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HARNESSING PLANT BIOMASS FOR BIOFUELS AND BIOMATERIALS

Plant surface lipid biosynthetic pathways and their utility formetabolic engineering of waxes and hydrocarbon biofuels

Reinhard Jetter1,2,* and Ljerka Kunst1

1Department of Botany, University of British Columbia, 6270 University Boulevard, Vancouver, BC V6T 1Z4, Canada, and2Department of Chemistry, University of British Columbia, 6174 University Boulevard, Vancouver, BC V6T 1Z3, Canada

Received 28 November 2007; revised 8 February 2008; accepted 13 February 2008.*For correspondence (fax +1 604 822 6089; e-mail [email protected]).

Summary

Due to their unique physical properties, waxes are high-value materials that are used in a variety of industrial

applications. They are generated by chemical synthesis, extracted from fossil sources, or harvested from a

small number of plant and animal species. As a result, the diversity of chemical structures in commercial waxes

is low and so are their yields. These limitations can be overcome by engineering of wax biosynthetic pathways

in the seeds of high-yielding oil crops to produce designer waxes for specific industrial end uses. In this review,

we first summarize the current knowledge regarding the genes and enzymes generating the chemical diversity

of cuticular waxes that accumulate at the surfaces of primary plant organs. We then consider the potential of

cuticle biosynthetic genes for biotechnological wax production, focusing on selected examples of wax ester

chain lengths and isomers. Finally, we discuss the genes/enzymes of cuticular alkane biosynthesis and their

potential in future metabolic engineering of plants for the production of renewable hydrocarbon fuels.

Keywords: cuticular waxes, fatty acid elongation, chain lengths, esters, hydrocarbons, industrial products.

Introduction

Primary plant surfaces are impregnated with waxes pro-

duced by epidermal cells (Riederer and Muller, 2006). These

cuticular waxes are complex mixtures of C20–C34 straight-

chain aliphatics derived from very-long-chain fatty acids

(VLCFAs), and in certain plant species also include alicyclic

and aromatic compounds such as triterpenoids, alkaloids,

phenylpropanoids and flavonoids. Plant cuticular waxes

serve as a protective barrier against water loss, UV light,

pathogens and insects. In addition, they are valuable raw

materials for a variety of industrial applications. Wax mix-

tures derived from different plant sources have unique

chemical compositions that determine their physical

properties, and therefore their potential applications and

industrial value.

At present, cuticular waxes are commercially harvested

from only a small number of plant species, so the structural

diversity of their wax constituents is limited. In addition,

these plant species are mostly grown in tropical areas and

are agronomically not well suited to commercial production.

These apparent shortcomings of plant surface wax produc-

tion can be circumvented through genetic engineering

approaches using established high-yielding oil crops as a

platform. By introducing wax biosynthetic pathways into

oilseeds, waxes with optimal chemical compositions for

various specialty markets could be produced, including

high-value lubricants, cosmetics and pharmaceuticals, as

well as high-energy fuels.

In this review, we present the chemical diversity of plant

cuticular wax mixtures and summarize our understanding of

the biosynthetic pathways involved in generating this

diversity; provide an overview of commercial sources and

uses of waxes, and of current limitations of wax production;

discuss how engineering of wax biosynthetic pathways in

target crops might be exploited for the production of novel

waxes with specific chain-length distributions in oilseeds;

and describe how wax biosynthetic pathways can be used in

metabolic engineering of plants for the production of

hydrocarbon biofuels. This information complements recent

670 ª 2008 The AuthorsJournal compilation ª 2008 Blackwell Publishing Ltd

The Plant Journal (2008) 54, 670–683 doi: 10.1111/j.1365-313X.2008.03467.x

reviews that have focused on the chemical composition

(Jetter et al., 2006), biosynthesis (Kunst et al., 2006) and

biological functions of plant cuticular waxes (Bargel et al.,

2006; Riederer and Muller, 2006).

Plant cuticular wax composition and biosynthesis

Cuticular wax composition varies between different species

and organs essentially in two respects: chain-length distri-

bution and compound class composition (Jetter et al., 2006).

This diversity is established during wax biosynthesis in

epidermal cells (Kunst et al., 2006), and involves two types

of pathways: those for the elongation of fatty acid wax pre-

cursors to assorted chain lengths and those for modifying

them into wax components with various functional groups.

Aliphatic compound classes ubiquitously present in cuticu-

lar wax mixtures are alkanes, primary alcohols, aldehydes

and fatty acids ranging in chain length between 20 and 34

carbons, as well as alkyl esters up to C60 in length (Figure 1).

The cuticular waxes from many plant species comprise

roughly equal amounts of the various compound classes,

with no particular class predominating. For example,

alkanes, aldehydes, primary alcohols, fatty acids and alkyl

esters each contribute 9–42% of the leaf wax of Zea mays

(Bianchi et al., 1984). In contrast, the wax mixtures from

many other plant species contain high percentages of a

single compound class. Hordeum vulgare leaf wax, for

example, contains 89% of primary alcohols, together with

only 0.2–9% of alkanes, aldehydes, fatty acids and alkyl

esters (Giese, 1975).

Compound chain length

Variation in the chain length of wax compounds is generated

during synthesis of VLCFA wax precursors. This process

involves several enzyme complexes in various cellular

compartments. The first phase, the de novo fatty acid syn-

thesis of C16 and C18 acyl chains, is catalysed by the soluble

fatty acid synthase (FAS) complex localized in the plastid

stroma (Ohlrogge and Browse, 1995; Ohlrogge et al., 1993),

and proceeds through a cycle of four reactions utilizing

intermediates attached to acyl carrier protein (ACP). In each

cycle, comprising the condensation of a C2 moiety origi-

nating from malonyl ACP to acyl ACP, the reduction of

b-ketoacyl ACP, the dehydration of b-hydroxyacyl ACP and

the reduction of trans-D2–enoyl ACP, the acyl chain is

extended by two carbons. Three different FAS complexes

participate in the production of C18 fatty acids in the plastid.

They differ in their b-ketoacyl-acyl carrier protein synthase

(KAS) condensing enzymes, which have strict acyl chain-

length specificities: KASIII (C2–C4; Clough et al., 1992), KASI

(C4–C16) and KASII (C16–C18; Shimakata and Stumpf, 1982).

The two reductases and the dehydratase have no particular

acyl chain-length specificity and are shared by all three

plastidial elongation complexes (Stumpf, 1984).

The second phase (Figure 2), the extension of the C16 and

C18 fatty acids to VLCFA chains, is carried out by fatty acid

elongases (FAE; von Wettstein-Knowles, 1982), multienzyme

complexes bound to the endoplasmic reticulum membrane

(Kunst and Samuels, 2003; Xu et al., 2002; Zheng et al.,

2005). To reach the ER-associated fatty acid elongation sites,

saturated C16 and C18 acyl groups must be hydrolysed from

the ACP by an acyl ACP thioesterase, exported from the

plastid, and esterified to CoA. Two classes of acyl ACP

thioesterases, designated FATA and FATB, have been

described in plants. The FATA class exhibits a strong

preference for 18:1 ACP in vitro, while the FATB thioester-

ases predominantly use saturated fatty acids (Voelker, 1996).

The involvement of the FATB thioesterase in cuticular wax

biosynthesis has been confirmed by analyses of the Arabid-

opsis fatb mutant, which exhibits a major reduction in its

wax load (Bonaventure et al., 2003). The specifics of fatty

acid export from the plastid, CoA esterification and transport

to the ER are not well understood. Fatty acids released from

ACP by a thioesterase in the plastid undergo conversion to

Figure 1. Structures of major components occurring in plant cuticular wax

mixtures.

(a) Ubiquitous compound classes lacking functional groups (alkanes) or with

primary functional groups. Typically, series of compounds with wide ranges

of chain lengths are present in these classes. n and m indicate the number of

methylene (CH2) groups, and can range from 18 to 32.

(b) Wax constituents with secondary functional groups accumulate to high

concentrations in the wax of certain plant species, usually with very narrow

chain-length and isomer distributions. Typical chain lengths and isomers are

shown for selected combinations of hydroxyl and carbonyl functionalities.

Metabolic engineering of waxes and hydrocarbon biofuels 671

ª 2008 The AuthorsJournal compilation ª 2008 Blackwell Publishing Ltd, The Plant Journal, (2008), 54, 670–683

acyl CoAs by a long-chain acyl CoA synthetase (LACS) in the

outer envelope membrane. Of the nine LACS genes anno-

tated in the Arabidopsis genome (Shockey et al., 2002), only

one, LACS9, has been demonstrated to encode a plastid

envelope enzyme (Schnurr et al., 2002). However, loss of

function of LACS9 does not result in reduced export of acyl

groups from the chloroplast, or a wax-deficient phenotype

(Schnurr et al., 2002), suggesting that the LACS isozyme

primarily responsible for CoA esterification of fatty acids en

route to wax biosynthesis has yet to be identified. Move-

ment of the fatty acyl group from the thioesterase to LACS

has been proposed to occur by some type of facilitated

diffusion (Koo et al., 2004), but the exact mechanism of

transfer is not known. An alternative model for fatty acid

export from the plastid was recently suggested by Bates

et al. (2007). Their radiolabelling studies revealed that 16:0

and 18:1 fatty acids synthesized de novo in the plastid can be

incorporated into phosphatidylcholine (PC), perhaps by

direct acylation of lyso-PC. The acyl groups removed from

PC by acyl editing may then be fed into the acyl CoA

pool. However, mechanistic details and the relevance of

this process for epidermal wax formation have not been

established.

Translocation of fatty acids to the ER, where additional

acyl chain elongation and modification of VLCFAs to diverse

aliphatic wax components take place, appears to involve

plastid-associated membranes (PLAMs; Andersson et al.,

2007). Physical manipulation of GFP-labelled ER strands,

using laser scalpels and optical tweezers, experimentally

verified the intimate connection between the plastid and the

ER of Arabidopsis leaf protoplasts. Therefore, PLAMs have

been proposed to be major routes for lipid transfer between

the two organelles.

Elongation of C16 and C18 fatty acids to VLCFAs

involves cycles of four consecutive enzymatic reactions

analogous to those of the FAS (Figure 2), and results in a

two-carbon extension of the acyl chain per cycle. The

chain lengths of aliphatic wax components are typically in

the range of 20–34 carbons, thus multiple elongation

cycles are needed to extend the acyl chain to its final

length. The differential effects of inhibitors on incorpora-

tion of radiolabelled precursors into wax components of

various chain lengths, and analyses of mutants with

defects in fatty acid elongation, demonstrated that

sequential acyl chain extensions are carried out by several

distinct FAEs with unique substrate chain-length specific-

ities (von Wettstein-Knowles, 1993). Specificity of each

elongation reaction resides in the condensing enzyme of

the FAE complex (Lassner et al., 1996; Millar and Kunst,

1997). Consistent with the requirement for fatty acyl

precursors of diverse chain lengths for the synthesis of

cuticular waxes, a family of 21 FAE condensing enzyme-

like sequences has been identified in the A. thaliana

genome (Dunn et al., 2004). An unrelated ELO-like gene

family of putative condensing enzymes, related to the

Saccharomyces cerevisiae condensing enzymes ELO1,

ELO2 and ELO3, has also been annotated (Dunn et al.,

2004). It is not known how many of these putative

condensing enzymes participate in wax production and

how many different condensing enzymes are needed

for the elongation of a C18 to a C34 fatty acyl CoA, as

single condensing enzymes may catalyse multiple elon-

gation steps. The only wax-specific condensing enzyme

characterized to date is CER6 (Fiebig et al., 2000; Hooker

et al., 2002; Millar et al., 1999), which is involved in the

elongation of fatty acyl CoAs longer than C22.

Figure 2. Wax biosynthetic pathways.

Repeated cycles of four enzymatic steps first

elongate acyl CoA precursors. They are then

modified by one of (up to) five different reactions

into various compound classes. Preferred chain

lengths are indicated by numbers. Characterized

enzymes catalysing key biosynthetic steps are

shown in blue (CER6, condensing enzyme¼b-ketoacyl CoA synthase; KCR, b-ketoacyl CoA

reductase; dehydratase, b-hydroxyacyl CoA de-

hydratase; CER10, enoyl CoA reductase; CER4,

fatty acyl CoA reductase; WSD1, wax ester

synthase; MAH1, mid-chain alkane hydroxylase).

672 Reinhard Jetter and Ljerka Kunst


Unlike the condensing enzymes, the other three enzyme

activities of the FAE complex, the b-ketoacyl reductase,

b-hydroxyacyl dehydratase and enoyl reductase, are shared

by all VLCFA elongase complexes. Thus, these three

enzymes have broad substrate specificities and generate a

variety of acyl products used to make different classes of

lipids (Millar and Kunst, 1997). Because genetic screens in

Arabidopsis did not result in isolation of mutants defective

in the reductases or the dehydratase, suggesting that these

enzymes are essential and/or functionally redundant (Millar

and Kunst, 1997), genes encoding the b-ketoacyl reductase

and enoyl reductase were cloned by homology to the

corresponding sequences from Saccharomyces cerevisiae

(Beaudoin et al., 2002; Kohlwein et al., 2001). Two b-ketoacyl

reductase (KCR) genes are present in both the A. thaliana

and maize (Zea mays) genomes. The maize genes, named

GL8A and GL8B (Dietrich et al., 2005; Perera et al., 2003; Xu

et al., 2002), are not only expressed in the epidermis, but

also in internal tissues. Attempts to generate double

mutants by crossing gl8a · gl8b failed because embryos

carrying both mutations were not viable. Thus, the KCR has

an essential function in plants, most likely in the production

of sphingolipids (Dietrich et al., 2005).

An A. thaliana single-copy gene was identified as an enoyl

reductase (ECR) candidate. Heterologous expression of the

putative plant ECR gene rescued the temperature-sensitive

lethality of yeast tsc13-1elo2D cells (Gable et al., 2004),

demonstrating that it encodes a functional ECR. The A. tha-

liana ECR gene is ubiquitously expressed, and the protein

physically interacts with the Elo2p and Elo3p condensing

enzymes when expressed in yeast (Gable et al., 2004). The

A. thaliana ECR was shown to be identical to CER10 (Zheng

et al., 2005), the protein defective in one of the original

A. thaliana eceriferum mutants isolated by Koornneef et al.

(1989). These eceriferum (literally ‘not bearing wax’)

mutants lack epicuticular wax crystals and therefore have

glossy green inflorescence stems that can easily be recog-

nized in visual screens. Biochemical analysis of the cer10

mutant demonstrated that the ECR gene product is involved

in the VLCFA elongation that is required for synthesis of all

the VLCFA-containing lipids, including cuticular waxes, seed

triacylglycerides and sphingolipids (Zheng et al., 2005).

Although the plant dehydratase remains unknown, recent

identification of the yeast b-hydroxyacyl dehydratase PHS1

(Denic and Weissman, 2007) should permit cloning and

characterization of this enzyme from plants.

Compound classes

In addition to variations in the chain-length distributions,

cuticular wax mixtures from diverse plants and plant

organs also contain various constituent compound classes.

These compounds vary in the nature and position of the

(typically oxygen-containing) functional groups, with the

Figure 3. Array of biosynthetic reactions leading to wax esters.

First, the variety of chain lengths is generated by elongation, leading to C22 fatty acid (‘ic’) precursors in seeds (black arrows) and including all chain lengths up to C32

in epidermal cells (orange arrows). Then, individual acyl precursors are reduced to the corresponding alcohols (‘ol’) (green arrows), and alcohols and acyl CoAs of

various chain lengths are combined into esters (blue arrows). Depending on the specificity of the elongase (KCS), acyl reductase (FAR) and ester synthase (WS)

enzymes, various mixtures of ester isomers and chain lengths can be generated. Arabidopsis stem surface wax contains esters with predominantly C16 acyl and C22–

C30 alkyl groups.



extreme case of hydrocarbons that are devoid of functional

groups (Jetter et al., 2006). Five or more parallel reactions

(or pathways), all competing for the VLCFA CoA precursors,

can be envisioned leading to these ubiquitous wax com-

ponents: (i) acyl reduction, (ii) esterification with an alkyl

alcohol, (iii) hydrolysis, (iv) aldehyde formation and (v)

alkane formation (Figure 2). Knowledge of all the wax

biosynthetic reactions will assist in their exploitation for

biotechnological production of individual compounds

and/or mixtures of compounds with specific combinations

of functional groups.

In virtually all vascular plants, wax compound classes

with predominantly even numbers of carbons are produced

by the so-called acyl reduction pathway (Figure 2; Kunst

et al., 2006). The most important of these compounds are

primary alcohols and alkyl esters. The latter are essentially

dimeric compounds, in which the primary alcohols are

bonded to acyl groups, most commonly C16, C18 or VLCFAs

(>C20; Figure 3). Primary alcohols are thus central metabo-

lites of wax biosynthesis, and their formation from VLCFA

CoA esters has been studied extensively.

Two reduction steps are required to transform acyl

precursors into primary alcohols, and aldehydes must

occur as intermediates of the reaction sequence. It has

been much debated whether both reduction steps are

catalysed by one fatty acyl reductase (FAR), or whether two

separate enzymes are necessary for alcohol formation.

There is currently substantial evidence for the existence of

a one-enzyme system in a number of plant species,

including the green alga Euglena gracilis (Kolattukudy,

1970) as well as the angiosperms jojoba (Simmondsia

chinensis; Pollard et al., 1979), pea (Pisum sativum; Vioque

and Kolattukudy, 1997) and A. thaliana (Rowland et al.,

2006). For example, functional expression of genes speci-

fying alcohol-forming FARs from jojoba (Metz et al., 2000)

and A. thaliana (Rowland et al., 2006) in heterologous

systems demonstrated that alcohol biosynthesis from

VLCFAs in these species is carried out by a single alcohol-

forming FAR. In contrast, biochemical feeding experiments

that allowed isolation of an aldehyde intermediate suggest

that the two-step process of alcohol formation operates in

Brassica oleracea (Kolattukudy, 1971). However, similar

biochemical evidence from other species and molecular

information supporting the two-step process in any system

is currently lacking.

It is generally assumed that primary alcohols serve as

precursors for ester biosynthesis. However, detailed analy-

ses of esterified and free alcohols of various mutants of

A. thaliana only recently demonstrated a clear correlation of

alcohol chain lengths in both types of compounds, indicat-

ing that the free alcohols are indeed incorporated into the

wax esters (Lai et al., 2007). In addition, this study revealed

that the levels of free alcohols are limiting for ester forma-

tion. Thus, a pool of primary alcohols, generated in the

A. thaliana epidermal cells, is available either for export

towards the cuticle or for esterification with an acyl CoA.

Other plant species exhibit large variations in compositions

of cuticular wax esters, characterized in some cases by broad

Figure 4. Diversity of acyl and alkyl composi-

tions of wax esters from three plant species.

Waxes were extracted from leaf surfaces and

analysed by GC-MS (n = 3). The relative acyl

composition for each ester chain length was

determined from the abundances of MS frag-

ments [RCO2H2]+, and used to calculate overall

acyl and alkyl distributions across ester chain

lengths.



distributions of acyl and/or alkyl moieties and in other cases

by relatively high preferences for certain isomers (Figure 4).

In higher plants, mammals and bacteria, ester biosynthe-

sis is catalysed by one of three classes of wax synthase (WS)

enzymes: jojoba-type WS, mammalian WS, and WS/DGAT

bifunctional enzymes. Jojoba-type WS uses a wide range of

saturated and unsaturated acyl CoAs ranging from C14 to

C24, with 20:1 as the preferred acyl and 18:1 as the preferred

alcohol substrate (Lardizabal et al., 2000). In A. thaliana,

there are 12 wax synthases with high homology to the jojoba

WS, but none have yet been characterized. Mammalian WS

enzymes do not have homologues in plants, and have

highest activities with C12–C16 acyl CoAs and alcohols

shorter than C20 (Cheng and Russell, 2004a,b). A bifunctional

WS/DGAT enzyme from Acinetobacter calcoaceticus has a

preference for C14 and C16 acyl CoA together with C14–C18

alcohols (Stoveken et al., 2005). Nearly a hundred WS/DGAT

homologues have been identified from over 20 other micro-

organisms so far (Waltermann et al., 2007), and ten

sequences in the A. thaliana genome have also been anno-

tated as WS/DGATs. One of these enzymes, WSD1, has been

characterized and shown to be responsible for the formation

of cuticular wax esters in A. thaliana stems (R.J., L.K., F. Li,

X. Wu and A.L. Samuels, University of British Columbia,

Canada, unpublished results). The enzyme utilizes mostly

saturated C16 acyl CoA precursors, showing that this

upstream precursor of wax production must be co-localized

in the cell with the primary alcohols, which are synthesized

far downstream in the wax biosynthetic pathway.

Two additional compound classes with predominantly

even-numbered chain lengths, aldehydes and free fatty

acids, are also found in the wax mixture of most plant

species, albeit usually at relatively low concentrations.

Currently, our knowledge on their biosynthesis is very

limited. Formation of free fatty acids must involve hydrolysis

of the elongated acyl CoA precursors (Figure 2). However, it

is not clear whether this reaction occurs spontaneously or

whether it is enzyme-catalysed. Aldehyde formation

requires reduction of acyl CoA precursors, and may occur

as an intermediate step during alcohol formation (see

above), during alkane formation (see below), or indepen-

dently of either of these pathways (Figure 2). Only a single

wax aldehyde-forming reductase enzyme has been partially

purified to date, and the gene encoding this enzyme has not

been identified (Vioque and Kolattukudy, 1997).

A separate set of wax biosynthetic reactions is responsible

for the formation of compounds with predominantly odd

numbers of carbons (Figure 2). Examples of such compound

classes include the alkanes, secondary alcohols and ketones

that occur together in many wax mixtures and typically

share similar chain-length distributions (Jetter et al., 2006).

Early biochemical experiments led to a model describing the

biosynthesis of these compounds as a two-stage process,

with a first set of reactions transforming VLCFA precursors

into alkanes and a second series of reactions modifying

them into secondary alcohols and ketones (Kolattukudy,

1965). Subsequent experiments confirmed the central role of

alkanes in this pathway (Kolattukudy, 1968; Kolattukudy and

Brown, 1974; Kolattukudy et al., 1974), either as intermedi-

ates en route to mid-chain functionalized compounds or as

end products if the downstream reactions are missing.

Overall, the second stage of the pathway is relatively

well characterized, whereas the first part remains poorly

understood.

Although conversion of VLCFA precursors into alkanes

could proceed directly in one reaction, the net acyl decar-

boxylation is apparently brought about by a sequence of

transformations. This multistep pathway is supported by the

fact that a number of different A. thaliana mutants with

alkane-deficient cuticular wax mixtures have been described

(Hannoufa et al., 1993; Jenks et al., 1995; Rashotte et al.,

2001, 2004). Cloning of several of these mutated genes

(CER1, CER2 and CER3/WAX2) revealed that the proteins

they encode contain motifs similar to known biosynthetic

enzymes (Aarts et al., 1995; Ariizumi et al., 2003; Chen et al.,

2003; Kurata et al., 2003; Negruk et al., 1996; Rowland et al.,

2007; Xia et al., 1996). While this suggests a potential

enzymatic role for these proteins, their exact function

remains unknown. Due to this lack of molecular information,

it is currently not possible to predict the exact number of

reaction steps involved in the conversion of acyl precursors

into alkanes, the nature of these steps or the resulting

intermediates.

Two alternative pathways have been proposed for the

conversion of acyl compounds into alkanes, which vary in

the central reaction in which a C1 unit is cleaved off (Bianchi,

1995; Bognar et al., 1984; Chibnall and Piper, 1934). The

difference lies in the nature of the immediate precursor from

which cleavage occurs and whether the C1 unit is CO or CO2

(decarbonylation versus decarboxylation). Only one model,

which describes alkane formation as the decarbonylation of

an aldehyde intermediate, has been tested experimentally to

some extent (Cheesbrough and Kolattukudy, 1984). How-

ever, conclusive molecular genetic and biochemical evi-

dence for either model is lacking, leaving alkane formation

as the least understood part of wax biosynthesis.

In A. thaliana leaves, alkanes are the major odd-num-

bered product, while a high level of secondary alcohols and

ketones accompanies alkanes in the stem wax, as well as in

wax from B. oleracea leaves (Baker, 1974; Jenks et al., 1995).

In these instances, a second stage of the pathway is

additionally involved, transforming alkanes first into sec-

ondary alcohols and then into ketones (Figure 2). This

reaction sequence is well-supported by chemical evidence

correlating chain-length and isomer compositions of all

three compound classes (Jenks et al., 1995), and by bio-

chemical evidence provided by feeding experiments and

detailed studies of label positions in resulting products



(Kolattukudy and Liu, 1970; Kolattukudy et al., 1971, 1973).

Recently, a reverse genetic approach led to the discovery of

a cytochrome P450 enzyme that is involved in secondary

alcohol and ketone formation in A. thaliana (Greer et al.,

2007). The protein is a mid-chain alkane hydroxylase (MAH1)

catalysing two consecutive reactions by first hydroxylating

the central CH2 group of alkanes, and then probably

re-binding the resulting secondary alcohol for a second

hydroxylation of the same carbon. Overall, this confirms the

original hypothesis that the pathway involves alkanes as

central intermediates that may be further oxidized depend-

ing on plant species and organ.

Waxes from certain taxa and/or organs can also contain

other compound classes (Figure 1), most prominently aro-

matic esters and compounds with two hydroxyl or carbonyl

functions (diols, ketols, ketoaldehydes and diketones; Jetter

et al., 2006). These wax constituents can be regarded as

downstream or side products of the ubiquitous biosynthetic

reactions forming the common product classes as described

above. This implies that additional enzymes, expressed at

high levels in certain plant species, can intercept intermedi-

ates and/or final products of the ubiquitous pathways before

they are exported to the cuticle. As these enzymes can

apparently handle the pre-formed wax compounds, they

could be added in a modular fashion to the standard

pathways in heterologous expression systems. This would

allow stepwise addition and modification of secondary

functional group(s), and substantially increase the chemical

diversity of biotechnologically produced wax mixtures. The

necessary biochemical and molecular genetic information

on the biosynthesis of these compound classes is currently

not available. However, cloning and characterization of the

genes involved may become possible in the near future,

once the standard wax biosynthetic pathways are better

understood in A. thaliana, so that the rapidly growing

genomic information from other species (Pennisi, 2007)

can be further exploited.

With the isolation and characterization of a number of key

genes involved in modification of VLCFA precursors into the

diverse wax compound classes, important information on

the intracellular localization of wax biosynthetic pathways

has emerged. The site of primary alcohol formation appears

to be the ER, as shown by localization of the alcohol-forming

Arabidopsis enzyme CER4 after expression in yeast (Row-

land et al., 2006). This is in contrast with mammalian FARs,

which are associated with peroxisomes (Burdett et al., 1991;

Cheng and Russell, 2004a,b), and therefore the localization

of the CER4 FAR will have to be verified in planta.

Meanwhile, the subsequent enzyme in the wax biosynthetic

pathway, the wax ester synthase WSD1, has been localized

to the ER, (R.J., L.K., F. Li, X. Wu and A.L. Samuels,

University of British Columbia, Canada, unpublished

results). Similarly, the mid-chain alkane hydroxylase MAH1

(CYP96A15), which catalyses the last two steps of the alkane-

pathway in A. thaliana stems, is also confined to the

ER (Greer et al., 2007). These two downstream pathway

enzymes are thus co-localized with the VLCFA-generating

FAEs (Kunst and Samuels, 2003; Xu et al., 2002; Zheng et al.,

2005), and it is very likely that the entire wax biosynthesis

process occurs in a single subcellular compartment. All the

wax biosynthetic enzymes and precursors are therefore

expected to be present in the ER of epidermal cells, leading

to accumulation of all intermediates and products in the

extensive membrane system of this organelle.

Current applications and commercial sources of waxes

Wax applications

From the applied perspective, waxes are defined as mixtures

of lipophilic compounds that are solid at room temperature,

range from transparent to opaque, and are ductile and easy

to polish (Illmann et al., 1983; Warth, 1956). Physical

parameters used to characterize waxes include hardness,

cure speed, melting point or range, pour point, viscosity,

(low) surface tension, adhesive strength, optical transpar-

ency and durability, and thermal expansion coefficient

(Anwar et al., 1999; Imai et al., 2001; Kim and Mahlberg,

1995; Kobayashi et al., 2005; McMillan and Darvell, 2000).

Due to their special properties, waxes are used as

lubricants, adhesives, coatings, sealants, impregnation

materials and adjuvants in formulations of (bio)active com-

pounds. A wide range of commercially important final

products rely on waxes, including automobiles, textiles,

papers and specialty inks, pesticides, candles, plastics

and wood–plastic composites, furniture and shoe polish,

household cleaners, cosmetics, dental treatment products,

drugs (lozenge coating) and food (chewing gum, cheese

packaging, confectionery coating).

Wax sources

To meet the demand for material applications, waxes are

currently generated by chemical syntheses, obtained from

geological deposits originating from past organisms (fossil

waxes) or obtained from living organisms (recent waxes)

(Illmann et al., 1983). The vast majority of these waxes are

based either on alkane or ester structures: synthetic waxes

are mainly generated by the Fischer–Tropsch process

(CO + H2) and olefin (ethylene, propylene) polymerization,

giving rise to mixtures of normal and branched alkanes (Ill-

mann et al., 1983; Schulz, 1999; Warth, 1956). Fossil waxes,

on the other hand, are extracted from crude oil and coal

deposits, yielding alkanes and alkyl ester mixtures (together

with the corresponding free acids and alcohols), respectively

(Illmann et al., 1983; Warth, 1956).

Beeswax and wool wax are the prime commodities of

recent waxes from animal sources (Tulloch, 1971; Warth,



1956), whereas the most important plant sources for com-

mercial production of waxes are carnauba (Copernicia

cerifera), candelilla (Euphorbia cerifera and E. antisyphiliti-

ca), ouricouri (Syagros coronata), sugar cane (Saccharum

sp.) and jojoba (Simmondsia chinensis). All these recent

waxes are relatively rich in aliphatic esters, with varying

overall chain lengths of both acyl and alkyl groups, and

contain characteristic admixtures of cinnamates, hydroxy-

esters and lactones, steryl esters, estolides and alkanes

(Basson and Reynhardt, 1988; Holloway, 1984; Illmann et al.,

1983; Lamberton and Redcliffe, 1960; Regert et al., 2005;

Vandenburg and Wilder, 1967; Warth, 1956).

Chemical diversity of commercial waxes

The chemical diversity that is currently available is relatively

broad for the wax alkanes, which include a wide variety of

isomeric branching patterns and chain lengths. For example,

alkane isomers with various patterns of methyl branches can

be synthesized through polymerization of propylene and/or

co-polymerization of propylene and ethylene (Illmann et al.,

1983; Warth, 1956). Furthermore, broad mixtures of (syn-

thetic and fossil) alkanes with diverse chain lengths can be

distilled into mixtures with desired chain-length ranges and

average carbon numbers, or even purified into single chain

lengths (Illmann et al., 1983; Warth, 1956).

The chemical nature of wax esters allows a similar

diversity of isomers and chain lengths through variation of

acyl and/or alcohol carbon numbers (Figure 3). However,

this diversity is presently not commercially exploited,

because the natural wax sources are characterized by ester

mixtures with relatively narrow ranges of chain lengths in

their acyl and alcohol moieties, and therefore also of overall

ester chain lengths (Illmann et al., 1983). To increase the wax

ester diversity and expand the array of ester applications,

chemical variations beyond those available in the current

sources will have to be explored. Examples of highly

desirable modifications in wax ester structures include

in-chain and x-terminal functional groups on the alkyl

and/or on the acyl chains, as well as further variations in

average chain lengths and chain-length distributions. The

target wax esters will also have to accumulate to high levels

in waxes of the source plants to make them viable industrial

raw materials. These goals can only be accomplished by

genetic engineering of wax biosynthetic pathways in oilseed

crops as described below.

Because of their special chain-length composition, some

of the wax ester mixtures from natural sources have proven

to be important commercial commodities. For example, wax

esters consisting of 20:1 fatty acid bonded to 20:1 and 22:1

alcohols are known to have outstanding lubrication proper-

ties combined with high resistance to hydrolysis and

oxidation (Carlsson, 2006). It has been estimated that there

is a market for millions of tonnes of these esters in

lubrication alone (Carlsson, 2006). However, these special

wax commodities can currently be commercially extracted

only from jojoba seeds, i.e. their production relies on the

agriculture of a single plant species (Carlsson, 2006; Purcell

et al., 2000; Yermanos, 1975). Jojoba cultivation is limited by

special growth conditions and low yields with respect to

time and agricultural area, resulting in the high cost of jojoba

oil and its almost exclusive use for high-value products such

as cosmetics and specialty lubricants. Genetic engineering

of the jojoba-type wax biosynthetic pathway in a conven-

tional oilseed crop would result in a new cost-effective

supply of these wax esters and enable their extensive use.

Exploitation of plant cuticular waxes

With the exception of the esters produced in jojoba seeds, all

other commercial plant waxes are harvested more or less

directly from plant surfaces, where they are deposited by the

epidermal cells. Cuticular wax biosynthesis is largely con-

trolled by developmental genetic programs, resulting in

fairly constant, specific compositions for each plant species

and organ. Plant cuticular waxes are therefore chemically

much more diverse than all the other wax sources, and this

greater chemical diversity goes hand in hand with the vari-

ations in wax physical properties that are desirable for

industrial applications. At present, however, the chemical

diversity of plant cuticular waxes is not being exploited

because waxes are commercially harvested from only a

small number of plant species. For example, the carnauba

palm (Copernicia cerifera) is grown exclusively for cuticular

wax production. Its large leaves are covered by an excep-

tionally thick layer of wax reaching a coverage of

300–1000 lg cm)2 of plant surface (Tulloch, 1976). This

greatly facilitates mechanical wax harvest, but yields only

10–100 kg per hectare (Da Silva et al., 1999; Johnson and

Nair, 1985). The vast majority of other plant species have leaf

wax coverages in the range of 1–100 lg cm)2 (Jetter et al.,

2006), but these low wax amounts can be offset by large

surface areas reached in crop fields (Gower et al., 1999). For

example, wheat fields are estimated to contain approxi-

mately 10–200 kg of wax per hectare (Austin et al., 1986;

Bianchi and Corbellini, 1977). However, substantial invest-

ments would be necessary to make harvesting this wax

source commercially viable. Sugar cane (Saccharum sp.) is

the only crop species from which cuticular wax is currently

exploited as a side-commodity, as wax is easily accessible

by extraction of the filter cakes from sugar production.

Approximately 40–240 kg of wax can be produced per

hectare of sugar cane, assuming average crop yields of

50 000 kg ha)1, with filter cakes amounting to 4% of the

mass and waxes to 2–12% of the filter cake (US patent

3931258; Paturau, 1982; Azzam, 2006; FAOSTAT, 2008).

In addition to the modest wax coverages and the lack of

structural diversity in the wax mixtures, wax utilization from



plant surfaces is also limited by the poor agronomic

properties and special growth conditions of plant species

currently used for wax production. In order to make the

surface wax a lucrative side-commodity in the future, the

wax coverage and composition of temperate plant species

would have to be genetically manipulated in a controlled

way. This is currently not feasible, however, because neither

the regulation of cuticle-forming genes nor the effect of

changed wax composition on the critical cuticle functions

have been investigated in any detail.

Potential for plant wax production in seeds

The apparent shortcomings of wax production on plant

surfaces can be circumvented by genetic engineering

approaches using established high-yielding oil crops as a

platform. By introducing wax biosynthetic pathways into

oilseeds, waxes with optimal chemical compositions for

various specialty markets could be produced, including

high-value lubricants, cosmetics and pharmaceuticals as

well as high-energy fuels. Potential wax yields from oilseed

engineering can be estimated based on the current yields for

major plant oil commodities. For example, Brassica seed oil

yields are in the range of 500–4000 kg ha)1, with typical

values for Canadian canola between 1500 and 1800 kg ha)1

(FAOSTAT, 2008). Even though wax yields from engineered

oilseed crops will probably be lower than the current oil

amounts, this would represent a several-fold increase over

current surface wax yields. That this estimated potential is

realistic can be seen from comparison with jojoba, the only

species known to accumulate wax in its seeds, which yields

75–750 kg of wax per hectare (Botti et al., 1998).

Metabolic engineering for wax ester production

Metabolic engineering of high-yielding oilseed species is a

rapid, cost-effective and rational approach for mitigating

current limitations in the chemical diversity and yield of

surface wax crops. Success will require the following indi-

vidual steps to be accomplished: (i) elucidation of wax bio-

synthetic pathways, (ii) reconstitution of selected pathways,

one at a time, in transgenic systems, (iii) modification of

other lipid biosynthetic pathways by up- or down-regulation

of certain enzymes, to control flux and to generate appro-

priate product mixtures, and (iv) integration of additional

unique modification steps into pathways. The first step in

this process is nearing completion, and the second step is

currently being attempted. The remaining steps can be

tackled in the near future. The following example illustrates

the whole process. (i) Wax ester biosynthesis is relatively

well understood, with genes cloned and characterized from

at least one plant species for each enzymatic step involved

(Lardizabal et al., 2000; Metz et al., 2000; Rowland et al.,

2006). As the early FAE steps of this pathway are shared with

formation of sphingolipids (Dietrich et al., 2005; Zheng et al.,

2005), which are essential membrane components of all

cells, it is likely that sufficient quantities of VLCFA precursors

for wax ester production will be available in all target species

and tissues. (ii) Heterologous overexpression of a fatty acyl

reductase (FAR) together with a wax ester synthase (WS)

should therefore lead to wax ester formation. (iii) Wax ester

formation can be manipulated to enhance flux or generate

novel products. For example, downregulation of triacyl-

glycerol biosynthesis competing for fatty acid percursors

should increase wax ester production. On the other hand,

up-regulation of steroid biosynthesis should increase the

levels of steryl alcohols and result in greater production of

steryl esters and/or mixtures of wax and steryl esters. (iv) To

further increase the wax ester diversity, additional enzymes

may be co-expressed that would lead to the hydroxylation,

desaturation or other modifications of the hydrocarbon

chains of either the acyl or alkyl moieties.

Proof of concept exists that jojoba-type wax esters (C38–

C44) can be produced at high levels by engineering of

oilseeds (Lardizabal et al., 2000). A recent study concluded

that production of wax esters by introduction of a three-

enzyme biosynthetic pathway in the crucifer Crambe abyss-

inica is a viable enterprise for the EU (Carlsson, 2006). This

may lead to high volume production of wax esters at

substantially reduced cost, and to their use for general

automotive lubrication applications, for example, as

transmission and hydraulic fluids.

Wax esters with a vast array of compositions of constit-

uent fatty acids and alcohols are present in various plant

species (Figures 3 and 4). Unfortunately, it is currently not

clear whether the chain-length compositions of esters from

various plant species are governed by the chain-length

specificities of the enzymes involved and/or by substrate

availability. Chemical evidence for A. thaliana stem wax

showed that epidermal ester biosynthesis was limited by

wax alcohol pools, but the study did not address enzyme

specificity (Lai et al., 2007). Biochemical characterization of

various wax ester synthases is currently under way. Once

the substrate specificities of these enzymes are known, it will

be possible to increase the chemical diversity of wax esters

through introduction of the desired enzymes in transgenic

crops. The various types of wax esters will have unique

properties and will serve as substrates for the production of

high-value specialty lubricants, cosmetics and pharmaceu-

ticals. Additional wax ester diversity can be generated by

engineering of artificial enzymatic steps into pathways that

do not normally occur in nature in a single species, to

introduce novel functional groups in either the acid or

alcohol moieties of the esters.

Examples of novel wax products with broad industrial

applications include esters containing acyl and/or alkyl

moieties with C=C double bonds, cyclopropane rings and

methyl branches. Interestingly, a recent study on wax



hydrocarbons in barley spikes showed that all three struc-

tural features are biosynthetically related (von Wettstein-

Knowles, 2007), implying that a small set of enzymes can

convert elongated wax precursors into these compounds. It

was hypothesized that a desaturase introduces a double

bond with high positional specificity, and then a cyclopro-

pane synthase and/or a methyl transferase generate(s)

branched structure(s). Although the exact nature of the

involved enzymes remains to be determined, their potential

biochemical function makes them very important targets

for cloning and future wax engineering studies.

Esters consisting of C20 and C22 alcohols and a hydroxy

fatty acid, for example ricinoleic acid (18:1 ) OH), are

another type of novel wax product. The presence of hydroxy

fatty acids disrupts the packing of hydrocarbon chains,

thereby reducing the melting temperature of the wax esters

and improving their lubrication properties at low tempera-

tures (Carlsson, 2006).

Wax ester fatty acid and alcohol components are also

valuable industrial raw materials, and wax esters containing

ricinoleic acid would be of particular interest due to high

demand for this chemical as an additive to base oils in

lubricant formulations, and as a feedstock for the manufac-

ture of nylon, surfactants, paints, cosmetics and biodegrad-

able polymers for medical applications (Ogunniy, 2006).

Castor oil, in which nearly 90% of acyl residues are ricinoleic

acid, is currently the only commercial source of hydroxy

fatty acids (Atsmon, 1989; Hogge et al., 1991). However,

castor beans are far from an ideal source, as they require

manual harvesting and contain complex allergens together

with the potent toxin ricin. Castor is also not a temperate

climate crop, making it necessary to import castor oil into

many countries from India and China, with irregular supplies

and fluctuating prices. Engineering of an existing oilseed

crop to replace castor bean as a major source of hydroxy

fatty acids is therefore highly desirable. To date, attempts to

produce oils rich in ricinoleic acid have not been successful

due to a general lack of understanding of the mechanisms

involved in channelling this unusual fatty acid from PC,

where it is synthesized, to storage triacylglycerols (Jaworski

and Cahoon, 2003). In contrast to triacylglycerol synthesis,

the enzymology of wax ester production is not as complex,

so engineering of crop plants that efficiently incorporate

ricinoleic acid into wax esters may be a viable alternative.

To further increase the structural diversity of genetically

engineered wax esters, biosynthetic genes from organisms

other than plants should be considered. Wax esters occur in

a wide variety of organisms (Kolattukudy, 1976), including

mammals (Jakobsson et al., 2006; Yen et al., 2005), birds

(Dekker et al., 2000; Haribal et al., 2005; Sweeney et al.,

2004), fungi (Cooper et al., 2000) and bacteria (Ishige et al.,

2002, 2003). Mammalian and bacterial ester formation is

fairly well understood, providing gene candidates for ester

synthases with various substrate/product chain-length pref-

erences (see description of ester biosynthesis above). Cor-

responding genes from birds, fungi and other organisms,

once they are identified and characterized, will help to

further broaden the repertoire of designer wax esters, for

example with methyl branched moieties. Similarly, genes

involved in the formation of cuticular alkanes of insects

(Howard and Blomquist, 2005) should be explored as

candidates for engineering hydrocarbons (see below).

The biosynthetic pathways for the production of novel

commercial wax esters can be engineered in oilseed crops

that are well suited for their synthesis and utilization. At

present, two crop species are being considered as platforms

for ester production, Crambe abyssinica and Brassica cari-

nata (Carlsson, 2006). These crops are not intended for food

production and will grow wherever other Brassica oilseed

crops are cultivated. C. abyssinica has an advantage over

B. carinata in that it is a high-yielding crop, and does not out-

cross with any other agricultural species (Carlsson, 2006).

However, despite its inferior seed yield and some

out-crossing with other Brassica crops, B. carinata may

have preference because it can be easily and efficiently

transformed.

Possible bottlenecks for wax production in oilseeds will

also have to be addressed for each species, including the

low germination rates of transgenic lines and the intracel-

lular autotoxicity of waxes accumulating in seed embryo

cells, whereas they would be exported to the plant surface

when produced in epidermal cells (Bird et al., 2007; Panik-

ashvili et al., 2007; Pighin et al., 2004). In addition, the

physical behaviour of VLCFA derivatives in seeds will have

to be tested in these transgenic crops. It has to be noted that

jojoba, the only currently available model for seed wax

accumulation, has wax esters with relatively short chain

lengths and a substantial amount of unsaturated acyl and

alcohol moieties. The resulting low melting points make

jojoba wax esters liquid at ambient temperatures. In con-

trast, longer-chain fully saturated esters are solid at room

temperature, and it is not clear whether this might affect

their accumulation in transgenic seeds.

Potential for biotechnological alkane production

Alkanes, the other large group of currently used very-long-

chain wax compounds, can be generated by chemical syn-

thesis and extracted from fossil sources with sufficient

chemical diversity and at very low cost (Schulz, 1999; Warth,

1956). It is therefore not commercially attractive to produce

alkane-rich waxes using biotechnological approaches.

Nevertheless, biosynthesis of cuticular alkanes has great

potential for application in commodities other than waxes.

One important future market for these hydrocarbons is in the

fuel sector, where gasoline and diesel are currently provided

by crude fossil oil consisting of various hydrocarbons. Much

of our transportation system relies on these hydrocarbons,



because the highly reduced carbon contained in them has

maximum chemical energy. Hydrocarbons can be replaced

to some extent by other compounds including ethanol and

biodiesel (Doran-Peterson et al., 2008; Dyer et al., 2008;

Pauly and Keegstra, 2008), but for some applications (e.g.

aircraft), high-energy hydrocarbon fuels will remain essen-

tial. Therefore, it is highly desirable to develop renewable

hydrocarbon sources.

Production of hydrocarbon-rich biofuels can be accom-

plished by biotechnological approaches harnessing photo-

synthetic organisms. To that end, a number of enzymes have

to be combined so that plant compounds, most importantly

fatty acids, can be utilized and transformed into the desired

products. While the enzymes necessary for the hydrocarbon

assembly have not been characterized from any organism to

date, they are known to be present in the epidermis of higher

plants, where they are capable of converting fatty acids into

the hydrocarbons that accumulate in the cuticular wax

deposited on the plant surface (see above). The cuticular

hydrocarbons are synthesized at too low a level for direct

industrial use. However, this system serves as an ideal

model for studying hydrocarbon formation, and can

be exploited for identification and isolation of genes

and enzymes for bio-gasoline production by genetic

engineering.

The central reaction of the alkane-forming pathway, i.e.

the transition step leading from even- to odd-numbered

carbon chains, is thought to involve the loss (rather than

addition) of one carbon atom from the acyl precursors (see

above). However, the biochemical details of this reaction

have not been determined. Several genes involved in alkane

formation have been cloned (CER1, CER2 and CER3/WAX2),

but all attempts to characterize their protein products have

failed so far. As multiple steps are probably required for

the conversion of fatty acids to hydrocarbons, additional

enzymes and corresponding genes might have to be iden-

tified to complete the pathway. Once all the genes/enzymes

have been established, the knowledge of this pathway can

be exploited for elucidation of analogous pathways leading

to the formation of short- and medium-chain, as well as

branched-chain, hydrocarbons.

Conclusions and perspectives

To overcome current limitations in the supply of waxes, to

generate wax commodities with new physical and chemical

properties that do not normally occur in a single species, and

to create entirely new arrays of wax-derived products with

desired chain lengths and functional groups for the chemical

industry, it will be necessary to harness the wax biosynthetic

diversity present in nature and genetically engineer wax

biosynthetic pathways capable of making such specialty

chemicals. These rationally designed wax biosynthetic

pathways will be introduced into the seeds of oil crops

dedicated to industrial use to avoid threat to the existing

food and feed systems, and will result in production of

renewable, high-value waxes that will be able to compete

with petroleum-based products, thus reducing our depen-

dency on fossil oils. In addition, a better understanding of

cuticular alkane biosynthesis might provide renewable

sources for high-energy hydrocarbons and lead to applica-

tions that do not rely on the physical properties of waxes.

Acknowledgements

This work has been supported by the Natural Sciences and Engi-neering Research Council (Canada), the Canada Research ChairsProgram, and the Canadian Foundation for Innovation.

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