Metabolic engineering of plant oils and waxes for use as industrial feedstocks

Abstract

Society has come to rely heavily on mineral oil for both energy and petrochemical needs. Plant lipids are uniquely suited to serve as a renewable source of high-value fatty acids for use as chemical feedstocks and as a substitute for current petrochemicals. Despite the broad variety of acyl structures encountered in nature and the cloning of many genes involved in their biosynthesis, attempts at engineering economic levels of specialty industrial fatty acids in major oilseed crops have so far met with only limited success. Much of the progress has been hampered by an incomplete knowledge of the fatty acid biosynthesis and accumulation pathways. This review covers new insights based on metabolic flux and reverse engineering studies that have changed our view of plant oil synthesis from a mostly linear process to instead an intricate network with acyl fluxes differing between plant species. These insights are leading to new strategies for high-level production of industrial fatty acids and waxes. Furthermore, progress in increasing the levels of oil and wax structures in storage and vegetative tissues has the potential to yield novel lipid production platforms. The challenge and opportunity for the next decade will be to marry these technologies when engineering current and new crops for the sustainable production of oil and wax feedstocks.

Full text

Review article
Metabolic engineering of plant oils and waxes for use as
industrial feedstocks
Thomas Vanhercke
1,
*, Craig C. Wood
1
, Sten Stymne
2
, Surinder P. Singh
1
and Allan G. Green
1
1
CSIRO Plant Industry, Canberra, ACT, Australia
2
Swedish University of Agricultural Science, Alnarp, Sweden
Received 3 September 2012;
revised 5 October 2012;
accepted 9 October 2012.
*Correspondence (Tel +61 2 6246 4913;
fax +61 2 6246 4950; email Thomas.
Vanhercke@csiro.au)
Keywords: fatty acids, oils, waxes,
metabolic engineering, industrial
feedstocks.
Summary
Society has come to rely heavily on mineral oil for both energy and petrochemical needs. Plant
lipids are uniquely suited to serve as a renewable source of high-value fatty acids for use as
chemical feedstocks and as a substitute for current petrochemicals. Despite the broad variety of
acyl structures encountered in nature and the cloning of many genes involved in their
biosynthesis, attempts at engineering economic levels of specialty industrial fatty acids in major
oilseed crops have so far met with only limited success. Much of the progress has been
hampered by an incomplete knowledge of the fatty acid biosynthesis and accumulation
pathways. This review covers new insights based on metabolic flux and reverse engineering
studies that have changed our view of plant oil synthesis from a mostly linear process to instead
an intricate network with acyl fluxes differing between plant species. These insights are leading
to new strategies for high-level production of industrial fatty acids and waxes. Furthermore,
progress in increasing the levels of oil and wax structures in storage and vegetative tissues has
the potential to yield novel lipid production platforms. The challenge and opportunity for the
next decade will be to marry these technologies when engineering current and new crops for the
sustainable production of oil and wax feedstocks.
Introduction
Modern society relies heavily and unsustainably on petroleum as a
source of many industrial products ranging from fuels and
lubricants to specialty chemicals and plastics. Petroleum reserves
are finite and nonrenewable, and their widespread use is
contributing heavily to undesirable increases in atmospheric
CO
2
levels (Le Que
´re
´et al., 2009). As environmental concerns
mount and supply constraints emerge, alternative bio-based raw
materials to support these industrial needs are gaining increasing
strategic importance.
Plant oils represent one of the main opportunities to provide
environmentally friendly, renewable and sustainable feedstocks
that can potentially substitute for petroleum in many industrial
applications. To date, most of the focus has been on using plant
oils for biodiesel, providing an alternative source of energy
particularly for liquid transport fuel applications. Unfortunately, in
spite of their technical suitability for this purpose, and thus their
usefulness as a transition technology, in the longer-term plant oils
cannot be produced on sufficient scale to make a major
contribution to the immense global demand for transport fuels
(approx 1.5 billion metric tonnes/year). In the long term, this need
must instead be addressed by a mix of other approaches,
including greater use of a range of non-carbon-based renewable
energy technologies (such as hydro, wind and solar) that are both
sustainable and scalable. Petroleum, however, is not only used for
energy but is also the primary source of a diverse array of carbon-
based molecules (petrochemicals) that are the foundation of the
industrial chemical and polymer sectors and that have much
greater unit value than hydrocarbon fuels. Diminishing petroleum
availability will also impact heavily on the manufacture of these
products, and biological carbon is the only alternative raw
material. Consequently, a strong resurgence in interest in bio-
based alternatives to petrochemicals is now well underway at
both the research and the commercial levels.
Crop plants offer substantial potential to provide renewable
sources of industrial chemicals through the synthesis and accu-
mulation of specific industrial target molecules in plant vegetative
or storage tissues (‘crop biofactories’), coupled with subsequent
postharvest thermochemical and/or enzymatic conversion (‘bior-
efineries’). Although a wide range of bio-based molecular
structures have potential industrial use, lipids—in particular fatty
acids, oils and waxes—are especially well suited as replacements
for petrochemicals because of the analogous linear carbon chain
structures, providing close molecular equivalents or functional
equivalents to many existing mainstay industrial feedstock
chemicals. Furthermore, global plant oil production, although
predominantly for food use, is currently approaching 160 million
metric tonnes annually (Oil World Annual, 2012), which equates
to around a third of the size of petrochemical production.
Importantly, there are now substantial technological opportuni-
ties, through metabolic engineering, to greatly increase plant oil
production beyond that required to meet future increased food
demand and thus to create a significant surplus for deployment
into industrial use (Carlsson et al., 2011). Industrial plant oils and
waxes could therefore, in the future, feasibly be produced on a
scale that could make significant inroads as renewable petro-
chemical replacements.
In this context, Carlsson et al. (2011) have recently framed a
technological challenge of trebling global plant oil production
over the next two decades to provide sufficient oil to replace 40%
of petrochemical usage, without compromising the supply of
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Plant Biotechnology Journal (2013) 11, pp. 197–210 doi: 10.1111/pbi.12023

plant oils for the food sector. To reach this goal, it will be
necessary to develop some of our major oil plants to produce
industrial oil compositions that go beyond their natural within-
species variation. It will also be necessary to greatly expand plant
oil production possibilities and levels, by dramatically increasing
oil productivity in current oil crops and by developing and
introducing novel oil production platforms. In each of these
pursuits, metabolic engineering of plant lipid biosynthesis will
clearly play a central role.
Current industrial use of plant oils and waxes
Plant oil production is dominated by the major food oil crops,
including oil palm, soybean, rapeseed (canola), sunflower,
cottonseed, peanut (groundnut), corn and olive, along with a
number of minor sources such as safflower, sesame, coconut and
linseed. The majority of these food oils are comprised of only five
fatty acids palmitic (C16:0), stearic (C18:0), oleic (C18:1
D9
),
linoleic (C18:2
D9,12
) and a-linolenic acid (C18:3
D9,12,15
). However,
although global plant oil production is heavily directed towards
food use, a significant proportion (around 20%) is already used
for industrial (nonfood) applications. One major industrial utiliza-
tion of plant oils involves medium-chain fatty acids, predom-
inantly laurate (C12:0) from palm kernel oil and coconut oil, in
surfactant applications, such as in soaps, detergents and related
personal care products. Other food oils that are highly unsatu-
rated, such as linseed and soybean, also find uses as renewable
alternatives for synthetic drying agents in surface coatings and
inks and are also processed into epoxygenated oils for use in
industrial resins and glues.
In addition to these multipurpose oils, there are a few specialty
industrial oils obtained from more minor oil plants, such as castor
oil, tung oil and high-erucic rapeseed oil. Castor oil is obtained
from the castor bean plant (Ricinus communis), a difficult crop to
manage because of the presence of the highly toxic ricin protein
and other allergenic compounds. Castor oil accumulates in the
endosperm and contains very high levels (90%) of the hydroxy
fatty acid, ricinoleic acid (12-hydroxy C18:1
D9
). Ricinoleic acid is
an increasingly important industrial feedstock as it can undergo
pyrolytic cleavage at the reactive D12 position to yield undecy-
lenic acid, an alpha olefin used for the production of the
polyamide Nylon 11. Castor oil also finds direct use for bio-based
polyols in the production of a range of products including
polyurethane and as a high-performance lubricant.
Tung oil (or China wood oil) is a highly valued drying oil
obtained from the nut of the tung tree (Vernicia fordii). Its unique
drying properties, highly valued for furniture protection, result
from its very high content (82%) of the conjugated fatty acid
a-eleostearic acid (C18:3
D9c,11t,13t
). As for castor, the prospects
for expansion of tung production are limited by its restricted
agronomic range.
Erucic acid (C22:1
D13
) is used to produce erucamide, an
important slipping agent used in the production of extruded
polyethylene and propylene films (Friedt and Luhs, 1998). Erucic
acid is now mainly derived from high-erucic acid rapeseed (HEAR)
(Brassica napus) that has 45%–55% erucic acid. Production of
HEAR will become increasing difficult with the continuing
expansion of food-grade canola (low-erucic rapeseed), because
the two crops are interfertile and HEAR must be grown in the
isolation of canola to prevent contamination with erucic acid.
These speciality fatty acids referred to above each have
tremendous opportunities for expanded utilization as industry
moves increasingly to bio-based products. However, the various
agronomic and production limitations that the current crop
sources suffer from, and their consequent high price, will present
significant constraints to this growth. For this reason, consider-
able attention has now been directed to exploring whether
production of these fatty acids could be engineered in high-
performance oilseed crops, rather than using their native sources.
Furthermore, the specialty industrial fatty acids currently in
production represent only the ‘tip of the iceberg’ compared with
the enormous chemical diversity that exists in nature for fatty acid
structure (Badami and Patil, 1980). Several of these novel
structures—such as hydroxylated, acetylenated, conjugated,
epoxidized, branched chain and cyclic fatty acids (Figure 1)—
are now fairly well studied, and their synthesis is generally under
relatively simple genetic control, usually involving a single catalytic
enzyme (Napier, 2007).
Although fatty acids and triacylglycerols (TAGs) clearly pre-
dominate as the plant lipids currently most used for industrial
applications, there is also specialized industrial use of a number of
plant-derived waxes. Waxes are comprised of a fatty acid
esterified to a fatty alcohol and generally range in overall length
from C40 to C60 (Figure 1). Most plants produce wax esters (WE)
as components of their surface lipid layers, where protection from
desiccation and pathogen entry is afforded by their solid state,
hydrophobicity and high resistance to hydrolytic degradation.
These unique physical properties of waxes are also especially
useful for industrial applications. The principal plant wax used
industrially is obtained from the leaves of the carnauba palm
(Copernicia prunifera). Carnauba wax is highly valued as a surface
protectant and high-shine polish with a multitude of industrial
uses. In contrast to leaf waxes, one plant, the desert shrub jojoba
(Simmondsia chinensis), is unique in being able to assemble
straight-chain WE in its seed where they serve as the predominant
storage lipid, accumulating at levels up to 50% of weight of the
jojoba bean. The jojoba WE are notably liquid at room temper-
ature and are highly valued ingredients in cosmetics and personal
care products.
The specific properties of WE are imparted by the overall chain
length and, in some cases, by the presence of functional side
groups within the molecule, such as methyl branches or di-esters
(Biester et al., 2012; Gamo and Saito, 1971). The ability to modify
these characteristics through metabolic engineering opens up the
potential to develop improved and specialized waxes designed for
particular industrial purposes. In particular, there is now the
prospect of engineering plant wax composition to better match
the physical and performance properties of modern-day synthetic
industrial lubricants, to provide viable renewable and biodegrad-
able alternatives to petrochemically derived products. However,
to realize their potential, waxes will need to be produced on
much larger scale and at much lower cost than from present
sources. The exemplar of WE accumulation in jojoba seed is now
providing considerable impetus to metabolic engineering of high-
yield oilseed crops to accumulate industrial quality WE. The recent
EU-FP7 ICON (Industrial Oil crops producing added value Oils for
Novel chemicals) project was established specifically to exploit this
potential (http://icon.slu.se).
Plant oil and wax synthesis and accumulation
Fatty acid and triacylglycerol synthesis
Rational and precise engineering of industrial oil and
wax production in plants will need to be predicated on a
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Thomas Vanhercke et al.198

comprehensive understanding of the metabolic pathways for the
biosynthesis of fatty acids and their accumulation into TAG and
WE storage lipids, and of the genetic control of these pathways.
Based on biochemical research over several decades, seed oil
biosynthesis had, until relatively recently, come to be viewed as
an essentially linear process comprised of sequential fatty acid
synthesis, modification and accumulation processes. These path-
ways and their component enzymes have been described in
significant detail in various previous reviews (Baud and Lepiniec,
2010; Li-Beisson et al., 2010; Napier and Graham, 2010;
Ohlrogge and Chapman, 2011; Snyder et al., 2009; Wallis and
Browse, 2010; Weselake et al., 2009).
In essence, the classical view has been that saturated or
monounsaturated acyl chains of various lengths generated via the
fatty acid synthesis pathway in the plastid (Figure 2) are exported
to the cytosolic compartment from where they are sequentially
assembled onto the sn-1, sn-2 and sn-3 positions of the glycerol
backbone by the reactions of the Kennedy pathway (Figure 3) to
form TAGs that are then sequestered into oil bodies (oleosomes)
to serve as energy reserves to support eventual seed germination.
Prior to their incorporation into TAG, the acyl chains exported
from the plastid are able to undergo a range of enzyme-mediated
modifications (Figure 2). The most common of these are the
further sequential desaturation at D12 and D15 positions to
produce the polyunsaturated fatty acids (PUFAs) linoleic and
a-linolenic. However, in many plants (and other organisms),
unusual enzymatic modifications can occur that result in diverse
fatty acid structures of potential industrial interest (such as shown
in Figure 1). A notable example is the divergent family of Fad2-
encoded enzymes that have evolved differing catalytic functions
from the ancestral D12-desaturase (Figure 4). These catalyse the
synthesis of a range of hydroxy, epoxy, acetylenic and conjugated
fatty acid structures (Broun et al., 1998a; Cahoon et al., 1999;
Dyer et al., 2002; Lee et al., 1998; van der Loo et al., 1995; Nam
and Kappock, 2007; Qiu et al., 2001; Sperling et al., 2000).
Enzymes for the conversion of oleic acid to cyclopropanoic acid
fatty acids have also been identified, for example in Sterculia (Bao
et al., 2002) and cottonseed (Yu et al., 2011). Also, there are
fatty acid elongase (FAE) systems that operate in the cytosol for
the elongation of oleic acid to eicosenoic acid (C20:1
D11
) and
erucic acid.
In recent years, however, the discovery and characterization of
several additional enzymes involved in acyl exchange within the
cytosolic compartment has challenged this essentially linear view
of TAG assembly. Newly identified acyl exchange enzymes appear
to be playing significant roles in the transfer of fatty acids from
the site of modification on phosphatidylcholine (PC) to deposition
on TAG molecules. They may also be providing specialized
(a) (b)
(c) (d)
Figure 1 Typical plant lipid structures including
(a) fatty acids commonly found in edible plant oils,
(b) some unusual fatty acids of industrial interest
found in minor crops and wild plants, (c)
triacylglycerol and (d) wax ester.
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Metabolic engineering of industrial plant oils and waxes 199

pathways for the channelling of unusual, and potentially dam-
aging, acyl groups away from membranes and into storage lipids
and thus have potentially significant consequences for attempts
to metabolically engineer unusual fatty acid production in plant
oils. In particular, a number of enzymes have now been
uncovered that mediate the transfer of acyl groups directly from
PC to TAG without passage through the acyl-coenzyme A (CoA)
pool. One such route involves the enzyme phosphatidylcholine
diacylglycerol acyltransferase (PDAT) that transfers an acyl group
from the sn-2 position of PC directly to the sn-3 position of TAG
in seed of Arabidopsis (Sta
˚hl et al., 2004). It has recently been
shown that PDAT and diacylglycerol acyltransferase (DGAT) are
the two main enzymes responsible for TAG synthesis in Arabid-
opsis seeds (Zhang et al., 2009). Mutation in one gene and
down-regulation by RNA interference (RNAi) of the other led to
seeds virtually lacking TAG, whereas double mutants were both
pollen and embryo lethal (Zhang et al., 2009). Similar to the
situation with DGAT, PDAT has been shown to be expressed both
in tissues that accumulate TAG (oil palm mesocarp) and in similar
tissues that are essentially devoid of TAG (date palm mesocarp)
(Bourgis et al., 2011). The relative contribution of DGAT and
PDAT in seed TAG synthesis can vary drastically between even
evolutionary closely related species, like sunflower and safflower
(W. Banas, A. Sanchez, A. Banas and S. Stymne, pers. commun.).
An alternative route, by which acyl groups present on PC can
be moved directly to the TAG backbone, is through exchange of
the polar head group between PC and diacylglycerol (DAG), the
immediate precursor of TAG. The enzyme responsible, phospha-
tidylcholine diacylglycerol cholinephosphotransferase (PDCT), was
recently described in Arabidopsis and shown to act by
transferring the complete phosphocholine head group to directly
form DAG, thereby effectively providing the entire glycerol
backbone from PC as a precursor of TAG (Lu et al., 2009).
Mutational inactivation of this gene results in the lowering of oil
content and polyunsaturation because of reduced flux of acyl
groups from PC to DAG. Furthermore, when the PDCT mutation
was combined with mutations in the lysophosphatidylcholine
acyltransferase genes (LPCAT1 and LPCAT2) that are responsible
for acyl loading and editing of PC, there was a dramatic
reduction in PUFA content in the seed TAG, down to one-third
the wild-type level. These results indicate that PC acyl editing and
phosphocholine head-group exchange between PC and DAG
control the majority of acyl fluxes through PC to provide PUFA for
TAG synthesis (Bates et al., 2012). Recent work also demon-
strates that the acyl-CoA-independent transfer of acyl groups
from PC to TAG catalysed by PDAT is dependent on an efficient
acylation of lysophosphatidylcholine, the coproduct of the PDAT
Figure 2 Principal fatty acid biosynthetic
pathways in plastidic and cytoplasmic
compartments of higher plant cells. Chain
elongation enzymes are shown as black arrows: 1
keto-acyl synthase III (KASIII), 2keto-acyl synthase
I (KASI), 3keto-acyl synthase II (KASII), 4Fatty
acid elongase (FAE1). Fatty acid desaturases are
shown as grey arrows: 5D9-desaturase (SAD), 6
D12-desaturase (FAD2), 7D15-desaturase
(FAD3). Acyl thioesterases are shown as white
arrows: 8Fatty acid thioesterase A (FATA), 9
Fatty acid thioesterase B (FATB).
Figure 3 Contemporary diagrammatic representation of the known
metabolic routes by which acyl groups from the acyl-PC and acyl-CoA
pools can be directly or indirectly channelled to triacylglycerol (TAG). The
traditional linear Kennedy pathway is shown in green and more recently
defined routes in blue. Acyl pools are shown as solid rectangular boxes,
and principal enzymes involved are shown as open oval boxes. ACS, acyl-
CoA synthase; DGAT, diacylglycerol acyltransferase; GPAT, glycerol-3-
phosphate acyltransferase; LPAAT, lysophosphatidic acid acyltransferase;
LPCAT, lysophosphatidylcholine acyltransferase; PAP, 3-sn-phosphatidate
phosphohydrolase; PDAT, phosphatidylcholine diacylglycerol
acyltransferase; PDCT, phosphatidylcholine diacylglycerol
cholinephosphotransferase; PLA, phospholipase A; PLC, phospholipase C;
PLD, phospholipase D.
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Thomas Vanhercke et al.200

reaction. In the absence of a functional DGAT1, the Arabidopsis
seed TAG is mainly synthesized by PDAT and disruption of
LPCAT2 activity caused drastic reduction in seed TAG content
and severely disturbed seed development (Xu et al., 2012).
Another related acyl-CoA-independent route to DAG involves
phospholipase D (PLD) that catalyses the removal of just the
choline group from PC to form phosphatidic acid (PA), which can
then be dephosphorylated by 3-sn-phosphatidate phosphohydro-
lase (PAP) to form DAG. Suppression of PLD in developing
soybean seeds has recently been shown to alter both the total
content and the level of unsaturation of soybean seed oil,
suggesting that it may be playing a role in the conversion of PC to
TAG during oil synthesis, either by a direct catalytic action to
produce phosphatidic acid (PA), or by exerting a regulatory
influence over other acyl exchange enzymes (Lee et al., 2011). In
both of these head-group removal reactions (PDCT and PLD), the
acyl groups at position sn-1 and sn-2 of PC retain their positioning
on the TAG backbone.
Taken overall, the relatively recent uncovering of PDAT and
PDCT has now provided clear evidence for the existence of
metabolic routes for direct (acyl-CoA independent) transfer of
acyl groups from PC to each of the three positions on TAG during
seed oil biosynthesis. These pathways presumably operate in
addition to the classical Kennedy pathway. However, it is
tempting to speculate that the full picture is still not elucidated,
especially given the significant numbers of uncharacterized
acyltransferase genes evident in recent and current genomic
and transcriptomic studies of several oil-bearing plants that
accumulate unusual fatty acids, such as flax (Wang et al., 2012),
Hiptage benghaliensis and Bernardia pulchella (A.G. Green,
S. Singh and X.-R. Zhou, unpublished data). Many of these gene
families could harbour additional enzymes for direct acyl
exchange either between PC and the various TAG intermediates,
or amongst the intermediates themselves. For example, there
have been biochemical indications of possible DAG/DAG acyl
exchange yielding a monoacylglycerol (MAG) and a TAG (Stobart
et al., 1997). However, no gene dedicated to such a function has
yet been identified, although it has been reported that a soluble
form of yeast PDAT enzyme has some low level of DAG/DAG
transacylation activity (Ghosal et al., 2007). Recent work could
not detect any DAG/DAG transacylase activity in sunflower or
safflower membranes, despite good PDAT activity (W. Banas,
A. Sanchez, A. Banas and S. Stymne, pers. commun.) casting
some doubts over any significant contribution of such enzyme
activity in TAG synthesis in oil seeds. Interestingly, PDAT can also
transfer acyl groups from sn-1 of PC to TAG, albeit at a lower
rate (about 30%) than with acyl groups from the sn-2 position
(W. Banas, A. Sanchez, A. Banas and S. Stymne, pers. commun.;
Sta
˚hl et al., 2004). As acyl groups at sn-1 position of PC can
undergo desaturation and structural modification at that position
(Bao et al., 2003; Stymne et al., 1992), it is possible that PDAT
and other enzymes might also be involved in channelling these
acyl groups from PC to TAG.
Thus, the picture of TAG synthesis that is now emerging is one
of a complex network of acyltransferase reactions mediating the
movement of acyl groups between pools of acyl-PC, acyl-CoA
and TAG precursors within the cytosolic compartment (Figure 3).
Acyl moieties exported from the plastid could take various
potential metabolic routes through these networks towards
assembly on TAG, the routes taken probably differing between
plant species, based on presence, activity and coordinated
regulatory control of alternative enzymatic steps. For example,
recent studies with soybean (Bates and Browse, 2011) and
Arabidopsis (Bates and Browse, 2012) have identified the
presence of kinetically distinct DAG pools during oil accumula-
tion. Diacylglycerol (DAG) used in TAG assembly was predomi-
nantly derived from PC, whereas DAG produced through de novo
synthesis (Kennedy pathway) was mainly used to produce PC. As
fatty acids esterified to PC are substrates for modifications, such
as desaturation, hydroxylation or epoxygenation, the fatty acid
composition of the PC-derived DAG pool may be markedly
different to that of the de novo DAG pool. The extent of this
shunting of DAG through PC, compared with direct conversion to
TAG, may vary significantly between different species and could
be a significant underlying factor in the varying ability of some
wild plants to incorporate unusual fatty acids into TAG, as well as
the difficulty in engineering some oilseeds to accumulate unusual
fatty acids.
Importantly, particular routes through this metabolic network
may have become specialized for particular unusual acyl groups in
certain plants. This feature could be based on customized
substrate specificity of the acyltransferases available, and there
is already evidence of divergent substrate specificities within a
number of acyltransferase families. For example, it is known that
DGAT enzymes in castor bean, Vernonia galamensis and tung
have strong preferences for DAG and CoA substrates containing
ricinoleoyl, vernoleoyl and eleostearoyl moieties, respectively
(Kroon et al., 2006; Shockey et al., 2006; Yu et al., 2006).
Wax ester synthesis
In contrast to the complex, and perhaps incompletely defined,
metabolic pathways leading to TAG synthesis, the pathways
resulting in WE are relatively simple and well understood
(Lardizabal et al., 2000). In essence, synthesis of a WE is a two-
Figure 4 Examples of unusual fatty acid
structures originating from modifications to the
structure of oleic acid (C18:1) in the acyl-PC pool
from the action of divergent Fad2 enzymes and
CPFA synthase. RA, ricinoleic acid; ESA,
eleostearic acid; VA, vernolic acid; CA, crepenynic
acid; DHSA, dihydrosterculic acid; C18:2, linoleic
acid.
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Metabolic engineering of industrial plant oils and waxes 201

step process involving, firstly, the conversion of a fatty acid to a
fatty alcohol by the action of a fatty acid reductase (FAR),
followed by the esterification of the fatty alcohol to a fatty acid by
the action of a wax synthase (WS). This final step in WE synthesis
can be viewed as being analogous to the final DGAT-mediated
step in the formation of TAG, in that the fatty acid donor
becomes esterified either through the primary OH group of the
fatty alcohol or the through sn-3 OH group of DAG. This
commonality has been reinforced by the discovery of DGAT
enzymes with dual-function WS/DGAT activity (Kalscheuer and
Steinbu¨ chel, 2003; Li et al., 2008) and able to utilize a variety of
acyl acceptors as substrates (Yen et al., 2005). Arguably, the best-
studied metabolic pathway for WE synthesis is that occurring
naturally in jojoba seed wax and its transgenic expression in oil-
forming seeds (Lardizabal et al., 2000). Jojoba WE synthesis is
located in the extra-plastidial compartments, and both FAR and
WS are membrane associated and utilize acyl-CoA (Lardizabal
et al., 2000; Metz et al., 2000). The structure of jojoba WE is
dominated by C20:1 and C22:1 acyl and alcohol chains, and this
is reflected in the substrate specificity of both the FAR and the WS
enzymes (Lardizabal et al., 2000; Metz et al., 2000).
Metabolic engineering of fatty acid synthesis and
accumulation
For most oleochemical applications of plant oils, it is desirable to
have as high a concentration (purity) as possible of the required
molecule, to maximize the recovery yield and to minimize
potentially difficult or expensive downstream processing and
separation costs. However, most of our oilseed species have been
chosen for their food use and nutritional value, and they tend to
instead have a balanced content of the main five fatty acids,
palmitic, stearic, oleic, linoleic and linolenic. The maximum levels
of individual fatty acids in these oils are suitable for the food use
of the oil, but fall short of very high purity levels preferred for
industrial feedstocks. Therefore, a major challenge of metabolic
engineering industrial fatty acids into major oil crops is not just to
achieve synthesis by introducing genes for the appropriate
catalytic enzymes, but also to engineer storage lipid assembly
pathways to enable high-level accumulation and purity of the
target molecule. We review below recent progress in these
endeavours that reveal differing levels of difficulty depending on
the type of fatty acid being engineered.
Monounsaturated fatty acids
Monounsaturated fatty acids have significant industrial potential
for two main reasons. Firstly, compared with the polyunsaturates
that are also present in most plant oils, monounsaturates are
relative resistant to oxidative attack and impart better stability for
direct use of the oil in industrial products such as biolubricants or
biodiesel. Secondly, they can be readily cleaved at their double-
bond sites by chemical processing to give rise to products in high
demand by the chemical industry, such as monomers for the
production of various nylons (polyamides). The predominant
monounsaturate present in most plant oils is oleic acid that can be
processed to yield azelaic acid (C9) monomers for bionylon
production (Ho
¨fer, 2003). High-oleic compositions have been
developed—using both conventional breeding and gene technol-
ogy—in several oilseeds (e.g. sunflower, safflower, canola,
soybean and peanut) to improve their stability and enable them
to be used as replacements for saturated and trans-fatty acid–
containing oils. Although the 75%–85% oleic acid levels in these
genetically improved oils are ideal for their food use, the
significant residual levels of polyunsaturates cause problems in
industrial and oleochemical applications.
However, a very recent development of super-high-oleic
safflower has now demonstrated the potential to metabolically
engineer oilseeds to achieve the sought after oleochemical purity
levels. RNAi-mediated gene silencing targeted against the seed-
expressed fatty acid thioesterase (FATB) and FAD2 genes has
resulted in the development of safflower oil containing up to 95%
oleic acid, with only 2% linoleic acid remaining and with no
apparent adverse effects on agronomic performance (C. Wood,
Q. Liu, J. Cao, X.-R. Zhou, S. Stephan, S. Singh and A. Green,
unpublished data). This achievement using seed-specific gene
silencing contrasts noticeably with previous efforts to raise oleic
acid using mutagenesis approaches that, although successful in
reaching levels around 90% (Skoric et al., 2008), have generally
been associated with reduced yield. This yield drag is considered to
be due to an undesirable reduction in PUFA content of membranes
in vegetative tissues resulting from the mutation of constitutively
expressed FAD2 genes (Clemente and Cahoon, 2009). The
development of safflower with seed-specific elevation of oleic
acid up to 95% should open the way for greater industrial
application of oleic acid as a chemical feedstock and also provide
an additional improvement in stability for direct industrial appli-
cations, such as in lubricants and in transformer fluids. Importantly,
this achievement now serves as a landmark in demonstrating the
potential to metabolically engineer highly pure oleochemicals in
plant oils. It is interesting that such very high levels of oleic acid and
also high levels of gamma-linolenic acid (C18:3
D6,9,12
) (Nykiforuk
et al., 2012) could be achieved by relatively straightforward
metabolic engineering in safflower, a species that can also
naturally accumulate up to 90% linoleic acid. This demonstrates
an extreme phenotypic plasticity for this species, at least with
respect to tolerance of unsaturated fatty acids.
Metabolic engineering of other monounsaturated fatty acids
with different chain lengths is also showing good signs of success
in generating quite high levels. Erucic acid is formed through the
sequential elongation of oleoyl-CoA by the extraplastidic elong-
ase system, but reaches only 45%–55% of total fatty acids in
naturally occurring HEAR, thus limiting the yield of this highly
valuable and versatile industrial fatty acid used, for example, in
erucamide production. This limitation is due to erucic acid being
found only at the outer positions (sn-1 and sn-3) of TAG in
rapeseed, because the rapeseed lysophosphatidic acid acyltrans-
ferase (LPAAT) enzyme, responsible for the insertion of fatty acids
in the sn-2 position, cannot use erucoyl-CoA as the acyl donor
(Frentzen, 1993). However, when a Limnanthes LPAAT gene that
readily utilizes erucoyl-CoA (Cao et al., 1990) was expressed
together with a rapeseed FAE in a mutant line having greatly
increased oleic acid substrate levels, erucic acid levels increased
up to 72% and the total amount of very long-chain fatty acids
accounted for up to 77% of all fatty acids (Nath et al., 2009).
Importantly, this achievement of high erucic acid content has
recently been emulated using a similar strategy in crambe
(Crambe abyssinica), to create a potential alternative high-erucic
crop source that is safely reproductively isolated from edible oil
rapeseed (Li et al., 2012b). It is possible that further gains in
erucic acid content might be achievable through blocking any
acyl-CoA-independent pathways (such as PDAT and PDCT) that
may be channelling the remaining oleate directly to TAG, as this
could result in this oleate being shunted instead through the acyl-
CoA pool and thus being made available for additional elongation
to erucic acid (Carlsson et al., 2011). In this context, it might be
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Thomas Vanhercke et al.202

noteworthy that the combination of blocks in PDCT and LPCAT in
Arabidopsis seeds led to a substantial increase in C20:1 in the
seed oil along with greatly decreased level of PUFAs (Bates et al.,
2012).
Two other monounsaturated fatty acids, palmitoleic acid
(C16:1
Δ9
) and its elongation product cis-vaccenic acid
(C18:1
Δ11
), that are normally present in seed oils at only very
low amounts are now of considerable industrial interest following
the recent breakthrough development of olefin metathesis, a new
organic chemistry process made possible through the develop-
ment of novel catalysts (Meier, 2009; Rybak et al., 2008).
Metathesis reactions utilizing either of these two x7-unsaturated
fatty acids can be used to generate an x-unsaturated fatty acid
and 1-octene that has great value as an industrial chemical
feedstock. To capitalize on this opportunity, metabolic engineer-
ing strategies for developing very high levels of x7-unsaturated
fatty acids have recently been explored with outstanding success
(Nguyen et al., 2010). By simultaneously blocking the ketoacyl
synthase II (KASII)-mediated elongation of C16:0 to C18:0 and
introducing two D9-desaturase genes capable of desaturating
C16:0 (one being operative in the plastid and the other in the
cytosol), combined levels of the two x7-fatty acids, palmitoleic
and vaccenic acid, of up to 71% of total fatty acids were obtained
in Arabidopsis. This matches the levels seen in Doxantha unguis-
cati, the plant with the highest recorded amount of x7-fatty acids
in its seed oil, with prospects for further increases through
engineering additional reduction in C18 unsaturates. A particular
feature of this work is that the introduced plastidic C16:0-acyl
carrier protein (ACP) D9-desaturase had been engineered to
convert C16:0 to C16:1
Δ9
with a more than 100-fold higher
specificity than that of the naturally occurring enzyme.
In spite of the significant recent success with engineering very
high levels of the aforementioned monounsaturates, not all
monounsaturate fatty acids have proved so amenable to meta-
bolic engineering. For example, C16:1
Δ6
and C18:1
Δ6
(petrose-
lenic acid) that constitute about 80% of all fatty acids in the seed
oils of Thunbergia alata and Coriander sativum, respectively, can
be split by ozonolysis to yield adipic acid (C6:0) and either capric
(C10:0) or lauric acid. Adipic acid is one of the building blocks in
6,6-nylon, which has an annual production of about 2.5 million
metric tonnes and is today made from petroleum. The acyl-ACP
desaturase enzymes responsible for the production of these
monounsaturated fatty acids have been identified some time ago
(Cahoon et al., 1992, 1994); however, when expressed in
transgenic plants, the synthesis of these fatty acids was very
low. It was concluded that the poor synthesis was probably due
to either the lack of, or the incorrect assembly of, a necessary
multicomponent enzyme association (Suh et al., 2002).
Medium-chain saturates
Medium-chain saturated fatty acids—in particular caprylic (C8:0),
capric and lauric acid—have a range of industrial applications.
Although palm kernel oil and coconut oil already provide
significant plant sources of lauric acid, there are no commercially
viable plant sources rich in caprylic acid and capric acid. These
fatty acids are currently fractionated out as minor components of
palm kernel and coconut fats, or synthesized from petroleum via
oxidation of the corresponding aldehydes. An oil crop producing
high amounts of caprylic or capric acid in its oil could be an
economically viable alternative to the organic chemical synthesis.
Medium-chain saturates are produced abundantly in seed oils
of some wild plants, notably in the genus Cuphea where they can
reach extremely high levels (Badami and Patil, 1980). They are
synthesized by termination of chain elongation during plastidial
fatty acid synthesis through the intervention of chain-length-
specific acyl-ACP thioesterases (Dehesh, 2001; Voelker et al.,
1992, 1997) followed by export to the cytosol for incorporation
into TAG. One of the earliest examples of metabolic engineering
of oil quality was the production of over 60% of lauric acid in
rapeseed (Voelker et al., 1992; Wiberg et al., 2000), achieved by
transgenically expressing a single gene encoding a C12:0-ACP-
specific thioesterase obtained from Californian bay tree. Similar
strategies were subsequently attempted in rapeseed using Cup-
hea thioesterase genes with specificities towards C8 and C10
acyl-ACPs, but resulted in much lower synthesis of the medium-
chain fatty acids (Wiberg et al., 2000). A range of biochemical
investigations (Bafor and Stymne, 1992; Bafor et al., 1990;
Eccleston and Ohlrogge, 1998; Larson et al., 2002; Poirier et al.,
1999; Wiberg et al., 1994) all suggested that the transgenic
seeds lacked the enzymes required to efficiently acylate the
medium-chain fatty acids onto TAG backbones, leading to a
build-up of these fatty acids in the acyl-CoA pool and their
resultant shunting to beta-oxidation. This contrasts with the
situation in wild Cuphea plants where the concerted action of
specialized glycerol-3-phosphate acyltransferase (GPAT) and
LPAAT allows the production of exclusively di-medium-chain
DAGs, which are preferentially used by DGAT to form TAG (Bafor
and Stymne, 1992; Bafor et al., 1990). It is interesting to contrast
the disappointing results obtained for C8 and C10 with the high-
level accumulation obtained for C12. It would appear that the
TAG assembly enzymes present in Brassicaceae plants, such as
Arabidopsis and rapeseed, may be competent on saturated fatty
acids in the range C12 to C18 but much less so with shorter chain
lengths such as C8 and C10. However, as for C16 and C18
saturated fatty acids, the C12 lauric acid is more or less excluded
from the sn-2 position of the TAG.
Unusual fatty acids
The aforementioned saturates and monounsaturates are relatively
innocuous fatty acids, and it is perhaps unsurprising that high
levels of purity are being achieved or approached. However,
engineering accumulation of many of the more unusual modified
fatty acids found in wild plants and other organisms is proving to
be a much more challenging endeavour. Fatty acids containing
reactive functional groups, such as hydroxy groups, epoxy bridges
or carbocyclic structures, or unusual bond structures, such as
acetylenic or conjugated bonds, represent valuable starting
materials for industrial chemistry. It is notable that in nature,
there are many instances where plants accumulate storage lipids
with very high concentrations of these unusual fatty acid
structures, for example 90% of ricinoleic acid in R. communis
(Badami and Patil, 1980) and 92% of vernolic acid (an epoxy fatty
acid) in Bernardia pulchella (Spitzer et al., 1996). Initial attempts
to engineer production of these, and other unusual fatty acids, in
oilseeds through transgenic expression of key biosynthetic (cat-
alytic) enzymes were relatively promising in achieving synthesis of
the target fatty acids. However, in all cases, the product levels fell
well short of achieving the ideal purity levels and generally failed
to even emulate the levels found in the organisms from which the
genes for the biosynthetic enzymes were obtained (Cahoon
et al., 2006; Dyer et al., 2008; Thomaeus et al., 2001).
Many of these modified fatty acids are known to be synthe-
sized from oleic acid or linoleic acid substrates esterified to PC on
the membranes (Figure 4), where their unusual or reactive
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Metabolic engineering of industrial plant oils and waxes 203

structure could be damaging to membrane integrity. Wild plants
that produce such fatty acids must therefore have developed
highly effective pathways for their removal from PC and
sequestration into inert storage lipids, presumably via the
concerted action and specialization of enzymes involved in PC
editing, PC to DAG conversion and TAG assembly that have
co-evolved with the catalytic enzyme (Napier and Graham, 2010).
It is therefore not surprising that the lipid metabolic machinery of
recipient oil crops that do not normally produce these unusual
fatty acids is not optimized for their handling.
Given the increased complexity of acyl exchange networks
responsible for this channelling now becoming apparent, it is
clear that alternative specialization strategies are possible, and
different organisms may have undergone different evolutionary
adaptations to handle these fatty acids. The metabolic engineer-
ing challenge therefore for achieving high-level synthesis and
accumulation of these unusual fatty acids in oil crops is to uncover
these specialized metabolic pathways and engineer them into the
crop platform alongside the required catalytic enzymes. Recent
progress in this regard has focussed primarily on the acyltrans-
ferases DGAT and PDAT (Figure 2) involved in the acylation at the
sn-3 position on DAG as they are considered to be two key
enzymes for TAG formation in seeds. It is therefore important that
these two enzymes are competent to handle engineered unusual
fatty acids in the target oilseed. Indeed, both a DGAT (RcDGAT2)
and a PDAT (RcPDAT1A) from castor have been shown to have a
preference for ricinoleic acid containing substrates and have been
implicated in the incorporation of high amounts (>90%) of this
fatty acid into TAG in castor. Not surprisingly, co-expression of
either of these enzymes alongside the fatty acid D12-hydroxylase
(FAH12) from castor led to significant increases in the accumu-
lation of ricinoleic acid in the seed oil of Arabidopsis, increasing
from 17% to around 28% in both cases (Burgal et al., 2008; van
Erp et al., 2011; Kim et al., 2011), but with reductions in total oil
content. However, co-expressing both RcPDAT1A and RcDGAT2
restored the oil content to nearly wild-type level and gave rise to a
major increase in the mass of hydroxy fatty acids accumulating in
the seed. Likewise, co-expression of DGAT1 and DGAT2 from
Vernonia galamensis, a plant that accumulates high levels of the
D12-epoxygenated vernolic acid in its oil, along with the D12-
epoxygenase resulted in a significant (fourfold) increase in the
accumulation of vernolic acid in soybean embryos and reversed
the depression in oil and protein content previously seen when
the D12-epoxygenase was expressed alone (Li et al., 2010b,c,
2012a).
Further upstream, the lack of efficient removal of the unusual
fatty acids from the site of synthesis on PC has also been
identified as a bottleneck in a number of instances. For example,
transgenic soybean and Arabidopsis seed expressing fatty acid
conjugases accumulated nearly 25% conjugated fatty acids in
their PC pool (where they are synthesized) compared with the
native tung tree that typically retains <1% of the conjugated fatty
acids in its PC pool (Cahoon et al., 2006). Similar results were also
reported in plants transformed with castor FAH12—the trans-
genics had more than twice the amount of hydroxylated fatty
acids in the PC pool than that found in castor (Bates and Browse,
2011; van Erp et al., 2011). Enzymes such as phospholipases C
and D, LPCAT and PDCT, sourced from native species that
accumulate high levels of unusual FAs, may be useful for their
removal from PC via acyl editing and exchange mechanisms
(Sta
˚lberg et al., 2009; Zheng et al., 2012). For example, when a
castor PDCT was co-expressed with FAH12 in Arabidopsis,
ricinoleic acid in PC was significantly reduced and accumulation
of hydroxylated fatty acids in the oil was doubled (Hu et al.,
2012). In addition, it has been observed that the overall fatty acid
synthesis rate of Arabidopsis seed expressing castor FAH12 is
reduced by >30% and it is possible that this could be due to the
feedback inhibition mechanism, linked with the plastidial acyl-
CoA carboxylase (ACCase), identified recently (Andre et al.,
2012) but also by the catabolism of DAG with ricinoleoyl groups
(Bates and Browse, 2011). Facilitating efficient removal and
channelling of unusual fatty acids to TAG might alleviate this
phenomenon and overcome reduced oil content in these lines.
To date, co-expression of specialized TAG assembly genes
seems to have been performed mainly as the addition of a
pathway rather than as a substitution for an endogenous
equivalent. As such the introduced specialized enzymes will be
competing against the endogenous network and thus may not
have the full opportunity to express their potential. It would be
interesting to see whether, for instance, silencing endogenous
DGAT in combination with introducing specialized DGATs will
further enhance the accumulation of unusual fatty acids.
Furthermore, such silencing might not just be restricted to the
counterpart endogenous gene(s), but could be used to eliminate
entire networks of activity operating on normal fatty acids, to
provide a ‘blank page’ for the expression of TAG assembly
pathways favouring accumulation of unusual fatty acids (Chap-
man and Ohlrogge, 2012). Such an approach has been success-
fully employed for the characterization of genes involved in TAG
synthesis in a yeast strain that is totally deficient in TAG assembly
genes (Sandager et al., 2002).
Another possible bottleneck to accumulation that is yet to be
fully explored is the level of expression of the fatty acid-modifying
enzyme in the transgenic plants. In some species that naturally
accumulate high levels of some unusual fatty acids, it has been
shown that mRNA levels for the fatty acid-modifying enzymes are
at similar high levels to those for storage proteins (Li et al., 2010a;
Nam and Kappock, 2007). It is unknown whether the levels of
expression of these genes in transgenic plants so far developed
are limiting to the accumulation of the unusual fatty acid
products. As the transgenes are often driven by seed storage
gene promoters, it is likely that expression levels are adequate, at
least for low-level synthesis, but might need to be further
increased to provide high levels of synthesis should impediments
to accumulation in storage lipids be successfully alleviated.
Another aspect that has to be taken into account is that these
enzymes need cytochrome b5 and b5 reductase for their activity.
It has been shown that down-regulating cytochrome b5 reduc-
tase lowers the hydroxylation of oleic acid to ricinoleic acid in
Arabidopsis seeds, although it has little effect on the desaturation
of oleic acid to linoleic, a reaction that also requires this reductase
(Kumar et al., 2006).
Engineering wax ester synthesis
Triacylglycerols have attractive physical and biodegradability
features for use as lubricants but have limited ability to replace
petroleum-derived lubricants because of their generally poorer
hydrolytic and oxidative stability. In contrast, WE have excellent
resistance to hydrolysis (Li et al., 2010a). In fact, WE harvested
from the spermaceti whale were widely used in high-pressure and
high-temperature lubricants before a global ban on hunting the
whale was introduced in 1972. Following this, the desert shrub
jojoba (Simmondsia chinensis) was investigated as an alternative
biological source of WE, because its seed oil is unique in being
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Thomas Vanhercke et al.204

composed almost entirely of WE. However, the jojoba plant is low
yielding and labour intensive, and therefore, its WE are very
expensive and unable to compete with cheaper petroleum-based
products in the lubricant market. Developments in gene technol-
ogy stimulated early interest to engineer oilseeds to provide a
lower-cost source of jojoba-type WE. In principle, a minimum of
only two additional enzymatic activities need to be introduced to
produce WE in seeds—a FAR to produce the fatty alcohol and a
WS to esterify the fatty alcohol to a fatty acid. This was confirmed
when expression in Arabidopsis of the jojoba FAR and jojoba WS
(in combination with an FAE to produce C20:1 to C24:1 fatty
acids) resulted in a major proportion of the seed TAG being
replaced by jojoba-type WE (Lardizabal et al., 2000). It is notable
that in these experiments, the introduced WE pathway was
operating in competition with the endogenous TAG assembly
pathway. It will be interesting to see the outcome of more
sophisticated approaches involving the simultaneous suppression
of TAG assembly, such as by silencing DGAT and PDAT activity.
Jojoba WE are mainly composed of monounsaturated C20 and
C22 fatty acid and alcohol carbon units and have too high a
melting point (about 9 °C) for widespread lubricant use, espe-
cially in cold climates. On the other hand, WE of very diverse
composition and melting points are found throughout nature,
and a vast pool of FAR and WS genes is therefore available to
enable the transgenic synthesis of other types of WE suited to a
range of applications and conditions (Rowland and Domergue,
2012). Metabolic engineering to achieve synthesis of WE with the
required properties is also likely to need engineering of the fatty
acid composition of the oil crop, to ensure that the required chain
lengths and functional modifications are inherent in the fatty acid
and fatty alcohol components of the WE. An important recent
development has been the demonstration that transgenic expres-
sion of engineered mouse FAR1 and WS genes in yeast and
Arabidopsis enabled synthesis of WE containing >65% oleoyl-
oleate, provided the genes were cotargeted to the oil body
membrane. This involved the removal of normal peroxisomal
targeting signal from the FAR1 gene and expressing it as an
oleosin fusion protein (Heilmann et al., 2012).
In addition to providing valuable biolubricants, WE can also be
hydrolysed to yield their component fatty acids and fatty alcohols,
of which the latter have substantial use in chemical industry and
an economic value about twice that of the fatty acid. As WE
biosynthesis, in contrast to TAG assembly, is a relatively simple
process and does not share any glycerolipid intermediate with
membrane lipid synthesis, it may ultimately prove to be a more
amenable system for engineering accumulation of unusual fatty
acids such as those that include hydroxy, epoxy or other
functionalities. This would still, however, require the unusual fatty
acid to be efficiently removed from PC and transferred to the acyl-
CoA pool to be available for processing by FAR and WS enzymes.
Other alternative pathways for engineered waxes may now be
possible. One such approach is suggested by the recent finding
that fatty alcohols are also produced by FAR enzymes located in
the chloroplast and operating on fatty acids bound to ACP (Doan
et al., 2012). This finding in combination with the identification
of chloroplast-localized WS genes (Lippold et al., 2012) opens up
the prospect for potential synthesis and deposition of WE within
the plastids, rather than the cytosol.
Protein engineering
Metabolic engineering is not limited to just the transgenic
expression of genes encoding naturally occurring biosynthetic
enzymes; there is also the potential to engineer the protein
structure of these enzymes to alter aspects such as substrate
specificity, double-bond positioning (regioselectivity) and reaction
outcome. This capability provides potential to improve effective-
ness in existing reaction mechanisms and even to engineer
enzymes that undertake reactions at novel positions in the carbon
chain. It also opens up the possibility of synthesizing novel fatty
acids with multiple functional groups, thereby paving the way for
designer lipids that currently lie beyond the reach of most
metabolic engineering strategies.
Fatty acid desaturases are prime targets for protein engineering
because of their central and well-defined role in the modification
of acyl chains within the plant cell. The resolution of the crystal
structures of the soluble plastidic C16:0-ACP D9-desaturase from
castor and C16:0-ACP D4-desaturase from Hedera helix (poison
ivy) resulted in a leap forward in the understanding of substrate-
binding pocket geometry as well as key residues that determine
chain length specificity of soluble desaturases (Guy et al., 2007;
Lindqvist et al., 1996). It also provided valuable insights into
structural determinants that govern the regioselectivity within the
acyl chain of the fatty acid substrate (Guy et al., 2011). Structure–
function informed strategies as well as directed in vitro evolution
have yielded new soluble desaturase variants displaying altered
catalytic reaction, regioselectivity and changes in substrate
specificity (Cahoon et al., 1997; Whittle and Shanklin, 2001;
Whittle et al., 2008). Acyl-ACP desaturases with engineered
substrate specificity are already finding their way into metabolic
engineering projects, such as in enabling synthesis of high levels
of palmitoleic acid in Arabidopsis as described earlier herein
(Nguyen et al., 2010).
In contrast to plastidial acyl-ACP desaturases, the current
structure–function understanding of membrane-bound fatty acid
desaturases remains extremely limited. Owing to the absence of a
crystal structure, protein engineering studies focussing on this
large and diverse class of fatty acid-modifying enzymes have
relied on domain swapping, the exchange of differentially
conserved amino acid residues between homologous members,
or more random approaches such as directed molecular evolu-
tion. This has provided some insights into residues and regions
that influence the reaction outcome (desaturation, hydroxylation
and acetylenation), regioselectivity, stereochemistry and chain
length specificity (Broadwater et al., 2002; Broun et al., 1998b;
Gagne et al., 2009; Hoffmann et al., 2007; Hongsthong et al.,
2004; Meesapyodsuk et al., 2007; Rawat et al., 2012; Vanhercke
et al., 2011). Similar to acyl-ACP desaturases, these studies have
also proven that in most cases, a limited number of substitutions
are sufficient to confer significant changes in the specificity of
membrane-bound desaturases. However, the lack of a detailed
structural model as well as the limited number of high-through-
put screening assays available mean that future progress in
engineering membrane-bound desaturases is likely to remain
limited for the foreseeable future.
Recently, acyltransferases have attracted significant attention
as another target for protein engineering. This interest has been
fuelled in part by the development of a high-throughput Nile Red
assay in yeast and an elegant yeast complementation selection
system that allows for the isolation of functional DGAT variants
within large random libraries (Siloto et al., 2009b). Both systems
have been used to improve the activity of the Brassica napus
DGAT1 acyltransferase in yeast (Siloto et al., 2009a) although
functional characterization and validation in model and target
plants is still needed. Other target enzymes and applications
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Metabolic engineering of industrial plant oils and waxes 205

envisaged include engineering PDAT acyltransferases, screening
cDNA libraries for novel TAG synthesizing enzymes and the
engineering of acyl donor specificity of DGAT acyltransferases
revealed by supplying different unsaturated and unusual fatty
acids during selection. A glimpse of the potential of engineered
acyltransferases can be found in the work of Xu et al. (2008)
where targeted mutagenesis of a putative serine/threonine kinase
site in the Tropaeolum majus DGAT1 acyltransferase increased
the activity in vitro and resulted in increased seed oil content
when expressed in Arabidopsis.
Engineering expanded oil production
The ability to engineer industrial fatty acid production in plants
will, on its own, be insufficient to achieve substantial replacement
of petrochemicals with bio-based alternatives. It will also be
necessary to greatly expand the global supply of plant oils to well
beyond the future food needs of an increasing population to
create the additional supply volume needed (Carlsson et al.,
2011). This will involve increasing the oil productivity of existing
oil crops, introducing additional oilseed crops and creating novel
oil production platforms. Metabolic engineering has a central role
to play in each of these pursuits and is already making significant
contributions to addressing these goals, as has been well
reviewed in recent years (Carlsson et al., 2011; Lu et al., 2011;
Ohlrogge and Chapman, 2011; Taylor et al., 2011; Weselake
et al., 2009). A wide array of metabolic engineering approaches
have been employed to increase the oil content in a variety of
plant tissues, including diverting carbon flow from starch to TAG,
up-regulating fatty acid synthesis, modifying expression of
individual TAG biosynthetic enzymes and transcription factors,
and slowing the turnover of TAG. These approaches have met
with various degrees of success, but it is fair to conclude that they
show a lot of promise for expanding the production of plant oils.
A promising new approach to engineer leaf oil has involved the
expression of a mammalian monoacylglycerol acyltransferase
(MGAT) in Nicotiana benthamiana leaves (Petrie et al., 2012a).
Based on in vitro yeast assays and expression results in N. benth-
amiana, the authors propose that co-expression of a MAG-
synthesizing enzyme such as Arabidopsis GPAT4 (Yang et al.,
2010b) and an MGAT or bifunctional MGAT/DGAT can result in
DAG and TAG synthesis from glycerol-3-phosphate via a route
that is independent and complementary to the endogenous
Kennedy pathway and other TAG synthesis routes. This possibility
of recruiting MAG as an alternative/additional substrate for TAG
biosynthesis presents a novel approach for engineering and
increasing oil in a range of plant tissues.
Overexpression of the AtLEC1 transcription factor, or its
rapeseed orthologs BnLEC1 and BnL1L, is known to raise fatty
acid levels in transgenic Arabidopsis plants, but with severe
developmental abnormalities. However, the use of truncated
napin A promoters, which retain the seed-specific expression
pattern but with a reduced expression level, to drive the
expression of BnLEC1 and BnL1L in rapeseed, resulted in increases
in the seed oil content by 2%–20% without any detrimental
effects on major agronomic traits (Tan et al., 2011). This
highlights the possibility of a similar approach to drive the ectopic
expression of other transcription factors like LEC2 whose expres-
sion has previously resulted in lipid increase but with associated
phenotypic abnormalities (Santos Mendoza et al., 2005; Stone
et al., 2001). Another lipid regulatory approach to raise oil
content is suggested by the recent identification of 18:1-ACP as
the signal molecule and plastidic ACCase as the target for
product feedback inhibition on fatty acid biosynthesis in rapeseed
(Andre et al., 2012). Recent studies have also pointed to other
novel approaches for directing more carbon flux to lipid synthesis
(Hua et al., 2012; Myer et al., 2012; Sanjaya et al., 2011; Shi
et al., 2012), including through the manipulation of genes not
normally associated with oil synthesis such as haemoglobin
(Vigeolas et al., 2011), opening up even further biotechnological
approaches for engineering oil increase in plants. Perhaps most
importantly, the ongoing additions of transcriptomes from a
growing list of oil-producing tissues from higher plants and algae
promise to continue to generate new insights into oil accumu-
lation (Brown et al., 2012; Guarnieri et al., 2011; Hajduch et al.,
2011; Jiang et al., 2012; Merchant et al., 2012; Miller et al.,
2010; Troncoso-Ponce et al., 2011; Yang et al., 2010a).
Future prospects
Recent progress in metabolic engineering of plant lipids has been
strong and sustained. In fact, the engineering of plants to
produce long-chain PUFA, involving the introduction of a
completely new pathway comprised of five additional enzymatic
steps, plus enhancement of endogenous precursor pathways
(Petrie et al., 2010, 2012b; Ruiz-Lo
´pez et al., 2012), is arguably
already the most complex metabolic engineering goal so far
achieved in plants. Importantly, metabolic engineering of indus-
trial oils and waxes, as well as other plant lipids, is now on the
verge of a significant acceleration as a result of several major
recent advances in underlying technologies.
Firstly, as evidenced herein, our understanding of biochemical
pathways governing fatty acid, oil and wax pathways is now
proceeding at a much more rapid rate, resulting from the
integration of genomic, transcriptomic, transgenic and biochem-
ical investigative approaches. This capability is built on recent
major increases in the efficiency of gene sequencing that has
made it feasible and affordable to generate full-sequence
information from any individual species in a relatively short
period of time. Whole genome sequencing can now be
conducted at a genotype level rather than a species level, and
comparative transcriptomics between contrasting tissues, geno-
types or species is being routinely used to uncover genetic
associations with lipid biosynthetic phenotypes.
Secondly, a number of highly effective transient assay systems
are now available that enable in planta metabolic engineering to
proceed on a rapid time cycle and high-throughput basis. In
addition to somatic embryo assay systems that have been applied
in soybean and rapeseed for some time, a significant new
capability has recently emerged through the development of the
Nicotiana benthamiana transient leaf assay system into a sophis-
ticated system for high-throughput experimental metabolic engi-
neering (Wood et al., 2009). This system was instrumental in
evaluating alternative multistep transgenic pathways for DHA
synthesis (Petrie et al., 2010) and is now in widespread use within
the plant lipid research community.
Thirdly, lipidomic analysis has been continually improved in
both sensitivity and reliability and now enables simultaneous
tracking of the multitude of intermediates involved in oil and wax
biosynthetic pathways (Hummel et al., 2011). Lipidomic technol-
ogies are currently undergoing some further fascinating devel-
opments including the use of mass spectroscopic (MS) imaging to
provide detailed lipid species composition across tissue sections,
or even individual cellular organelles or oil drops isolated from oil-
bearing tissues (Horn and Chapman, 2012; Horn et al., 2011,
ª2012 CSIRO
Plant Biotechnology Journal ª2012 Society for Experimental Biology, Association of Applied Biologists and Blackwell Publishing Ltd, Plant Biotechnology Journal, 11, 197–210
Thomas Vanhercke et al.206

2012). These developments will provide increased precision
for unravelling the interplay of pathways with developmental
biology.
Coupled together, these significant advances now enable, for
the first time, the application of combinatorial metabolic
engineering strategies in oil plants, to maximize efficiencies and
specificities of component biosynthetic enzyme pathways. This
technological step change will enable a move from the slow and
laborious step-by-step testing and assembly of relatively simple
transgenic pathways that has characterized the gene discovery
period, to a rigorous, high-throughput and multistep combina-
torial strategy. This may herald a move towards synthetic biology
approaches (French, 2009) with the promise of further significant
advances in engineering of plant-based oils and waxes for future
industrial use.
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