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Progress in plant metabolic engineering
Teresa Capell and Paul Christou
Over the past few years, there has been a growing realization
that metabolic pathways must be studied in the context of the
whole cell rather than at the single pathway level, and that even
the simplest modifications can send ripples throughout the
entire system. Attention has therefore shifted away from
reductionist, single-gene engineering strategies and towards
more complex approaches involving the simultaneous
overexpression and/or suppression of multiple genes. The use
of regulatory factors to control the abundance or activity of
several enzymes is also becoming more widespread. In
combination with emerging methods to model metabolic
pathways, this should facilitate the enhanced production of
natural products and the synthesis of novel materials in a
predictable and useful manner.
Addresses
Fraunhofer Institute for Molecular Biology and Applied Ecology (IME),
Grafschaft, Auf dem Aberg 1, 57392 Schmallenberg, Germany
e-mail: [email protected]
Current Opinion in Biotechnology 2004, 15:148–154
This review comes from a themed issue on
Plant biotechnology
Edited by Takuji Sasaki and Paul Christou
0958-1669/$ – see front matter
ß 2004 Elsevier Ltd. All rights reserved.
DOI 10.1016/j.copbio.2004.01.009
Abbreviations
PHA
polyhydroxyalkanoate
RNAi RNA interference
Introduction
Metabolic engineering in plants involves the modification of endogenous pathways to increase flux towards
particular desirable molecules. In some cases the aim is
to enhance the production of a natural product, whereas
in others it is to synthesize a novel compound or macromolecule. Over the past few years, significant advances
have been made in metabolic engineering through the
application of genomics and proteomics technologies to
elucidate and characterize metabolic pathways in a holistic manner, rather than on a step-by-step basis [1]. This
has been supported by direct studies at the level of the
metabolome [2]. Further work has been carried out on
the modeling of metabolic pathways on a genomic scale
[3]. Such studies have shown that metabolic pathways
are controlled at multiple levels and any form of perturbation can have wide-ranging effects at the wholesystem level.
Current Opinion in Biotechnology 2004, 15:148–154
As a result, it has been realized that the manipulation of
single genes is of only limited value in metabolic engineering, and attention has shifted towards more complex
and sophisticated strategies in which several steps in a
given pathway are modified simultaneously to achieve
optimal flux. In this review, we look at recent examples of
single and multiple gene metabolic engineering in primary and secondary metabolism that demonstrate how
this new knowledge of metabolic systems is being applied
in plants.
Strategies for metabolic engineering in
plants
There are three basic goals of metabolic engineering in
plants [4]: the production of more of a specific desired
compound, the production of less of a specific unwanted
compound, and the production of a novel compound (i.e.
a molecule that is produced in nature, but not usually
in the host plant, or a completely novel compound).
Strategies for achieving these goals include the engineering of single steps in a pathway to increase or decrease
metabolic flux to target compounds, to block competitive
pathways or to introduce short cuts that divert metabolic
flux in a particular way (Figure 1a). However, this
approach has only limited value, because the effects of
modulating single enzymatic steps are often absorbed by
the system in an attempt to restore homeostasis. Targeting multiple steps in the same pathway could help to
control metabolic flux in a more predictable manner.
This might involve upregulating several consecutive
enzymes in a pathway, upregulating enzymes in one
pathway while suppressing those in another competing
pathway, or using regulatory genes to establish multipoint control over one or more pathways in the cell
(Figure 1b). Examples are provided below to show
how such strategies are applied to manipulate the production of different classes of compound.
Engineering primary metabolic pathways
Carbohydrate metabolism
Carbohydrate metabolism in plants centers on the reversible conversion of sugars, the direct products of photosynthesis, into storage and structural carbohydrates such
as starch and cellulose. Starch is a storage carbohydrate
that accumulates transiently in leaves and stably in seeds,
tubers and roots. It is a staple source of dietary carbohydrate for animals and also has a large number of industrial
uses. Metabolic engineering has been used in an attempt
to increase starch yields and to modify the properties of
starch by changing the relative proportions of its two
components, amylose and amylopectin. There has also
been some interest in producing novel starches using
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Progress in plant metabolic engineering Capell and Christou 149
Figure 1
(a)
Over-expression
Feedback insensitive
Over-expression
Feedback insensitive Higher substrate affinity
Unidirectional
Import
e5
4
3
e2
e1
1
2
e4
e3
Import shunt
5
Antisense/RNAi
Knockout
Dominant negative
(b)
Transcription factor
(coordinate upregulation
of multiple steps)
TF
RNAi against
competing
pathway
T
Import of
heterologous
enzyme
Polycistronic antisense RNA
(coordinate downregulation
of multiple steps)
Current Opinion in Biotechnology
Approaches to plant metabolic engineering. (a) Strategies for single-step metabolic engineering. To increase production of a desired compound (3)
or a novel compound (4) genes encoding endogenous enzymes in the biosynthetic pathway can be over-expressed (e1, e2) and novel enzymes
can be imported (e5). Further increases in flux can be achieved by using feedback-insensitive enzymes or those modified to favor unidirectional
catalysis in reversible reactions (e1). At branch points, over-expression of key enzymes or their modification to out-compete other enzymes using the
same substrate (e2) can divert flux into appropriate pathways, while competing enzymes can also be inhibited directly (e3). If possible, competing
pathways can be short-circuited by the import of enzyme shunts (e4) that redirect flux in the appropriate direction. Solid arrows represent endogenous
metabolism and broken arrows represent imported (heterologous) metabolism. (b) Instead of modulating individual steps, the future of metabolic
engineering will probably involve holistic approaches in which multiple steps are targeted simultaneously. In this complex pathway to a desired
compound T, a transcription factor is being used to coordinately upregulate several enzymes, a polycistronic antisense RNA is being used to
suppress several enzymes in a competing pathway, a novel enzyme is being expressed to extend the endogenous pathway beyond its normal
end point, and other competing pathways are being suppressed by RNAi. Solid arrows represent endogenous metabolism and the broken arrow
represents imported (heterologous) metabolism.
bacterial enzymes to change the nature and frequency of
branching [5]. Recent examples of metabolic engineering
to increase the quantity of starch include overexpressing
the potato sucrose transporter in transgenic pea seeds to
accelerate sugar uptake [6] and boosting the adenylate
pool in potato tubers, resulting in a 60% increase in the
level of starch in combination with an increased tuber
yield [7]. Smidansky and colleagues [8,9] have shown
that wheat and rice plants transformed with a modified
form of the maize gene for ADP-glucose pyrophosphorylase (shrunken2) utilize their spare capacity for starch
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biosynthesis and produce 40% and 20% more seed
biomass, respectively, than wild-type controls.
Many of the enzymes that control starch synthesis and
branching have been modulated in potatoes to produce
high-amylose starch (e.g. for improved frying or for
industrial use as gelling agents and thickeners) and
high-amylopectin starch (e.g. for improved freeze thaw
characteristics or for improved paper quality or adhesive
manufacture). More recently, similar experiments have
been carried out in other starch-producing crops. For
Current Opinion in Biotechnology 2004, 15:148–154
150 Plant biotechnology
example, a low-amylose rice grain has been produced by
expressing an antisense Waxy gene [10] and rice grains
with modified amylopectin have been produced by antisense inhibition of the gene encoding isoamylase [11].
Several novel strategies for starch metabolic engineering
have been explored, including the use of starch-binding
domains to target recombinant enzymes to starch granules
during starch biosynthesis [12].
content of Arabidopsis seeds can be increased either
by expressing a bacterial, feedback-insensitive dihydrodipicolinate synthase (DHPS) transgene or by knocking
out the lysine catabolism pathway, resulting in twelvefold or fivefold gains in lysine, respectively. However,
where both the transgene and knockout are combined in
the same plant line, an 80-fold increase over wild-type
levels can be achieved [20].
The cellulose biosynthetic pathway is also a useful target
for metabolic engineering, because cellulose is valuable
as pulp, fiber and as a starting point for the synthesis of
commercially important polymers. Although the cellulose
biosynthesis pathway is not fully understood, several
enzymes required for cellulose synthesis have been identified, including sucrose synthase, the CesA family of
cellulose synthases, and Korrigan cellulases. The inhibition of sucrose synthase in cotton has been shown recently
to inhibit fiber formation [13], whereas antisense and
dominant negative experiments involving CesA genes
in Arabidopsis have also demonstrated roles for several
of the CesA proteins in primary and secondary cell-wall
formation [14,15].
Polyamines are small aliphatic amines that are derived
from the amino acids ornithine and arginine by decarboxylation. They have several important roles in plant
physiology and development. The three major polyamines found in plants are putrescine, spermidine and
spermine. Recent analysis of the polyamine biosynthesis
pathway has focused on tobacco and rice, with many
enzymatic steps being studied by overexpression or
antisense suppression. Rice plants expressing S-adenosylmethionine decarboxylase (SAMDC) from carrot
showed increased levels of the higher polyamines (spermine and spermidine) in seeds, while only spermine
levels were affected in leaves [21]. When a yeast SAMDC
gene was expressed in tomato, higher levels of spermine
and spermidine were detected in fruits, as well as higher
levels of lycopene, increasing the nutrient value of the
tomato juice [22]. Inhibition of the arginine decarboxylase (ADC) gene in rice using an oat antisense ADC
construct reduces the levels of putrescine and spermine
in transgenic plants, but does not effect downstream
genes in the pathway [23].
The fructans are another group of storage carbohydrates
that are important targets for metabolic engineering, as
they have a similar texture to fats and can substitute for
them in many foods, yet contain far fewer calories. At the
present time the only commercially available fructan is
inulin from chicory, but bacterial and plant enzymes for
fructan biosynthesis have been introduced into several
crops to increase fructan levels to levels suitable for
extraction [16]. Another use envisaged for fructans is
the protection of plants from abiotic stress. This application is shared with sugars such as trehalose, as well as
other small-molecule compounds like glycine betaine,
mannitol and sorbitol, all of which have been subject
to metabolic engineering to produce improved plant
varieties [17].
Amino acid and polyamine metabolism
Amino acid synthesis pathways have been targets for
metabolic engineering predominantly to increase the
abundance of essential amino acids (lysine, threonine,
methionine and tryptophan) in food and feed crops [18].
Other amino acids, such as proline, are important in
plant stress responses and their modulation provides a
way to engineer stress tolerance in transgenic plants
[17]. Recent examples of amino acid pathway engineering demonstrate the impact of RNA interference (RNAi)
on metabolic engineering and the value of multiple
point intervention. A new version of the maize opaque2
mutant has been generated by RNAi, using a construct
targeting the 22 kDa zein genes [19]. Like the original
mutant, the new version of opaque2 has an increased
lysine content, but unlike the original it behaves in a
dominant rather than recessive manner. The lysine
Current Opinion in Biotechnology 2004, 15:148–154
Lipid metabolism
The modification of lipid metabolism to change the
quantity and quality of fatty acids in plants has important
applications in the food industry, as well as in the production of detergents, fuels, lubricants, paints and plastics
[24,25]. The metabolic engineering of seed oils is attractive, because even extensive modifications do not affect
the growth and development of the vegetative parts of the
plant [26]. Recent notable studies include experiments in
which oilseed rape (Brassica napus) was transformed with
multiple cDNAs encoding enzymes for the desaturation
and functional modification of long-chain fatty acids. Liu
et al. [27] expressed 18:1 D12 desaturase either alone or in
combination with 18:2 D6 desaturase. With the 18:1 D12
desaturase alone, the seeds produced up to 46% linoleic
acid, whereas with both enzymes the seeds produced up
to 43% g-linolenic acid. Han et al. [28] expressed a
combination of b-ketoacyl-CoA synthase and 22:1 acylCoA:lysophosphatidic acid acyltransferase in oilseed rape,
yielding 60% erucic acid. More recently, the a-linolenic
acid content of rice grains has been increased tenfold
by expressing the soybean FAD3 gene encoding O3
fatty acid desaturase [29]. Cahoon et al. [30], expressing
a cytochrome P450 isolated from seeds of Euphorbia
lagascae, have produced up to 7% epoxy-fatty acids
without inhibiting oleate desaturation.
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Progress in plant metabolic engineering Capell and Christou 151
Engineering secondary metabolic pathways
Alkaloids
consecutive enzymatic steps catalyzed by e-cyclase and
b-cyclase [37].
The alkaloids are perhaps the largest group of secondary
products synthesized by plants and the pathways,
enzymes and regulatory genes involved in their synthesis
have been extensively reviewed [31]. Several distinct
types of alkaloids can be distinguished, including the
isoquinoline, indole and tropane classes. Park et al. [32]
have recently demonstrated antisense suppression of the
berberine bridge enzyme in poppy cells, reducing the
amount of benzophenanthridine alkaloids but increasing
the levels of several amino acids. There have been many
attempts to modulate the indole alkaloid biosynthetic
pathway, which produces important pharmaceutical compounds such as the anticancer drugs vinblastine and
vincristine [33].
The principal example of carotenoid metabolic engineering in plants is of course the synthesis of b-carotene in rice
endosperm (Golden Rice), which normally accumulates
GGPP but lacks the subsequent enzymes in the pathway.
Since publication of the original reports, further ‘Golden
Rice’ lines have been generated containing only two
foreign enzymes (the daffodil phytoene synthase and a
bacterial phytoene desaturase [38]). Hoa et al. [39]
reported the production of indica Golden Rice varieties
that are amenable to deregulation and also assure freedom
to operate in developing countries due to the removal of
contraints brought about by intellectual property and
regulatory issues.
Terpenoids
Terpenoids (isoprenoids) are structurally diverse polymers constructed from the basic building block isoprene. They are classified according to the number of
isoprene units they contain, which increase by fives.
Thus, hemiterpenes contain five isoprene units, monoterpenes contain 10, sesquiterpenes contain 15 and
diterpenes contain 20. Carotenoids, another important
group of plant secondary metabolites, are terpenoids
with 40 isoprene units. The largest polymers contain
thousands of units and include natural products such as
rubber. Despite their diversity, all terpenoids are
synthesized from the common precursors dimethylallyl
pyrophosphate and isopentyl pyrophosphate. This
occurs through two distinct pathways, the mevalonate-independent pathway and the mevalonate pathway, both of which have been targets for metabolic
engineering [34].
Thus far, there have been few examples of monoterpenoid metabolic engineering, but see Update. In one
study, petunia was transformed with the (S)-limonene
synthase gene from Clarkia breweri resulting in the production of linalool, which repels aphids [35]. In another,
the same gene was introduced into tomato, under the
control of a fruit-specific promoter, resulting in the accumulation of linalool in tomato fruits [36].
Much more attention has been paid to the carotenoid
metabolic pathway, which produces pigments with roles
in light harvesting and photoprotection, and which form
vital components of human and animal diets. In plants,
the first step in carotenoid biosynthesis is the coupling of
two geranyl phosphate molecules to yield geranylgeranyl
pyrophosphate (GGPP). This is converted into phytoene
by the enzyme phytoene synthase and then to z-carotene
by the enzyme phytoene desaturase. Further desaturation
results in the formation of lycopene, which is converted
either into b-carotene (provitamin A) by the enzyme
b-cyclase, or into d-carotene and then a-carotene by
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Transgenic tomatoes have also been described expressing
phytoene desaturase or phytoene synthase [40]. In the
former case, there was no overall increase in carotenoid
content, although b-carotene levels were three times
higher than normal; in the latter case, the total carotenoid
content was up to fourfold normal levels [40].
Flavonoids
The flavonoids are phenolic compounds derived ultimately from phenylalanine, and thus share a common
metabolic source with lignins and lignans (see below).
The first committed step in flavonoid biosynthesis is the
conversion of the precursor 4-coumaroyl-CoA into chalcone by the enzyme chalcone synthase. Chalcone is then
derivatized in a series of enzymatic steps to produce a
variety of molecules that act as pigments, defense chemicals (phytoalexins) and regulatory molecules [41].
Flavonoids have been a favorite target for metabolic
engineering since the early 1990s, because the result of
different modifications can be determined through alterations in flower color [42]. As for other pathways, the
number of experiments involving multiple gene transfer
strategies is beginning to increase. A recent example is
the stacking of dihydroflavonol 4-reductase and anthocyanidin synthase transgenes in forsythia by sequential
transformation [43]. Increasing the levels of flavonoids in
food plants can provide health benefits, as these molecules often have antioxidant activity. This has been
demonstrated in the case of tomatoes by overexpressing
the petunia gene for chalcone isomerase, leading to an
80-fold increase in the flavonoid content of the tomato
peel and a corresponding 20-fold increase in the flavonoid
level in tomato paste [44]. In addition, chalcone synthase
and flavonol synthase transgenes were found to act synergistically to upregulate flavonol biosynthesis significantly
in the flesh of tomato fruits [45].
Lignins
Lignins, like flavonoids, are derived from phenylalanine
and can be classed as phenylpropanoid compounds. They
Current Opinion in Biotechnology 2004, 15:148–154
152 Plant biotechnology
are complex polymers composed of caffeic, ferulic and
sinapic acid monomers joined together and to the cell
wall. The metabolic engineering of lignins has been
reviewed comprehensively very recently [46–48] and
we will only mention here a few notable examples that
demonstrate contemporary metabolic engineering approaches. One interesting example is the use of the
LIM regulatory protein from tobacco to control several
of the metabolic enzymes involved in the lignification
pathway coordinately [49]. Similarly, two Myb-like transcription factors have been isolated from Eucalyptus gunni,
which are expressed in the xylem and recognize sites in
the hydroxycinnamoyl-CoA reductase (CCR) and cinnamyl alcohol dehydrogenase (CAD) genes which encode
sequential enzymes in the synthesis of p-coumaryl alcohol
[48]. Recent reports have also demonstrated the feasibility of multiple gene transfer to modify several lignin
characteristics simultaneously in trees [50] and the
simultaneous suppression of multiple lignin biosynthetic
genes in tobacco [51,52].
Quinones and other benzoic acid derivatives
Quinones are cyclic compounds derived from benzoic
acid and often act as electron transporters in plants.
Thus far, three major metabolic pathways that begin
with benzoic acid have been modified in plants — those
leading to salicylic acid, shikonin and a-tocopherol
(vitamin E) production. Vitamin E synthesis in plants
has obvious implications for the nutritional value of
foods and recent work has aimed to increase the levels
of this molecule in the model plant Arabidopsis thaliana.
For example, Savidge et al. [53] have overexpressed
A. thaliana homogentisate prenyltransferase, producing
twice the level of vitamin E found in normal seeds.
A very recent study [54] described the expression
of the A. thaliana VTE3 and VTE4 genes, encoding
2-methyl-6-phytylbenzoquinol methyltransferase and
g-tocopherol methyltransferase, in transgenic soybean.
The transgenic seeds showed a greater than eightfold
increase in a-tocopherol and a fivefold increase in
vitamin E levels. Other strategies for increasing vitamin
E levels in transgenic plants are discussed in a recent
review [55].
Engineering novel metabolic pathways
Although metabolic engineering is often used to modify
or extend existing pathways in the host plant, there are
some examples where multigene engineering has been
used to introduce completely novel pathways and thus to
produce completely alien products. In one such experiment two multifunctional cytochrome P450 enzymes
and a uridine diphosphate glucose (UDPG)-glucosyltransferase were transferred from sorghum (Sorghum
bicolor) into A. thaliana, resulting in the production of
the cyanogenic glucoside dhurrin (which confers resistance to the flea beetle Phyllotreta nemorum) [56].
These investigators also generated transgenic plants
Current Opinion in Biotechnology 2004, 15:148–154
carrying one or both of the cytochrome P450 enzymes,
but lacking the UDPG-glucosyltransferase, and demonstrated the accumulation of appropriate metabolic
intermediates.
Another example of this general approach is the production of polyhydroxyalkanoate (PHA) polymers in transgenic plants [57]. PHAs are polyesters of hydroxyacids
synthesized by a range of bacteria, which can be used as
biodegradable plastics. The best-studied PHA, poly(3hydroxybutyrate) (PHB), can be made to accumulate in
the plastids of transgenic A. thaliana plants at levels
exceeding 40% dry shoot weight. This is achieved
through the introduction of three bacterial genes, encoding the enzymes 3-ketothiolase, acetacetyl-CoA reductase and PHA synthase [58]. PHB is a brittle plastic with
few potential uses. The copolymer poly(3-hydroxybutyrate-co-3-hydroxyvalerate) is much more useful, and is
marketed under the trade name Biopol. This has been
produced in A. thaliana and rapeseed through the coordinated expression of four bacterial genes: the three mentioned above plus Escherichia coli threonine deaminase
[59]. Although high-level accumulation of these plastics is
generally detrimental to plant growth (by draining the
pools of acetyl- and propionyl-CoA), both plastics have
recently been produced at relatively high levels in alfalfa
with no adverse effects [60].
Conclusions
Multipoint metabolic engineering is now beginning
to supersede single-point engineering as the best way
to manipulate metabolic flux in transgenic plants. As
discussed above, several points in a given metabolic
pathway can be controlled simultaneously either by
overexpressing and/or suppressing several enzymes or
through the use of transcriptional regulators to control
several endogenous genes. Applied genomics, proteomics and metabolomics are continuing to expand our
knowledge of metabolic pathways, while advances in
systems biology help us to model the impact of different
modifications more accurately. We are fast approaching a
time when sophisticated strategies for the multipoint
manipulation of metabolic pathways can be modeled
and implemented by multiple gene transfer, therefore
facilitating the production of desirable molecules in
transgenic plants.
Update
Since this article was written, Lucker et al. [61] have
reported the transformation of tobacco plants with three
different monoterpene synthases from lemon. When the
three transgenes were stacked in one transgenic line, the
tobacco plants produced more terpenoids and displayed a
different terpenoid profile to wild-type plants. Incidentally, this is also the first report of transgenic plants
expressing multiple foreign enzymes competing for the
same substrate.
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Progress in plant metabolic engineering Capell and Christou 153
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
1.
Jacobs DI, van der Heijden R, Verpoorte R: Proteomics in
plant biotechnology and secondary metabolism research.
Phytochem Anal 2000, 11:277-287.
2.
Sumner LW, Mended P, Dixon RA: Plant metabolomics:
large-scale phytochemistry in the functional genomics era.
Phytochemistry 2003, 62:817-836.
3.
Papin JA, Price ND, Wiback SJ, Fell DA, Palsson BO: Metabolic
pathways in the post-genome era. Trends Biochem Sci 2003,
28:250-258.
4.
Verpoorte R, Van der Heijden R, Memelink J: Engineering the
plant cell factory for secondary metabolite production.
Transgenic Res 2000, 9:323-343.
5.
Kok-Jacon GA, Ji Q, Vincken J-P, Visser RGF: Towards a more
versatile a-glucan synthesis in plants. J Plant Physiol 2003,
160:765-777.
6.
Rosche E, Blackmore D, Tegeder M, Richardson T, Schroeder H,
Higgins TJV, Frommer WB, Offler CE, Patrick JW: Seed-specific
overexpression of the potato sucrose transporter increases
sucrose uptake and growth rates of developing pea
cotyledons. Plant J 2002, 30:165-175.
7.
Regierer B, Fernie AR, Springer F, Perez-Melis A, Leisse A,
Koehl K, Willmitzer L, Geigenberger P, Kossmann J: Starch
content and yield increase as a result of altering
adenylate pools in transgenic plants. Nat Biotechnol 2002,
20:1256-1260.
The authors demonstrate that by increasing the activity of plastidial
adenylate kinase in transgenic potato plants, a larger pool of adenylates
becomes available that can be used to fuel several metabolic pathways.
Not only was there an increase in starch levels, but there was also a twoto fourfold elevation in the levels of several amino acids.
8.
Smidansky ED, Clancy M, Meyer FD, Lanning SP, Blake NK,
Talbert LE, Giroux MJ: Enhanced ADP-glucose
pyrophosphorylase activity in wheat endosperm increases
seed yield. Proc Natl Acad Sci USA 2002, 99:1724-1729.
9.
Smidansky ED, Martin JM, Hannah LC, Fischer AM, Giroux MJ:
Seed yield and plant biomass increases in rice are conferred by
deregulation of endosperm ADP-glucose pyrophosphorylase.
Planta 2003, 216:656-664.
10. Liu QQ, Wang ZY, Chen XH, Cai XL, Tang SZ, Yu HX, Zhang JL,
Hong MM, Gu MH: Stable inheritance of the antisense Waxy
gene in transgenic rice with reduced amylose level and
improved quality. Transgenic Res 2003, 12:71-82.
11. Fujita N, Kubo A, Suh DS, Wong KS, Jane JL, Ozawa K, Takaiwa F,
Inaba Y, Nakamura Y: Antisense inhibition of isoamylase alters
the structure of amylopectin and the physicochemical
properties of starch in rice endosperm. Plant Cell Physiol 2003,
44:607-618.
12. Vincken QJJP, Suurs LCJM, Visser RGF: Microbial starch-binding
domains as a tool for targeting proteins to granules during
starch biosynthesis. Plant Mol Biol 2003, 51:789-801.
The authors describe an interesting study in which a starch-binding
domain from potato granule-bound starch synthase I was used to target
luciferase to the inside of the starch granule. This is a promising method
for directing enzymes into the starch granule for starch metabolic engineering.
13. Ruan YL, Llewellyn DJ, Furbank RT: Suppression of sucrose
synthase gene expression represses cotton fiber cell initiation,
elongation, and seed development. Plant Cell 2003, 15:952-964.
14. Zhong RQ, Morrison WH, Freshour GD, Hahn MG, Ye ZH:
Expression of a mutant form of cellulose synthase AtCesA7
causes dominant negative effect on cellulose biosynthesis.
Plant Physiol 2003, 132:786-795.
15. Burn JE, Hocart CH, Birch RJ, Cork AC, Williamson RE: Functional
analysis of the cellulose synthase genes CesA1, CesA2, and
CesA3 in Arabidopsis. Plant Physiol 2002, 129:797-807.
www.sciencedirect.com
16. Ritsema T, Smeekens S: Fructans: beneficial for plants and
humans. Curr Opin Plant Biol 2003, 6:223-230.
17. Chen THH, Murata N: Enhancement of tolerance of abiotic
stress by metabolic engineering of betaines and other
compatible solutes. Curr Opin Plant Biol 2002, 5:250-257.
18. Galili G, Hofgen R: Metabolic engineering of amino acids and
storage proteins in plants. Metab Eng 2002, 4:3-11.
19. Segal G, Song RT, Messing J: A new opaque variant of maize by
a single dominant RNA-interference-inducing transgene.
Genetics 2003, 165:387-397.
20. Zhu XH, Galili G: Increased lysine synthesis coupled with a
knockout of its catabolism synergistically boosts lysine
content and also transregulates the metabolism of other
amino acids in Arabidopsis seeds. Plant Cell 2003,
15:845-853.
This study demonstrates multipoint engineering of lysine metabolism,
combining the overexpression of a bacterial enzyme resistant to lysine
inhibition together with the knockout of the lysine catabolism pathway,
resulting in much higher lysine levels than produced by either strategy
alone.
21. Thu-Hang P, Bassie L, Safwat G, Trung-Nghia P, Christou P,
Capell T: Expression of a heterologous S-adenosylmethionine
decarboxylase cDNA in plants demonstrates that changes in
S-adenosyl-L-methionine decarboxylase activity determine
levels of the higher polyamines spermidine and spermine.
Plant Physiol 2002, 129:1744-1754.
22. Mehta RA, Cassol T, Li N, Ali N, Handa AK, Mattoo AK: Engineered
polyamine accumulation in tomato enhances phytonutrient
content, juice quality, and vine life. Nat Biotechnol 2002,
20:613-618.
A yeast gene encoding S-adenosylmethionine decarboxylase was
expressed in tomato under the control of the ripening-inducible E8
promoter to increase levels of the polyamines spermidine and spermine
specifically in tomato fruit during the ripening process. This resulted in
the enhanced production of lycopene, a longer vine life and nutritionally
improved fruit juice.
23. Trung-Nghia P, Bassie L, Safwat G, Thu-Hang P, Lepri O, Rocha P,
Christou P, Capell T: Reduction in the endogenous arginine
decarboxylase transcript levels in rice leads to depletion of the
putrescine and spermidine pools with no concomitant changes
in the expression of downstream genes in the polyamine
biosynthetic pathway. Planta 2003, 218:125-134.
24. Murphy DJ: Biotechnology and the improvement of oil crops –
genes, dreams and realities. Phytochem Rev 2002, 1:67-77.
25. Drexler H, Spiekermann P, Meyer A, Domergue F, Zank T,
Sperling P, Abbadi A, Heinz E: Metabolic engineering of fatty
acids for breeding of new oilseed crops: strategies, problems
and first results. J Plant Physiol 2003, 160:779-802.
26. Thelen JJ, Ohlrogge JB: Metabolic engineering of fatty acid
biosynthesis in plants. Metab Eng 2002, 4:12-21.
27. Liu J-W, Huang Y-S, DeMichele S, Bergana M, Bobik E Jr,
Hastilow C, Chuang L-T, Mukerji P, Knutzon D: Evaluation of the
seed oils from a canola plant genetically transformed to
produce high levels of c-linolenic acid. In g-Linolenic Acid:
Recent Advances in Biotechnology and Clinical Applications.
Edited by Hunag Y-S, Ziboh VA. AOCS Press; Champaign, Illinois;
2001:61–71.
28. Han J, Lühs W, Sonntag K, Zähringer U, Borchardt DS, Wolter FP,
Heinz E, Frentzen M: Functional characterization of b-ketoacylCoA synthase genes from Brassica napus L. Plant Mol Biol 2001,
46:229-239.
29. Anai T, Koga M, Tanaka H, Kinoshita T, Rahman SM, Takagi Y:
Improvement of rice (Oryza sativa L.) seed oil quality through
introduction of a soybean microsomal O-3 fatty acid
desaturase gene. Plant Cell Rep 2003, 21:988-992.
30. Cahoon EB, Ripp KG, Hall SE, McGonigle B: Transgenic
production of epoxy fatty acids by expression of a cytochrome
P450 enzyme from Euphorbia lagascae seed. Plant Physiol 2002,
128:615-624.
31. Hashimoto T, Yamada Y: New genes in alkaloid metabolism and
transport. Curr Opin Biotechnol 2003, 14:163-168.
Current Opinion in Biotechnology 2004, 15:148–154
154 Plant biotechnology
32. Park SU, Yu M, Facchini PJ: Antisense RNA-mediated
suppression of benzophenanthridine alkaloid biosynthesis in
transgenic cell cultures of California poppy. Plant Physiol 2002,
128:696-706.
33. Verpoorte R, Memelink J: Engineering secondary metabolite
production in plants. Curr Opin Biotechnol 2002, 13:181-187.
34. Broun P, Somerville C: Progress in plant metabolic engineering.
Proc Natl Acad Sci USA 2001, 98:8925-8927.
35. Lucker J, Bouwmeester HJ, Schwab W, Blaas J, van der Plas LH,
Verhoeven HA: Expression of Clarkia S-linalool synthase in
transgenic petunia plants results in the accumulation of
S-linalyl-b-D-glucopyranoside. Plant J 2001, 27:315-324.
36. Lewinsohn E, Schalechet F, Wilkinson J, Matsui K, Tadmor Y,
Nam KH, Amar O, Lastochkin E, Larkov O, Ravid U et al.: Enhanced
levels of the aroma and flavour compound S-linalool by
metabolic engineering of the terpenoid pathway in tomato
fruits. Plant Physiol 2001, 127:1256-1265.
37. Sandmann G: Genetic manipulation of carotenoid biosynthesis:
strategies, problems and achievements. Trends Plant Sci 2001,
6:14-17.
38. Beyer P, Al-Babili S, Ye X, Lucca P, Schaub P, Welsch R, Potrykus I:
Golden rice: introducing the b-carotene biosynthesis pathway
into rice endosperm by genetic engineering to defeat vitamin A
deficiency. J Nutr 2002, 132:506S-510S.
39. Hoa TT, Al-Babili S, Schaub P, Potrykus I, Beyer P: Golden Indica
and Japonica rice lines amenable to deregulation. Plant Physiol
2003, 133:161-169.
40. Fraser PD, Romer S, Shipton CA, Mills PB, Kiano JW, Misawa N,
Drake RG, Schuch W, Bramley PM: Evaluation of transgenic
tomato plants expressing an additional phytoene synthase
in a fruit-specific manner. Proc Natl Acad Sci USA 2002,
99:1092-1097.
Fruit-specific expression of the Erwinia uredovora crtB gene was
achieved using the tomato polygalacturonase promoter, and the recombinant protein was directed to the chromoplast using the tomato phytoene synthase-1 transit sequence. Total fruit carotenoids were found to
be two- to fourfold higher than in wild-type plants.
41. Winkel-Shirley B: Flavonoid biosynthesis. A colorful model for
genetics, biochemistry, cell biology, and biotechnology.
Plant Physiol 2001, 126:485-493.
42. Forkmann G, Martens S: Metabolic engineering and application
of flavonoids. Curr Opin Biotechnol 2001, 12:155-160.
43. Rosati C, Simoneau P, Treutter D, Poupard P, Cadot Y, Cadic A,
Duron M: Engineering of flower color in forsythia by expression
of two independently-transformed dihydroflavonol
4-reductase and anthocyanidin synthase genes of flavonoid
pathway. Mol Breed 2003, 12:197-208.
44. Muir SR, Collins GJ, Robinson S, Hughes S, Bovy A, De Vos CHR,
Van Tunen AJ, Verhoeyen ME: Overexpression of petunia
chalcone isomerase in tomato results in fruits containing
increased levels of flavonoids. Nat Biotechnol 2001, 19:470-474.
45. Verhoeyen ME, Bovy A, Collins G, Muir S, Robinson S, de Vos CHR,
Colliver S: Increasing antioxidant levels in tomatoes through
modification of the flavonoid biosynthetic pathway. J Exp Bot
2002, 53:2099-2106.
46. Anterola AM, Lewis NG: Trends in lignin modification: a
comprehensive analysis of the effects of genetic
manipulations/mutations on lignification and vascular
integrity. Phytochemistry 2002, 61:221-294.
47. Boerjan W, Ralph J, Baucher M: Lignin biosynthesis.
Annu Rev Plant Biol 2003, 54:519-546.
48. Boudet AM, Kajita S, Grima-Pettenati J, Goffner D: Lignins and
lignocellulosics: a better control of synthesis for new and
improved uses. Trends Plant Sci 2003, 8:576-581.
49. Kawaoka A, Ebinuma H: Transcriptional control of lignin
biosynthesis by tobacco LIM protein. Phytochemistry 2001,
57:1149-1157.
Current Opinion in Biotechnology 2004, 15:148–154
50. Li L, Zhou YH, Cheng XF, Sun JY, Marita JM, Ralph J, Chiang VL:
Combinatorial modification of multiple lignin traits in trees
through multigene cotransformation. Proc Natl Acad Sci USA
2003, 100:4939-4944.
In this study, the investigators transformed aspen with an antisense
construct against 4-coumarate-CoA ligase (4CL) and a sense construct
encoding coniferaldehyde 5-hydroxylase (CAld5H). Plants expressing
each gene alone, and both genes together, were recovered. The effects
of the two constructs were additive. In the dual transgenic plants, there
was half as much lignin, a 64% higher syringyl/guaiacyl ratio, and 30%
more cellulose than in wild-type controls.
51. Chabannes M, Barakate A, Lapierre C, Marita JM, Ralph J, Pean M,
Danoun S, Halpin C, Grima-Pettenati J, Boudet AM: Strong
decrease in lignin content without significant alteration of plant
development is induced by simultaneous down-regulation of
cinnamoyl CoA reductase (CCR) and cinnamyl alcohol
dehydrogenase (CAD) in tobacco plants. Plant J 2001,
28:257-270.
52. Abbott JC, Barakate A, Pincon G, Legrand M, Lapierre C, Mila I,
Schuch W, Halpin C: Simultaneous suppression of multiple
genes by single transgenes. Down-regulation of three
unrelated lignin biosynthetic genes in tobacco. Plant Physiol
2002, 128:844-853.
A new strategy is described in which three different genes are suppressed
using a single chimeric antisense construct incorporating partial sequences for all three genes. The authors show that this strategy gives
more predictable and consistent results than the use of three separate
antisense constructs.
53. Savidge B, Weiss JD, Wong YHH, Lassner MW, Mitsky TA,
Shewmaker CK, Post-Beittenmiller D, Valentin HE: Isolation and
characterization of homogentisate phytyltransferase genes
from Synechocystis sp. PCC 6803 and Arabidopsis. Plant Physiol
2002, 129:321-332.
54. Van Eenennaam AL, Lincoln K, Durrett TP, Valentin HE,
Shewmaker CK, Thorne GM, Jiang J, Basziz SR, Levering CK,
Aasen ED et al.: Engineering vitamin E content: from
Arabidopsis mutant to soy oil. Plant Cell 2003, 15:3007-3019.
This report describes the map-based cloning of an Arabidopsis gene
affecting the ratio of d- and g-tocopherol in seeds and the identification of
its biochemical function. The transformation of soybean with this gene in
combination with another gene active in the same metabolic pathway
was shown to increase the amount of a-tocopherol in soy oil from 10% to
over 90%.
55. Herbers K: Vitamin production in transgenic plants.
J Plant Physiol 2003, 160:821-829.
56. Tattersall DB, Bak S, Jones PR, Olsen CE, Nielsen JK, Hansen ML,
Hoj PB, Moller BL: Resistance to an herbivore through
engineered cyanogenic glucoside synthesis. Science 2001,
293:1826-1828.
57. Snell KD, Peoples OP: Polyhydroxyalkanoate polymers and their
production in transgenic plants. Metab Eng 2002, 4:29-40.
58. Bohmert K, Balbo I, Kopka J, Mittendorf V, Nawrath C, Poirer Y,
Tischendorf G, Trethewey RN, Willmitzer L: Transgenic
Arabidopsis plants can accumulate polyhydroxybutyrate at up
to 4% of their fresh weight. Planta 2000, 211:841-845.
59. Slater S, Mitsky TA, Hourniel KL, Hao M, Reiser SE, Taylor NB,
Tran M, Valetin HE, Rodriguez DJ, Stone DA et al.: Metabolic
engineering of Arabidopsis and Brassica for poly(3hydroxybutyrate-co-3-hydroxyvalerate) copolymer
production. Nat Biotechnol 1999, 17:1011-1016.
60. Saruul P, Srienc F, Somers DA, Samac DA: Production of a
biodegradable plastic polymer, poly-b-hydroxybutyrate, in
transgenic alfalfa. Crop Sci 2002, 42:919-927.
In this report, the expression of multiple bacterial enzymes in alfalfa results in the accumulation of polyhydroxybutyrate granules at levels up
to 1.8 g kg 1 dry weight in transgenic leaves, with apparently no adverse
effects on plant growth or development.
61. Lucker J, Schwab W, van Hautum B, Blaas J, van der Plas LHW,
Bouwmeester HJ, Verhoeven HA: Increased and altered
fragrance of tobacco plants after metabolic engineering using
three monoterpene synthases from lemon. Plant Physiol 2004,
134:510-519.
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