2. fatty acids is tolerant to changes in chemical structure and is
a good target for genetic manipulations that are unlikely to
disturb the physiology of the plant. Second, up to one-third
of plant oil is already used for nonfood applications and the
chemical industry is familiar with fatty acid chemistry and
applications. Third, as noted above, over 200 different
fatty acid structures with attractive functional properties
occur in plants. In many cases the pathways that produce
these structures have been identified (review: Voelker and
Kinney, 2001). Finally, rising costs of imported petroleum
coupled with efforts to move toward renewable resources
suggest good long-term prospects for increased use of plant
oils to provide biobased alternatives to petroleum.
Because plant oils have broad uses in both food and
nonfood applications the goals of plant oilseed biotech-nologists
are diverse. The major goals can be summarized
as:
• Increase content of ‘‘healthy’’ fatty acids and reduce
‘‘unhealthy’’ fatty acids.
• Improve oil stability to expand applications and
reduce the need for hydrogenation.
• Expand the repertoire of fatty acids available at low
cost and high volume through exploitation of genetic
diversity and enzyme engineering.
• Increase oil content to reduce production costs.
Some success has been achieved in reaching all of these
goals. In at least two cases, this has led to new commercial
crops and thus oilseed engineering has led the way toward a
new generation of agricultural products whose traits have
been enhanced through metabolic engineering. In other
cases attempts to modify plant oils have had disappointing
outcomes that reveal our ignorance of lipid biochemistry
and seed metabolism. This review discusses some recent
advances toward the goal of engineering qualitative and
quantitative fatty acid traits in plants and some of the
challenges that have emerged.
Overview of Fatty Acid and Triacylglycerol Biosynthesis
in Plants
In plants, the reactions for de novo fatty acid synthesis
(FAS)2 are located in plastids (Ohlrogge et al., 1979), which
are plant-specific organelles bound by an envelope double
membrane. Priming and elongation of nascent acyl chains
requires acetyl- and malonyl-CoA, respectively, as direct
precursors (Fig. 1A). The fatty acid synthase machinery is
2 Abbreviations used: ACP, acyl carrier protein; ACCase, acetyl-CoA
carboxylase; BC, biotin carboxylase; CHD, coronary heart disease; ER,
endoplasmic reticulum; FAS, fatty acid synthesis; KAS, 3-ketoacyl-ACP
synthase; LPAAT, lysophosphatidic acid acyltransferase; TAG, triacyl-glycerol.
Metabolic Engineering 4, 12–21 (2002)
doi:10.1006/mben.2001.0204
similar to prokaryotes in that the enzymatic components are
separable polypeptides rather than large multifunctional
polypeptides as found in animals and fungi. The series of
reactions necessary for de novo synthesis of fatty acids, up to
18 C in length, has been elucidated and is discussed in
detail elsewhere (Schultz and Ohlrogge, 2001; Voelker and
Kinney, 2001). The first desaturation step for fatty acids is
catalyzed by a plastidial stearoyl-acyl carrier protein (ACP)
desaturase. Termination of plastidial fatty acid chain
elongation is catalyzed by acyl-ACP thioesterases, which
hydrolyze acyl chains from ACP. After termination, free
fatty acids are activated to CoA esters, exported from the
plastid, and assembled into glycerolipids at the endoplasmic
reticulum (ER). In addition, further modifications (desatu-ration,
hydroxylation, elongation, etc.) occur in the ER
while acyl chains are esterified to glycerolipids or CoA
(Fig. 1B). In developing seeds, the flux of acyl chains in the
ER eventually leads to esterification on all three positions of
glycerol to form triacylglycerol (TAG). The low polarity of
TAG is believed to result in the accumulation of this lipid
between bilayer leaflets leading to the budding of storage
organelles termed oil bodies.
TAILORING OFCOMMONFATTY ACIDS
IN OILSEED CROPS
Modification of naturally occurring common fatty acids
found in oilseed crops has led to major technical achieve-ments
and a commercial product in transgenic high-oleic
soybean oil. Simply by overexpressing or suppressing single
genes it has been possible to make large compositional
changes (Table 1). Because seed-specific promoters are used
the changes have been restricted to the storage oils of seeds,
which appear to tolerate a wide range of oil physical
properties.
Current medical understanding indicates a strong impact
of dietary fatty acids on cardiovascular disease and
human health (Hu et al., 2001). Consequently, there is
much interest in tailor-producing healthier vegetable oils
and such products may help to balance consumer opposi-tion
to ‘‘GMO’’ foods. One health concern regarding
vegetable oil-derived food is the presence of trans-unsa-turated
fatty acids. Most vegetable oil used for food
applications is partially or fully hydrogenated during pro-cessing
to make the oil semisolid for spreads and also
to increase oxidative stability during storing or frying
(Kinney, 1996). Industrial hydrogenation increases satu-rated
fatty acid content and also results in production of
trans-isomers of unsaturated fatty acids that are normally
not found in vegetable oils and have been associated with
coronary heart disease (Broun et al., 1999). For many
13
Fatty Acid Biosynthesis
3. FIG. 1. Fatty acid synthesis, modification, and assembly into triacylglycerols in plants. Numbers refer to reactions that have been modified in
transgenic plants and are described in Table 1. (A) Simplified scheme of reactions of plastid fatty acid synthesis. In oilseeds, fatty acid synthesis is
terminated by acyl-ACP thioesterases (FatA and FatB classes), which release free fatty acids, allowing their export from the plastid and reesterifica-tion
to CoA at the plastid envelope. (B) Simplified scheme of reactions for modification of fatty acids in oilseeds and their assembly into triacylgly-cerols.
After activation to CoA, fatty acids formed in the plastid can be sequentially esterified directly to glycerol 3-phosphate (G-3-P) to produce
lysophosphatidic acid (LPA), phosphatidic acid (PA), diacylglycerol, and triacylglycerol. However, in most oilseeds the major flux of acyl chains
involves movement through phosphatidylcholine (PC) pools where modifications such as further desaturation and hydroxylation occur. Only in jojoba
or plants transformed with jojoba genes are wax esters formed in seeds.
food applications, vegetable oils with a reduced amount of
trans-unsaturated fatty acids are desirable to improve
human health. This has been achieved using strategies
such as cosuppression, antisense, and RNA interference to
down-regulate endogenous stearoyl-ACP desaturase genes
in soybean, cotton, and Brassica oilseeds (Table 1). In
these plants, levels of stearic were increased up to 40% to
provide a semisolid margarine feedstock without the need
for hydrogenation.
An oxidatively stable liquid oil low in saturated fatty
acids was also produced in soybean by suppression of the
oleoyl desaturase (Kinney, 1996). Oleic acid content was
increased up to 86%, 18:2 content was reduced from 55%
to less than 1%, and saturated fatty acids were reduced to
10%. This oil has been produced commercially and is
extremely stable for high-temperature frying applications.
In addition, its stability matches that of mineral oil-derived
lubricants and therefore nonfood uses as bio-degradable
lubricants are under way. An added benefit to
consumers from future use of engineered high-oleic oils in
foods may be a reduction in coronary heart disease (CHD)
associated with high omega-6 fatty acid consumption. In
recent years evidence has accumulated that the balance of
omega-3 and omega-6 unsaturated fatty acids in diets
influences the risk of CHD (Hu et al., 2001). The domi-nance
of plant oils with high omega-6 18:2 in many diets
has led to omega-6/omega-3 consumption ratios near
10:1, whereas populations that consume ratios near 1:1
(e.g., Greenland, Japan) have strikingly lower incidence of
CHD.
ENGINEERING OF UNUSUAL FATTY ACIDS
IN OILSEED CROPS
Among the approximately 200 fatty acid structures
produced by plants are several that might find wide use if
available in high quantity and at low cost. Included in this
list are hydroxy, epoxy, conjugated, and acetylenic fatty
acids, all of which result from the action of enzymes
closely related to the ubiquitous oleoyl desaturase (Broun
et al., 1998). These fatty acids have interest because they
provide a second reactive functional group to a hydro-carbon
chain and offer opportunities for polymerizations
or other chemical modifications. Therefore, considerable
interest has developed in engineering high-yielding oilseeds
to produce these or other specialty fatty acids and in a few
Metabolic Engineering 4, 12–21 (2002)
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Thelen and Ohlrogge
4. TABLE 1
Selected Examples of Fatty Acid Engineering in Transgenic Plants
Engineered reaction(s)
Metabolic Engineering 4, 12–21 (2002)
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Engineered Transgenic Max. produced Gene
fatty acid plant (mol %) Reaction (number) Gene source regulation Reference
Caprylic, capric Brassica napus 38 Acyl-ACP thioesterase (1) Cuphea Up Dehesh et al., 1996
Lauric Brassica napus 58 Acyl-ACP thioesterase (2) California bay Up Voelker et al., 1996
Lauric Arabidopsis 24 Acyl-ACP thioesterase (2) California bay Up Voelker et al., 1992
Palmitic Arabidopsis 39 Acyl-ACP thioesterase (3) Arabidopsis Up Dormann et al., 2000
Palmitic Brassica napus 34 Acyl-ACP thioesterase (3) Cuphea Up Jones et al., 1995
Stearic Soybean 30 Stearoyl-ACP D-9(5) and Soybean Down Kinney, 1996
oleoyl-D-12 desaturase (7)
Stearic Brassica napus 40 Stearoyl-ACP D-9 desat (5) Brassica Down Knutzon et al., 1992
Stearic Cotton 38 Stearoyl-ACP D-9 desat (5) Cotton Down Liu et al., 2000
Stearic Brassica napus 22 Acyl-ACP thioesterase (4) Mangosteen Up Hawkins and Kridl, 1998
Petroselinic Tobacco 4 Palmitoyl-ACP D-4 desat (6) Coriander Up Cahoon et al., 1992
Oleic Soybean 86 Oleoyl-D-12 desaturase (7) Soybean Down Kinney, 1996
Oleic Brassica napus 89 Oleoyl-D-12 desaturase (7) Brassica Down Stoutjesdijk et al., 2000
Oleic Cotton 77 Oleoyl-D-12 desaturase (7) Cotton Down Liu et al., 2000
Oleic Brassica juncea 73 Oleoyl-D-12 desaturase (7) Brassica Down Stoutjesdijk et al., 2000
Oleic Arabidopsis 54 Oleoyl-D-12 desaturase (7) Arabidopsis Down Okuley et al., 1994
c-Linolenic (18:3 w-6) Brassica napus 47 Oleoyl-D-6 and D-12 desat (7) Mortierella apina Up Ursin et al., 2000
c-Linolenic acid Tobacco 1 Oleoyl-D-6 desaturase (7) Cyanobacteria Up Reddy and Thomas, 1996
Eleostearic, parinaric Soybean 17 Conjugase (11) Momordica Up Cahoon et al., 1999
D-5 Eicosenoic Soybean 18 b-Ketoacyl-CoA synthase (8), Meadowfoam Up Cahoon et al., 2000
acyl-CoA desaturase (9)
Hydroxy fatty acids Arabidopsis 30 Oleate-12-hydroxylase (10) Castor, Lesquerella Up Smith et al., 2000
Ricinoleic Arabidopsis 17 Oleate-12-hydroxylase (10) Castor Up Brown and Somerville, 1997
Acetylenic Arabidopsis 25 Acetylenase (11) Crepis Up Lee et al., 1998
12, 13-Epoxy-cis-9-oleic Arabidopsis 15 Epoxygenase (11) Crepis Up Singh et al., 2000
Wax esters Arabidopsis 70 b-ketoacyl synthase (12), Jojoba Up Lardizabal et al., 2000
acyl-CoA reductase (13),
wax synthase (14)
Note. The numbers after the names of engineered reactions refer to Fig. 1A and 1B. Reactions 1 to 6 occur in the plastid and reactions 7 to 14
occur at the ER or other nonplastidial membrane.
cases such engineering efforts have been successful (sum-marized
in Table 1). In the following we discuss selected
examples of the engineering of novel fatty acids in plants.
Engineering of Fatty Acid Chain Length
Plants that accumulate short- to medium-chain (C8 to
C14) fatty acids in seed triacylglycerol have seed-specific
acyl-ACP thioesterase activities toward the corresponding
acyl-ACPs (Pollard et al., 1991; Davies, 1993). For example,
California bay and Cuphea seeds accumulate up to 90%
short chain saturated fatty acids in triacylglycerol. In
groundbreaking studies, expression of a California bay
thioesterase in the seeds of non-laurate (12:0)-accumu-lating
plants, Arabidopsis and Brassica napus (rapeseed),
resulted in the ‘‘short-circuiting’’ of acyl chain elongation
to produce laurate up to 24 and 58% of total seed fatty
acids, respectively (Table 1; Voelker et al., 1992, 1996).
Position analysis of TAG revealed that laurate was present
at both sn-1 and sn-3 positions but not the sn-2 position.
Lack of laurate at the sn-2 position was attributed to the
high specificity of lysophosphatidic acid acyltransferase
15
Fatty Acid Biosynthesis
5. (LPAAT). Further increases in laurate yield seemed pos-sible
if all three positions of TAG were acylated with
laurate. The introduction of a laurate-specific coconut
LPAAT into rapeseed containing the California bay
thioesterase resulted in further increases in laurate levels,
up to 67% total fatty acid, by catalyzing laurate acylation
at the sn-2 position of TAG (Knutzon et al., 1999). This
level of laurate is higher than observed in palm kernel, a
commercial source of laurate. Applications of high-laurate
rapeseed oil include detergents and soaps, a large market
that is currently met by imported palm kernel and coconut
oils.
The previous example demonstrates that the transfer of
a single gene into rapeseed confers laurate accumulation at
levels very similar to California bay seed. However, such
success with a single gene may be the exception rather
than the rule. For example, when a medium-chain thio-esterase
from Cuphea hookeriana was introduced into
rapeseed, caprylate (8:0) accumulated to only 12% in
transgenic rapeseed, while Cuphea contains 50% caprylate
(Dehesh et al., 1996). In another investigation, expression
of elm or nutmeg FatB thioesterases in rapeseed did not
result in seed containing 65% caprate (10:0) or 80%
laurate as observed in these two respective plants but
rather 4% caprate and 20% laurate, respectively (Voelker
et al., 1997). In these examples short-chain fatty acids were
significantly lower in transgenic hosts compared to donor
species. One explanation for these differences is the low
availability of short-chain acyl-ACP pools for thioesterase
termination in non-short-chain-accumulating plants. This
was addressed by crossing plants expressing condensing
enzymes (3-ketoacyl-ACP synthase, KAS) from Cuphea
that have unique specificity for 6:0-(caproic) and 8:0-acyl-
ACPs with lines carrying Cuphea FatB thioesterases
(Leonard et al., 1998; Dehesh et al., 1998). All lines carry-ing
both a Cuphea KAS and a Cuphea thioesterase had
higher levels of short-chain fatty acids than the single-transgene
parents. Enhancement of short-chain fatty acid
accumulation was attributed to the short-chain specificity
of the Cuphea KAS, effectively increasing 10:0- and 12:0-
acyl-ACP pool sizes for short-chain thioesterase cleavage.
Thus obtaining significant amounts of short-chain fatty
acids in TAG may require multiple genes which increase
the substrate pools for the thioesterase as well as short-chain-
specific acyltransferases which can assemble the
novel fatty acids into TAG.
Plants Sometimes Fight Back against Metabolic
Engineering Schemes
An unexpected lesson learned from the study of laurate-producing
transgenic plants described above was that high-level
production of novel fatty acids can induce a futile
cycle of fatty acid synthesis and degradation (Fig. 2).
By analyzing hundreds of independent transgenic lines,
Voelker et al., (1996) found that laurate production in
canola seeds increased linearly up to about 35 mol% with
increased lauroyl-ACP thioesterase expression. However,
to achieve 58 mol% laurate required 10-fold higher
levels of the introduced enzyme, raising the question of
what limits higher laurate accumulation. Eccleston and
Ohlrogge (1998) examined these high-laurate canola seeds
and found that enzymes for medium-chain fatty acid
b-oxidation were increased severalfold, as were malate
dehydrogenase and isocitrate lyase, which participate in
the glyoxylate cycle for fatty acid carbon reutilization.
These and other results led to the conclusion that high
production of unusual fatty acids in transgenic hosts can
induce pathways for their breakdown. Surprisingly, seed
oil yield was not reduced, which led to the additional
discovery that the FAS pathway was also induced,
presumably to compensate for the loss by oxidation of
medium-chain fatty acids.
Production of Waxes
Long-chain wax esters were once harvested from sperm
whales and were a major ingredient of industrial lubri-cants
and transmission fluids. Banning of whale harvests
led to searches for alternative biological sources of such
structures. Jojoba, a desert shrub found in the American
southwest, is the only plant species known to accumulate
waxes (up to 60% dry weight) rather than TAG as a seed
FIG. 2. Scheme for a futile cycle of production and oxidation of
lauric acid in transgenic canola, based on results of Eccleston and
Ohlrogge (1998). Transgenic seeds that produce 58 mol% lauric acid
were found to have increased activity of lauric acid b-oxidation,
isocitrate lyase, and malate dehydrogenase. In addition, up to 50% of
[14C]acetate added to seeds was recovered in malate, sucrose, and other
water-soluble metabolites. These results suggest that up to half the lauric
acid produced is degraded and returned to intermediate pools in a futile
cycle of fatty acid synthesis and turnover.
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Thelen and Ohlrogge
6. storage reserve. These waxes are mostly derived from
C20–C24 monounsaturated fatty acids and alcohols and
are synthesized by the elongation of oleate followed by
reduction to alcohols by a fatty acid reductase (Metz
et al., 2000). The wax storage lipid is formed by a fatty acyl-
CoA:fatty alcohol acyltransferase, also referred to as wax
synthase. The reductase and acyltransferase were purified
from jojoba and the corresponding cDNAs cloned (Metz et
al., 2000; Lardizabal et al., 2000). Coordinated expression
of three genes—a Lunaria annua long-chain acyl-CoA
elongase and the jojoba reductase and acyltransferase—in
Arabidopsis resulted in wax production at up to 70% of the
oil present in mature seeds (Lardizabal et al., 2000).
The high levels of accumulation indicated that all the genes
necessary for this trait were identified. If this trait can be
successfully transferred to commercial crops this would
represent a large potential source of waxes for a variety of
applications, including cosmetics and industrial lubricants.
Production of Novel Monoenoic Fatty Acids
Introduction of the first double bond in fatty acids occurs
in plastids by a soluble desaturase specific for acyl-ACP
substrates. The location of this double bond can vary
depending upon specificity of the plastidial acyl-ACP desa-turase.
Typically the double bond is inserted between
carbons 9 and 10 of a stearoyl-ACP substrate. However,
seed-specific plastidial acyl-ACP desaturases that intro-duce
double bonds at the D4, D6, or D9 position of
palmitoyl-ACP have been identified from coriander,
black-eyed Susan vine (Thunbergia alata), and cat’s claw,
respectively, which accumulate these unusual monoenes
up to 80% in seed oil (Cahoon et al., 1992, 1994a, 1998).
Double-bond position on palmitate and stearate alters the
physical properties such that unusual monoenes have
potentially different commercial uses including monomer
feedstocks for specific nylon polymer applications or as
higher melting unsaturated fatty acids for margarines.
Since monomers for most nylons are derived from
the petrochemical industry there is interest in plants as
renewable sources for these precursors. To achieve wide use
of such fatty acids it will be essential to move the unusual
monoene trait into high-yielding oilseed crops from
which the oil can be produced at low cost. However, intro-duction
of a coriander D4 16:0-ACP desaturase or a
Thunbergia D6 16:0-ACP desaturase into tobacco callus
and Arabidopsis seed, respectively, resulted in less than
10% accumulation of these unusual fatty acids (Cahoon
et al., 1992; Schultz and Ohlrogge, 2001). The reason for the
low levels of unusual monoene production in non-native
plants remains unknown and represents a major challenge
Metabolic Engineering 4, 12–21 (2002)
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in our understanding of plant lipid synthesis. Some evidence
suggests specific isoforms of the cofactors, ferredoxin and
ACP, may be important for production of unusual monoenes
(Suh et al., 1999; Schultz et al., 2000). In addition,
coriander and Thunbergia unusual monoenes are incor-porated
into phosphatidylcholine pools prior to accu-mulation
into TAG (Cahoon et al., 1994b; Schultz and
Ohlrogge, 2000). Coriander also expresses KAS (Mekhedov
et al., 2001), thioesterase (Dörmann et al., 1994), and
acyltransferase (Dutta et al., 1992) activities specific for
these unusual fatty acids, which are likely important for
their accumulation in TAG.
In a recent investigation, transgenic expression of an
engineered castor D9 18:0-ACP desaturase (with improved
specificity toward 16:0-ACP) in Arabidopsis seed resulted
in 13% of total seed fatty acids as 16:1D9 and elonga-tion
products 18:1D11 and 20:1D13 (Cahoon and Shanklin,
2000). Expression of this same desaturase in fab1 Arabi-dopsis
mutants containing a lesion in KAS II, which cata-lyzes
the elongation of 16:0-ACP to 18:0-ACP, resulted in
up to 30% accumulation of the same three fatty acids.
Thus availability of 16:0 ACP substrate is likely one limi-tation
for unusual monoene production. In addition, this
study suggests that novel acyl-ACP desaturases produced
by protein engineering strategies may be more effective
than enzymes derived from wild species.
Product Yield: The New Challenge in Oilseed
Metabolic Engineering
Identification of key genes as described earlier and their
transfer into transgenic crops have occupied many aca-demic
and industrial laboratories for the past 10–15 years.
However, in many cases this is not the central problem
in oilseed modification. For a new oil to be economic,
the desired fatty acid almost always must be the major
constituent to avoid expensive purification costs. Despite
impressive successes with medium-chain fatty acids and
wax esters, in most cases in which a newly identified gene
has been transferred into another oilseed, the proportion
of the desired product in the transgenic host has been
considerably lower than in the wild species from which
the gene was obtained. The activity of the introduced
enzyme has generally not been limiting, so it is necessary to
determine what other factors limit product accumulation.
Accumulation of unusual fatty acids to levels found
naturally will likely require introduction of activities in
addition to those directly responsible for synthesizing the
unusual fatty acid. One possible explanation for this is the
presence of a redundant set of biosynthetic enzymes for
novel fatty acids in seeds. Such a scenario would explain
17
Fatty Acid Biosynthesis
7. differences in substrate specificity between seed-specific
lipid biosynthetic enzymes and those involved in general
cell lipid synthesis. Presumably this is because most
unusual fatty acids possess physical properties distinctly
different from fatty acids commonly found in membranes,
and thus plants must possess ‘‘editing’’ or exclusion
mechanisms to prevent the accumulation of these fatty
acids in lipid bilayers (reviewed in Volker and Kinney,
2001). Addressing these issues will require more knowl-edge
of the cellular biochemistry in oil-accumulating
tissues than is currently available.
PROGRESS TOWARD INCREASING SEED
OIL CONTENT
For both edible and industrial uses, an increase in seed
oil content is desirable and has been a major goal of
oilseed engineering. However, to be economically useful,
such a change must not come at the expense of overall
seed yield or at the loss of other high-value components.
For example, soybean is the largest source of vegetable oil,
comprising 30% of the world market, and now consti-tutes
over 80% of all dietary vegetable oils in the United
States. Although termed an oilseed, soybean contains only
18–22% oil on a seed dry-weight basis and is grown prin-cipally
as a high-protein meal for animal feeds. Thus,
increasing oil in soybean will in most cases not be useful if
it comes at the expense of high-value soy protein that
drives the crop’s economics. By comparison, other oilseed
crops (except cotton) are grown primarily for their oil and
produce seeds with 40–60% oil. The wide range of seed oil
percentage observed in nature suggests that this pathway
might be amenable to metabolic engineering, particularly
in ‘‘low-oil’’ oilseeds, provided the key mechanisms which
control oil content are identified.
Production of Malonyl-CoA by Acetyl-CoA Carboxylase
Is a Key Regulatory Step
The committed step for de novo FAS is the production
of malonyl-CoA catalyzed by acetyl-CoA carboxylase
(ACCase) (Fig. 1). Malonyl-CoA production appears to be
a potential control point for this pathway, based upon
analysis of acyl-CoA and acyl-ACP pool sizes (Post-
Beittenmiller et al., 1991, 1992; Roughan, 1997). Since
malonyl-CoA levels in plastids are very low (less than
10%) compared to acetyl-CoA, it seemed likely that up-regulating
ACCase activity would increase flux to fatty
acids. This has been clearly shown to be the case in
Escherichia coli (Davis et al., 2000). The plastidial ACCase
from most plants is a complex comprising four different
subunits. One early effort to increase ACCase was to
overexpress the biotin carboxylase (BC) subunit using a
CaMV 35S promoter in tobacco. Although BC protein
increased threefold in leaves, there was no accompanying
increase in the amount of other ACCase subunits
(Shintani et al., 1997) and no effect on fatty acid content
or composition. Thus, for ACCase—unlike some other multi-enzyme
complexes—overexpressing just one subunit does
not increase the amount of the remaining subunits.
Evidence that increased malonyl-CoA pools could
increase fatty acid production was obtained by targeting a
homomeric ACCase to rapeseed plastids (Roesler et al.,
1997). Under the control of a seed-specific promoter this
chimeric protein resulted in higher ACCase activities and
increased oil yield by 3–5% on a seed dry-weight basis.
These data provided the first evidence that seed oil could
be quantitatively enhanced by increasing the pool size of
malonyl-CoA precursor. However, the small increase
pointed toward additional control points for FAS.
Overexpression of Several Individual Fatty Acid Synthase
Enzymes Does Not Increase Flux through Fatty Acid
Biosynthesis
Increasing malonyl-CoA precursor pools for FAS
resulted in only slight increases in seed oil yield. Such a
modest improvement would suggest that another step(s)
might be limiting. Could fatty acid synthase activities also
be limiting FAS? Several labs have addressed this question
by overexpressing enzymes downstream of malonyl-CoA
production. The conclusion from these investigations is
that up-regulation of any one enzyme does not increase
flux through FAS. Indeed, overexpression of some activi-ties
actually decreased FAS and fatty acid content as
observed with the overexpression of a condensing enzyme.
Condensation of acetyl-CoA with malonyl-ACP is
catalyzed by KAS III. Recently, a spinach KAS III
was expressed in tobacco and resulted in approximately
50-fold increases in activity above control levels. Rather
than an increase in fatty acid content a 5–10% decrease
was observed (Dehesh et al., 2001). In the same report, a
Cuphea KAS III expressed in rapeseed seed embryos
resulted in a 9% decrease in fatty acid content. An
interesting and unexpected consequence of KAS III
overexpression was an increase in ACP protein levels in
tobacco leaves, although other fatty acid synthase activi-ties
were unaffected. Decreases in fatty acid content as a
result of KAS III overexpression were attributed to
decreased rates of de novo FAS most likely by reducing
malonyl-CoA pools for subsequent KAS condensation
reactions. In a related study, targeting of an E. coli
malonyl-CoA:ACP trans-acylase to rapeseed leucoplasts
Metabolic Engineering 4, 12–21 (2002)
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Thelen and Ohlrogge
8. increased this plastid activity up to 45-fold but did not
increase fatty acid content (Verwoert et al., 1994).
Based upon the aforementioned and other studies it
seems unlikely that the up-regulation of any single fatty
acid synthase enzyme will have a major positive effect on
FAS flux. Although not all fatty acid synthase enzymes
have been overexpressed to determine the effect on FAS,
substantial increases in flux will likely require up-regula-tion
of multiple activities. This conclusion has stimulated
more comprehensive efforts to identify transcriptional,
protein kinase, or other regulatory factors that might
up-regulate the entire pathway (Girke et al., 2000).
Preliminary studies suggest that reactions late in the
TAG biosynthetic pathway may provide increased sink
strength that could stimulate increased fatty acid produc-tion.
Overexpression of a yeast long-chain sn-2 acyltrans-ferase
resulted in > 50% (dry mass/seed) increases in seed
oil content of Arabidopsis and rapeseed (Zou et al.,
1997). Field trials of the transgenic rapeseed gave increases of
8.1–13.5% (Katavic et al., 2000). Recently, Jako et al.
(2001) reported that overexpression of an Arabidopsis
diacylglycerol acyltransferase in Arabidopsis seeds can also
increase seed oil content as well as seed weight. Together,
these studies suggest that increased flux into oil may be
more easily achieved by strategies targeted at the later
steps in the pathway. It is important to note that despite
intense efforts in this area, commercial varieties with con-sistently
increased oil yield per hectare have not been
achieved through transgenic means.
CONCLUSIONS
Engineering of FAS has progressed rapidly in the past 5
years and has led to the commercialization or field trial of
several modified oilseed crops. Although the engineering
of fatty acid chain length and degree and location of fatty
acid desaturation has at least been demonstrated in prin-ciple,
engineering plants with increased flux through FAS
has been difficult. This is likely due to the complexity
associated with the engineering of primary carbon meta-bolism
and an unclear picture of how this pathway is
regulated in vivo. One of the challenges that lie ahead is to
understand the mechanism for feedback inhibition of fatty
acid production in vivo (Shintani and Ohlrogge, 1995).
Although plants with increased seed oil and those con-taining
nutritional supplements may have an immediate
market niche, plants engineered to accumulate industrial
‘‘specialty oils’’ may encounter problems and will need to
be cost-evaluated on an individual basis (Hitz, 1999).
Some of these potential problems include expensive pro-cessing
costs, loss of value associated with toxicity of the
meal by-product (a particular problem with high-value
Metabolic Engineering 4, 12–21 (2002)
doi:10.1006/mben.2001.0204
soybean meal), and occasional undesirable side-effects
resulting from major alterations in fatty acid profiles
(Knutzen et al., 1992; Miquel et al., 1993; Miquel and
Browse, 1994). Nevertheless, the long-term forecast of
vegetable oils as an alternative to petroleum for chemical
feedstocks is not fanciful. With rapid advances occurring
in plant lipid biotechnology and the increasing cost of
petroleum, vegetable oils will eventually provide new
cost-effective raw materials.
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