genetics. This achievement was facilitated by the fact that
flower colour is an easily screenable phenotype. Other
secondary metabolite pathways that have extensively been
studied at the level of intermediates and enzymes mainly
lead to pharmaceutically important products such as indole
and isoquinoline alkaloids [6,7,8•]. From these pathways,
however, only a relatively small number of genes have
been identified so far.
Genetic engineering of a secondary metabolic pathway
aims to either increase or decrease the quantity of a certain
compound or group of compounds [2,3•,4•,6,8•,9]. To
decrease the production of a certain unwanted (group of)
compound(s) several approaches are possible. An enzymatic
step in the pathway can be knocked out, for example, by
reducing the level of the corresponding mRNA via anti-
sense, cosuppression or RNA interference technologies, or
by overexpressing an antibody against the enzyme. The
antisense gene approach has been successfully used for
changing flower colours [10]. Other approaches include
diversion of the flux into a competitive pathway or an
increase in the catabolism of the target compound.
More often, the goal is to increase the production of certain
compounds in the normal producing plant species or to
transfer (part of) a pathway to other plant species or other
(micro)organisms. Also, there is interest in the production
of novel compounds not yet produced in nature by plants.
To increase the production of a (group of) compound(s),
two general approaches have been followed. Firstly, methods
have been employed to change the expression of one or a
few genes, thereby overcoming specific rate-limiting steps
in the pathway, to shut down competitive pathways, and to
decrease catabolism of the product of interest. Secondly,
attempts have been made to change the expression of
regulatory genes that control multiple biosynthesis genes.
Recent examples of the different approaches are reviewed
here, covering several major secondary metabolite groups.
Flavonoids and anthocyanins
Biosynthesis genes
Flavonoid and anthocyanin biosynthesis was the first target
for genetic engineering, as the biosynthetic pathway was
well known and the results could easily be observed by
changes in flower colour [4•,5,11]. Numerous experiments
have been performed involving the overexpression of
various pathway genes, aiming, for example, to produce new
flower colours by introducing new compounds in the plant.
Because of their antioxidant activity, higher levels of antho-
cyanins and flavonoids in food are an interesting objective.
Much work has focused on tomato. Chalcone isomerase
(CHI), an early enzyme of the flavonoid pathway, was found
to be the key to increase flavonol production [12•].
182 Plant biotechnology
Overexpression of the Petunia CHI gene led to a 78-fold
increase of flavonoid levels in the tomato peel. Upon
processing such tomatoes, a 21-fold increase of flavonols in
tomato paste was achieved, if compared with non-transgenic
controls. Except for the flavonoid content, neither trans-
genic plants nor the paste of the transgenic tomatoes could
be distinguished from their respective controls. Thus show-
ing that it is feasible to increase the level of compounds that
are beneficial for health in tomato-based products.
Isoflavones in legumes act as phytoalexins, that is, the
biosynthesis of these antimicrobially active compounds is
induced by microbial infection. These compounds could be
produced in Arabidopsis, tobacco plants and maize, which
normally lack the ability to synthesize these compounds, by
overexpression of isoflavone synthase, a cytochrome P450
enzyme [13,14]. The production of the isoflavones depends
on the availability of precursors from the phenylpropanoid
pathway. The induction or genetic engineering of this path-
way is therefore important to further improve isoflavone
biosynthesis in heterologous plant species.
Regulatory genes
An alternative to engineering the expression of single
pathway genes is the use of transcription factors that
control multiple pathway genes. In maize kernels, antho-
cyanin biosynthesis is regulated by a combination of two
transcription factor species, R and C1. The R protein
shares homology with the basic helix–loop–helix protein
encoded by the vertebrate proto-oncogene c-MYC, whereas
the C1 protein has homology with the proto-oncogene
cMYB product. Induction of the complete flavonoid
pathway has been achieved by the overexpression of the
transcription factors R and C1 in undifferentiated maize
cells cultured in vitro [15]. Also, in rice the overexpression
of the maize transcription factors C1 and R in combination
with the chalcone synthase gene resulted in activation of
anthocyanin biosynthesis, causing an increased resistance
to fungi [16]. In Arabidopsis a single MYB-type transcription
factor (PAP1) was identified, which upon overexpression
led to plants with intense purple pigmentation throughout
development [17]. These examples show that the stringent
genetic control of natural product accumulation during
plant development can be overcome by overexpression of
one or a few transcription factors, even in heterologous
plant species.
Transcription factors can also act as repressors of natural
product accumulation. Knocking out the gene for the
MYB-type transcription factor MYB4 in Arabidopsis led to
enhanced levels of sinapate esters in the leaves, and to
increased tolerance of UV-B irradiation [18•]. Similarly,
overexpression in tobacco of the MYB-type protein
FaMYB1 from strawberry led to a reduction in flower
pigmentation and decreased levels of anthocyanin and
flavonol compounds, suggesting that in strawberry fruit
FaMYB1 acts as a repressor of certain steps in the
flavonoid pathway [19]. These results indicate that optimal
pathway engineering using transcriptional regulators
requires detailed knowledge of the regulatory control circuit.
Alkaloids
Indole alkaloids: biosynthesis genes
The terpenoid indole alkaloid pathway has been the target
of numerous genetic engineering attempts, owing to the
fact that about 15 terpenoid indole alkaloids are industrially
important, including the antitumour alkaloids vinblastine,
vincristine and camptothecin. These alkaloids share the
pathway leading to the intermediate strictosidine, and
from this point the pathways in various alkaloid-producing
plant species diverge. Most efforts have been concentrated
on mapping the early part of the pathway and on over-
expression of early genes, aiming to increase the metabolic
flux into the alkaloid pathway. In particular, the genes
encoding tryptophan decarboxylase (TDC) and strictosidine
synthase (STR) have been studied extensively in
Catharanthus roseus cell cultures [20]. Overexpression of
TDC resulted in higher levels of the immediate product
tryptamine, but not in increased levels of alkaloids; in the
case of STR higher levels of alkaloids were noted [21]. Feeding
such cell lines with tryptophan and terpenoid intermedi-
ates showed that they have the capacity for high alkaloid
production (up to 1100 µmol/L) [22], indicating that the
terpenoid branch of the pathway is limiting. Such studies
indicate that there may be multiple rate-limiting steps.
TDC and/or STR were also expressed in various other
non-alkaloid-producing plants [23,24]. Feeding transgenic
tobacco cell cultures with secologanin [24,25] led to the
production of strictosidine, but this glucoalkaloid is excreted
to the medium instead of being stored in the vacuole as in
the indole-alkaloid-producing C. roseus. This illustrates the
importance of physiological aspects in secondary metabolism
besides biosynthesis. Thus, not only genes that encode
enzymes catalysing biosynthetic steps are involved, but also
genes involved in, for example, pH regulation and transport.
Cell cultures of Weigelia ‘Styriaca’, capable of secologanin
biosynthesis, overexpressing the TDC and STR genes
produce small amounts of ajmalicine and serpentine. This
shows that indole alkaloid biosynthesis in otherwise non-
alkaloid-producing plants is feasible via expression of one
or a few heterologous pathway genes [24].
In contrast with the above mentioned studies using
Agrobacterium-mediated transformation, Leech et al. [26]
used the particle gun to introduce the TDC and STR genes
into tobacco plants. Although both genes were integrated
into all 150 analysed tobacco plants, in 26% of the plants
both transgenes were silenced, in 41% either one of the
two genes was silenced, and in 33% both genes were
expressed. No clear correlation could be observed between
the number of integration events and the levels of
accumulated transcripts. In the transgenic tobacco
seedlings a 24-fold and 110-fold variation were found in
TDC and STR activity, respectively.
 Engineering secondary metabolite production in plants Verpoorte and Memelink
Indole alkaloids: regulatory genes
Two transcription factors, ORCA2 and ORCA3, were charac-
terized that control several steps of the alkaloid biosynthetic
pathway in C. roseus [27,28,29••]. Overexpression of ORCA3
did not result in increased alkaloid production, however,
owing to the fact that it did not control the gene G10H
encoding a cytochrome P450 enzyme that catalyzes the first
step in secologanin biosynthesis. A threefold increase in
alkaloid production was observed compared with control cells
only after feeding the secologanin precursor loganin [29••].
Both ORCA2 and ORCA3 are involved in jasmonate-
responsive expression of terpenoid indole alkaloid biosynthesis
genes [27,28,29••]. The fact that they do not control G10H,
although the gene is jasmonate-responsive [30], indicates
that other jasmonate-responsive transcription factors control
a subset of pathway genes. These studies clearly show that
regulatory genes can be used to increase the level of a series
of enzymes in a pathway, thus avoiding the need to over-
express each individual pathway gene.
Isoquinoline alkaloids
Another pharmaceutically important group of plant secondary
metabolites comprises the isoquinoline alkaloids, which
include, among others, the important medicines morphine
and codeine. The pathways to several of these alkaloids have
been elucidated in the past few years, opening the way for
metabolic engineering. Yamada and co-workers [31•] hypo-
thesized that overexpression of an enzyme at a branchpoint in
a pathway should lead to an increased flux through the
affected branch. In the biosynthesis of berberine, the enzyme
(S)-scoulerine 9-O-methyltransferase (SMT) is such an
enzyme that might control the ratio of coptisine:berberine
plus columbamine in Coptis japonica cells [31•]. Over-
expression of this gene resulted in a 20% increase in enzyme
activity, with an increase of berberine and columbamine from
79% of the total alkaloid content in wild-type cells to 91% in
transgenic cells. This observation proves that fluxes at a
branchpoint can be changed by metabolic engineering.
Overexpression of the C. japonica SMT gene in a plant cell
culture of Eschscholzia californica, a plant lacking this enzyme,
resulted in the production of columbamine, which is normally
not found in this species. Opening up a new pathway at the
intermediate scoulerine apparently channelled the flux away
from the sanguinarine branch, resulting in considerably lower
levels of this alkaloid.
An interesting approach for the production of new com-
pounds in a plant might be to introduce enzymes with a
different substrate specificity (combinatorial biochemistry).
Recombining Thalictrum tuberosum O-methyltransferase
subunits to form heterodimeric enzymes with a substrate
specificity different from that of the homodimers was
proposed as a possible way to generate new alkaloids [32].
Tropane alkaloids and pyrrolidine alkaloids
Quite some research has already been done on the genetic
engineering of the pharmaceutically important tropane
alkaloids [33]. In particular, the conversion of hyoscyamine
183
to the much more valuable scopolamine is the major goal
of these studies. The enzyme hyoscyamine-6β-hydroxylase
(H6H) catalyzes this conversion. By the overexpression of
the gene encoding H6H in Hyoscyamus muticus hairy root
cultures, a 100-fold increase of scopolamine levels could be
reached compared with controls that produced hyoscyamine
as the major alkaloid [34]. The hyoscyamine level (about
10-fold higher than for scopolamine in the transgenic roots)
was similar in transgenic and control cell lines.
Recent efforts have been aimed at increasing the flux
through the biosynthetic pathways [31]. The tobacco
putrescine N-methyltransferase (PMT) gene was over-
expressed in Atropa belladonna and Nicotiana sylvestris, with
the aim of increasing the production of tropane alkaloids
and pyrrolidine alkaloids, respectively. For both types of
alkaloids the formation of methylputrescine is the first
committed step, withdrawing putrescine from the
polyamine biosynthetic metabolite pool. Although a modest
increase of PMT activity of up to 3.3-fold was found in
the transgenic A. belladonna plants, no increase in alkaloid
levels was observed and only the level of the methyl-
putrescine was increased. In some transgenic N. sylvestris
plants the PMT activity was increased four- to eightfold,
whereas in others co-suppression was noted. The trans-
genic lines showed a 40% increase in nicotine level,
whereas in the case of co-suppression the nicotine level
was only 2% of wild type. The latter plants had a clear
increase in polyamine levels. A certain step in a pathway
might appear to be rate-limiting, but overexpression of the
encoding gene will, in most cases, immediately reveal new
rate-limiting steps.
Terpenoids
Monoterpenes and sesquiterpenes
Terpenoids are by far the largest group of plant secondary
metabolites. Following the recent discovery of the role of
the 2-C-methyl-D-erythritol-4-phospate (MEP) pathway in
the biosynthesis of plastidial terpenoids, such as the
carotenoids and monoterpenes and diterpenes, several
genes of this pathway have been cloned [35,36••,37,38].
Modifying the MEP pathway is potentially useful for a wide
range of applications. A co-suppression and an antisense
strategy used to knock out a cytochrome P450 enzyme in
tobacco trichome glands, for example, conferred an increased
resistance to aphids [39]. There was a clear shift in the cem-
branoid spectrum, with a 19-fold increase of the diterpenoid
cembratriene-ol and a decrease in its oxidation product
cembratriene-diol. In another example, overexpression of a
chimeric farnesyl diphosphate synthase gene in Artemisia
annua was reported to increase the flux in the sesquiter-
penoid biosynthetic pathway leading to a twofold to
threefold increase in the antimalaria drug [40]. In tomatoes,
over-expression of an S-linalool synthase transgene increased
many fold the production of the monoterpenoid flavor com-
pound S-linalool, compared with control plants, although no
changes in the levels of other terpenoids were observed [41].
184 Plant biotechnology
Possibilities for genetic engineering of essential oil
production in mint have been reviewed [37]. Over-
expression of the gene encoding deoxyxylulose
phosphate reductoisomerase (DXR) in mint resulted in
plants that had twofold to fourfold higher DXR activities
[36••]. The plants had a normal phenotype and an almost
50% increase in essential oil (monoterpenoid) production.
In plants in which co-suppression was observed growth was
reduced as well as monoterpenoid production. Although
most plants had the normal oil composition, some did show
qualitative and quantitative differences, which was thought
to be due to insertional effects of the transgenes.
Increasing menthol levels in mint oil might be achieved by
cutting one of the competitive branches in the mono-
terpenoid metabolic network leading to menthofuran
[36••]. An attempt was made to block the branch that
channels pulegone away from the menthol pathway by
using the antisense gene of menthofuran synthase. Most
transgenic plants did not show any effect, but a few had
decreased levels of menthofuran (35–55%) and more
menthol than wild-type plants. Surprisingly, these plants
also had a lower pulegone level.
Carotenoids
For several reasons the carotenoid biosynthetic pathway is
a very interesting target for genetic engineering. The
carotenoids are important colour compounds in flowers,
food and fruits, they are also antioxidants, and last, but not
least, vitamin A is formed from β-carotene [42]. Vitamin A
deficiency is widespread. The introduction of β-carotene
biosynthesis into the major staple food rice, by over-
expression of phytoene synthase, phytoene desaturase and
lycopene β-cyclase, is thus an important achievement
[43••]. β-Carotene (provitamin A) levels of 2 mg/kg dry rice
endosperm were achieved (see the article by Zimmermann
and Hurrell pp 142–145). In contrast to rice, where over-
expression of a plant phytoene synthase (originating from
narcissus) only results in the production of phytoene, in
canola seed specific overexpression of the bacterial gene
results in a 50-fold increase of β-carotene levels in seeds
[44]. In tomato, β-carotene content was increased up to
threefold by overexpression of a bacterial phytoene
desaturase in tomato plastids. However, the total carotenoid
content, including the direct product of the enzyme
lycopene, was decreased [45]. Several of the carotenoid
enzymes were upregulated. The decrease of total
carotenoid content is probably due to feedback inhibition
somewhere in the pathway. The expression of a bacterial
phytoene synthase in tomato fruits resulted in a twofold to
fourfold increase of total carotenoids. Levels of other
plastidial isoprenoids were not affected, neither were the
activities of various enzymes in the pathway [46•].
Overexpression of the lycopene β-cyclase gene (βLcy)
using a specific promoter increased levels of the direct
product of the enzyme, β-carotene, in tomatoes sevenfold
[47]. By introduction of an algal gene encoding β-carotene
ketolase into tobacco, astaxanthin production was achieved
in chromoplasts, mainly in the nectaries. The total
carotenoid level was increased in the flowers of the
transgenic plants [48••].
From these results it is obvious that the carotenoid pathway
is amenable to genetic engineering, and thus the nutritional
value of various food plants might be improved.
Interestingly, from the engineering of different steps in
carotenoid biosynthesis it becomes clear that changing later
steps in the pathway results in a larger flux into the total
carotenoid pool. This might be due to the fact that the
normal products are channelled away into new products,
and are therefore no longer able to cause feedback inhibition.
Vitamin E levels have also been genetically modified.
Levels were increased 10-fold in Arabidopsis seed oil by
overexpression of γ-tocopherol methyltransferase [49].
Benzoic acid derivatives
Salicylic acid (SA) is an important signalling molecule in
plants involved in systemic acquired resistance after
challenge with plant pathogens. Although the evidence is
incomplete, it has long been thought that this compound was
formed via phenylalanine. More recently, it was discovered
that SA produced in response to pathogen infection is formed
from chorismate via conversion to isochorismate by isochoris-
mate synthase (ICS) [50]. Microorganisms produce SA from
chorismate via conversion to isochorismate by ICS followed
by the splitting off of the pyruvate group by isochorismate
pyruvate lyase (IPL). Plants overexpressing the bacterial
enzymes ICS and IPL in the plastids showed a normal pheno-
type, but an increased resistance against viral and fungal
infections [51••]. The SA level was increased 1000-fold com-
pared with wild-type plants, but growth was not affected. It
seems that the second enzyme, IPL, is the rate-limiting step
for SA formation. A fused protein of two bacterial enzymes
has been constructed and was introduced into Arabidopsis
[52]. The plants with the fused enzyme in the cytosol showed
a two- to threefold increase in SA levels, whereas after expres-
sion in plastids a 20-fold increase was observed. The plants
clearly showed reduced growth, which might have been due
to depletion of isochorismate for phylloquinone production.
In the transgenic tobacco IPL activity was limiting for SA pro-
duction [51,53], therefore, the overexpressed ICS produced
sufficient isochorismate for both SA and phylloquinone
biosynthesis. This example shows that engineering of two or
more steps in a pathway requires a correct balance between
the activity of the overexpressed enzymes, to avoid affecting
negatively the precursor availability for other pathways.
Similar to SA, 4-hydroxybenzoic acid (4HB) can also be
formed via phenylalanine or directly from chorismate. Heide
and co-workers [54•] introduced the microbial ubiC gene
encoding chorismate pyruvate lyase into Lithospermum
erythrorhizon hairy root cultures, which normally produce the
naphtoquinone shikonin via the phenylalanine pathway. The
chorismate-derived 4HB contributed to 20% of the overall
4HB production in the hairy roots. No overproduction of
naphtoquinones was observed, although 4HB from both
 Engineering secondary metabolite production in plants Verpoorte and Memelink
pathways was incorporated. Instead, the levels of menisdaurin,
a nitrile glucoside, were increased fivefold. This is yet another
example to demonstrate that increasing the level of an inter-
mediate may lead to the production of unexpected products.
Cyanogenic glucosides
An example of the expression of a complete secondary
metabolite biosynthesis pathway in a heterologous plant
species is provided by the expression of cyanogenic glucoside
biosynthesis genes from Sorghum bicolor in Arabidopsis [55••].
Sorghum contains the cyanogenic glucoside dhurrin, which is
hydrolysed by a β-glucosidase upon tissue damage. The
resulting cyanide release is an effective pest deterrent and
insecticide. Dhurrin is synthesised from tyrosine via the action
of two multifunctional cytochrome P450 enzymes (CYPs) and
a specific UDPG-glucosyltransferase. Overexpression of the
first enzyme (CYP79A1) of the pathway in Arabidopsis led to
the formation of p-hydroxybenzylglucosinolates, which are
not native to this plant species [56,57]. Arabidopsis plants over-
expressing both Sorghum CYP genes produced various
glucosides of p-hydroxybenzoic acid, formed after decomposi-
tion of the nitrile, but no dhurrin. Apparently, none of the
many glucosyltransferases occurring in Arabidopsis was able
to glucosylate p-hydroxymandelonitrile to form dhurrin. The
overexpression of the specific Sorghum glucosyltransferase in
combination with the two CYP genes resulted in dhurrin
production in Arabidopsis. The dhurrin-producing transgenic
Arabidopsis released high levels of cyanide upon tissue dam-
age, indicating that dhurrin is hydrolysed by an endogenous
β-glucosidase. Leaf tissue from the transgenic Arabidopsis
plants was rejected by larvae of the flea beetle Phyllotreta
nemorum, and larvae feeding on transgenic leaves died. High
levels of a foreign metabolite were thus produced in a plant
species without negative effects on growth and with positive
effects on resistance against pests. The results show that an
entire biosynthetic pathway can be transferred to a hetero-
logous plant species, and suggest that it may be possible to
transfer other pathways in the future.
Conclusions
In the past few years, several secondary metabolism genes
have been overexpressed in the original plant or in other
plant species. In some cases, overexpression resulted in an
improved production of the desired products, whereas in
other cases only an increase in the level of the direct product
of the overexpressed enzyme was achieved. However, from
these results no general conclusions can be drawn about the
chance that a certain approach will be successful. This is
partly because the differences in transgene expression levels
in individual transformants are not well understood, and
partly because of differences observed between different
plant species. In several cases, overexpression results in the
production of unexpected products, demonstrating the com-
plexity of the metabolic networks and our lack of knowledge
of these networks and their regulation.
The picture that evolves from the studies on biosynthetic
pathways and metabolic engineering is that once the plant cell
185
factory has been assembled, based on the genetic information
present in the cell and on its environment, the cells can
produce all kind of compounds, without the need for further
genetic regulation. The fluxes through the pathways are
controlled to a great extent at the level of enzymes and intra-
cellular and intercellular transport. Fluxes through metabolic
pathways are thus not only determined by gene expression
levels, but also by post-translational regulation of enzyme
activity and enzyme and metabolite compartmentation and
transport. The major challenge for the coming years is to
obtain more information about regulation at all these
levels — genes, enzymes, compartmentation, transport and
accumulation. This will open the way for successful strategies
for altering the accumulation of certain compounds. Our
challenge will be to integrate proteomics and metabolomics
with the genetic information in order to map and subsequently
better understand metabolic networks. A thorough characteri-
sation of the enzymes will also be of great value. The intricate
and highly complex metabolic networks present in cells
involve numerous enzymes. These enzymes can, for example,
be either highly specific or non-specific, might have positive
and negative feedback controls, and may require activation or
cofactors. Detailed knowledge at the level of the enzymes will
help us to develop models simulating the fluxes through
the metabolic networks. These models will be important to
identify targets for metabolic engineering [46•,58].
Presently, a major constraint in engineering plant secondary
metabolite production is that only a few genes of these path-
ways are known. As per definition, plant secondary metabolism
is species-specific and the knowledge of the sequence of the
Arabidopsis genome is of limited value for cloning genes of sec-
ondary metabolite pathways in other plants. On the other hand,
regulatory genes have been shown to be more generally applic-
able as useful tools to identify genes from secondary metabolite
pathways and for up- or downregulation of a pathway, or part of
a pathway, in different plants.
The most important conclusion is that the knowledge of
secondary metabolite pathways is very limited, and is the
major constraint for successful application of metabolic engi-
neering. Despite the little knowledge presently available
some very interesting results have already been obtained.
The examples discussed in this review show the enormous
potential for the genetic engineering of plant secondary
metabolism. Unravelling plant secondary metabolite path-
ways is the challenging way to successful applications in
fields such as molecular farming, health food, functional
food, and plant resistance.
References and recommended reading
Papers of particular interest, published within the annual period of review,
have been highlighted as:
• of special interest
•• of outstanding interest
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186 Plant biotechnology
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An interesting application to improve the level of antioxidants in a food plant.
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This paper is of general interest as transcription factors also appear to be
involved in suppressing pathways. Consequently suppression of transcrip-
tion factors can also be used as an approach to alter fluxes through
biosynthetic pathways.
19. Aharoni A, De Vos CHR, Wein M, Sun Z, Greco R, Kroon A, Mol JNM,
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27. Memelink J, Verpoorte R, Kijne JW: ORCAnisation of jasmonate-
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28. Menke FLH, Champion A, Kijne JW, Memelink J: A novel jasmonate-
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29. Van der Fits L, Memelink J: ORCA3, a jasmonate-responsive
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An interesting example of the identification of a regulatory gene in a pathway
and how one can make use of such a transcription factor for upregulating
several genes active in a secondary metabolite pathway.
30. Collu G, Unver N, Peltenburg-Looman AMG, van der Heijden R,
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31. Sato F, Hashimoto T, Haciya A, Tamura K, Choi K-B, Morishige T,
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The effect of engineering at a branch point in secondary metabolite
pathways is extensively studied, and the implications for pathway engineer-
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32. Frick S, Ounaroon A, Kutchan TM: Combinatorial biochemistry in
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33. Oksman-Caldentey KM, Arroo R: Regulation of tropane alkaloid
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34. Jouhikainen, Lindgren L, Jokelainen T, Hiltunen R, Teeri TH, Oksman-
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sion of an early terpenoid pathway enzyme, leading to an increased flux
through the pathway, and by expression of an antisense gene resulting in
suppression of menthofurane synthase activity. It also shows that with anti-
sense one can block a certain step, but that this does not necessarily result
in an increased level of the desired compound.
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38. Lange BM, Rujan T, Martin W, Croteau RB: Isoprenoid biosynthesis:
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39. Chen DH, Ye HC, Li GF: Expression of a chimeric farnesyl
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plants via Agrobacterium tumefaciens-mediated transformation.
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40. Lewinsohn E, Schalechet F, Wilkinson J, Matsui K, Tadmor Y,
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enhances natural-product-based aphid resistance. Nat Biotechnol
2001, 19:371-374.
By changing the spectrum of terpenoids in trichomes, through suppression
of a cytochrome P450 enzyme involved in the later steps of the biosynthesis,
increased resistance against aphids could be achieved in tobacco.
42. Giuliano G, Aquilani R, Dharmapuri S: Metabolic engineering of
plant carotenoids. Trends Plant Sci 2000, 5:406-409.
43. Ye X, Al-Babili S, Kloti A, Zhang J, Lucca P, Beyer P, Potrykus I:
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into (carotenoid-free) rice endosperm. Science 2000,
287:303-305.
This paper is an excellent example of the enormous potential that metabolic
engineering of plant secondary metabolism has for improving the nutritional
value of food.
44. Shewmaker CK, Sheehy JA, Daley M, Colburn S, Ke DY: Seed
specific overexpression of phytoene synthase: increase in
carotenoids and other metabolic effects. Plant J 1999,
20:401-412.
45. Roemer S, Fraser PD, Kiano JW, Shiptn CA, Misawa N, Schuch W,
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tomato plants. Nat Biotechnol 2000, 18:666-669.
46. Fraser PD, Romer S, Shipton CA, Mills PB, Kiano JW, Misawa N,
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plants expressing an additional phytoene synthase in a fruit-
specific manner. Proc Natl Acad Sci USA 2002, 99:1092-1097.
An analysis of the effect of the overexpression of a gene of the pathway on
the overall flux through the affected isoprenoid pathways.
47. Rosati C, Aquilani R, Dharmapuri S, Pallara P, Marusic C, Tavazza R,
Bouvier F, Camara B, Giuliano G: Metabolic engineering of
β-carotene and lycopene content in tomato fruit. Plant J 2000,
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187
48. Mann V, Harker M, Pecker I, Hirschberg J: Metabolic engineering of
•• astaxanthin production in tobacco flowers. Nat Biotechnol 2000,
16:888-892.
This paper demonstrates that the carotenoid pathway in plants can be
modified to make new compounds, for example, to introduce new colours in
flowers or food.
49. Shintani D, DellaPenna D: Elevating the vitamin E content of plants
through metabolic engineering. Science 1998, 282:2098-2100.
50. Wildermuth MC, Dewdney J, Wu G, Ausubel FM: Isochorismate
synthase is required to synthesize salicylic acid for plant defence.
Nature 2001, 414:562-565.
51. Verberne M, Verpoorte R, Bol J, Mercado-Blanco J, Linthorst HJM:
•• Overproduction of salicylic acid in plants by bacterial transgenes
enhances pathogen resistance. Nat Biotechnol 2000, 18:779-783.
This study demonstrates the feasibility of using microbial genes to overpro-
duce a signal compound in a plant, thus improving resistance against viral
and fungal infection.
52. Mauch F, Mauch-Mani B, Gaille C, Kull B, Haas D, Reimmann C:
Manipulation of salicylate content in Arabidopsis thaliana by the
expression of an engineered bacterial salicylate synthase. Plant J
2001, 25:67-77.
53. Nugroho LH, Verberne MC, Verpoorte R: Salicylate produced by
isochorismate synthase and isochorismate pyruvate lyase in
various parts of constitutive salicylic acid producing transgenic
tobacco plants. Plant Sci 2001, 161:911-915.
54. Sommer S, Köhle A, Yazaki K, Shimomura K, Bechthold A, Heide L:
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of Lithospermum erythrorhizon transformed with the bacterial
ubiC gene. Plant Mol Biol 1999, 39:683-693.
Another example of introducing a microbial pathway in plant cells, in this
case to create a new pathway different from the normal plant pathway,
leading to a precursor needed for naphtoquinone biosynthesis.
55. Tattersall DB, Bak S, Jones PR, Olsen CE, Nielsen JK, Hansen ML,
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cyanogenic glucoside synthesis. Science 2001, 293:1826-1828.
A good example of the potential for engineering plant secondary metabolism
to increase resistance of plants against herbivores, by transferring a
secondary metabolite pathway from one plant to another.
56. Bak S, Olsen CE, Petersen BL, Lindberg Moeller B, Halkier BA:
Metabolic engineering of p-hydroxybenzylglucosinolate in
Arabidopsis by expression of the cyanogenic CYP79A1 from
Sorhghum bicolor. Plant J 1999, 20:663-671.
57. Petersen BL, Andreasson E, Bak S, Agerbirk N, Halkier BA:
Characterization of transgenic Arabidopsis thaliana with
metabolically engineered high levels of
p-hydroxybenzylglucosinolate. Planta 2001, 212:612-618.
58. Wiechert W: Modeling and simulation: tools for metabolic
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