Plant Surface Lipid Biosynthetic Pathways and Their Utility for Metabolic Engineering of Waxes and Hydrocarbon Biofuels

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

HARNESSING PLANT BIOMASS FOR BIOFUELS AND BIOMATERIALS Plant surface lipid biosynthetic pathways and their utility for metabolic 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, and 2Department 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- These apparent shortcomings of plant surface wax produc- duced by epidermal cells (Riederer and Mu¨ ller, 2006). These tion can be circumvented through genetic engineering cuticular waxes are complex mixtures of C20–C34 straight- approaches using established high-yielding oil crops as a chain aliphatics derived from very-long-chain fatty acids platform. By introducing wax biosynthetic pathways into (VLCFAs), and in certain plant species also include alicyclic oilseeds, waxes with optimal chemical compositions for and aromatic compounds such as triterpenoids, alkaloids, various specialty markets could be produced, including phenylpropanoids and ﬂavonoids. Plant cuticular waxes high-value lubricants, cosmetics and pharmaceuticals, as serve as a protective barrier against water loss, UV light, well as high-energy fuels. pathogens and insects. In addition, they are valuable raw In this review, we present the chemical diversity of plant materials for a variety of industrial applications. Wax mix- cuticular wax mixtures and summarize our understanding of tures derived from different plant sources have unique the biosynthetic pathways involved in generating this chemical compositions that determine their physical diversity; provide an overview of commercial sources and properties, and therefore their potential applications and uses of waxes, and of current limitations of wax production; industrial value. discuss how engineering of wax biosynthetic pathways in At present, cuticular waxes are commercially harvested target crops might be exploited for the production of novel from only a small number of plant species, so the structural waxes with speciﬁc chain-length distributions in oilseeds; diversity of their wax constituents is limited. In addition, and describe how wax biosynthetic pathways can be used in these plant species are mostly grown in tropical areas and metabolic engineering of plants for the production of are agronomically not well suited to commercial production. hydrocarbon biofuels. This information complements recent

670 ª 2008 The Authors Journal compilation ª 2008 Blackwell Publishing Ltd Metabolic engineering of waxes and hydrocarbon biofuels 671 reviews that have focused on the chemical composition with no particular class predominating. For example, (Jetter et al., 2006), biosynthesis (Kunst et al., 2006) and alkanes, aldehydes, primary alcohols, fatty acids and alkyl biological functions of plant cuticular waxes (Bargel et al., esters each contribute 9–42% of the leaf wax of Zea mays 2006; Riederer and Mu¨ ller, 2006). (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 Plant cuticular wax composition and biosynthesis example, contains 89% of primary alcohols, together with Cuticular wax composition varies between different species only 0.2–9% of alkanes, aldehydes, fatty acids and alkyl and organs essentially in two respects: chain-length distri- esters (Giese, 1975). bution and compound class composition (Jetter et al., 2006). This diversity is established during wax biosynthesis in Compound chain length epidermal cells (Kunst et al., 2006), and involves two types of pathways: those for the elongation of fatty acid wax pre- Variation in the chain length of wax compounds is generated cursors to assorted chain lengths and those for modifying during synthesis of VLCFA wax precursors. This process them into wax components with various functional groups. involves several enzyme complexes in various cellular Aliphatic compound classes ubiquitously present in cuticu- compartments. The ﬁrst phase, the de novo fatty acid syn- lar wax mixtures are alkanes, primary alcohols, aldehydes thesis of C16 and C18 acyl chains, is catalysed by the soluble and fatty acids ranging in chain length between 20 and 34 fatty acid synthase (FAS) complex localized in the plastid carbons, as well as alkyl esters up to C60 in length (Figure 1). stroma (Ohlrogge and Browse, 1995; Ohlrogge et al., 1993), The cuticular waxes from many plant species comprise and proceeds through a cycle of four reactions utilizing roughly equal amounts of the various compound classes, intermediates attached to acyl carrier protein (ACP). In each

cycle, comprising the condensation of a C2 moiety originating 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-

Figure 1. Structures of major components occurring in plant cuticular wax ases predominantly use saturated fatty acids (Voelker, 1996). mixtures. The involvement of the FATB thioesterase in cuticular wax (a) Ubiquitous compound classes lacking functional groups (alkanes) or with biosynthesis has been confirmed by analyses of the Arabid- 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 opsis fatb mutant, which exhibits a major reduction in its methylene (CH2) groups, and can range from 18 to 32. wax load (Bonaventure et al., 2003). The specifics of fatty (b) Wax constituents with secondary functional groups accumulate to high acid export from the plastid, CoA esterification and transport concentrations in the wax of certain plant species, usually with very narrow chain-length and isomer distributions. Typical chain lengths and isomers are to the ER are not well understood. Fatty acids released from shown for selected combinations of hydroxyl and carbonyl functionalities. ACP by a thioesterase in the plastid undergo conversion to

ª 2008 The Authors Journal compilation ª 2008 Blackwell Publishing Ltd, The Plant Journal, (2008), 54, 670–683 672 Reinhard Jetter and Ljerka Kunst

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 enzyme¼ b-ketoacyl CoA synthase; KCR, b-ketoacyl CoA reductase; dehydratase, b-hydroxyacyl CoA dehydratase; CER10, enoyl CoA reductase; CER4, fatty acyl CoA reductase; WSD1, wax ester synthase; MAH1, mid-chain alkane hydroxylase).

acyl CoAs by a long-chain acyl CoA synthetase (LACS) in the Elongation of C16 and C18 fatty acids to VLCFAs outer envelope membrane. Of the nine LACS genes anno- involves cycles of four consecutive enzymatic reactions tated in the Arabidopsis genome (Shockey et al., 2002), only analogous to those of the FAS (Figure 2), and results in a one, LACS9, has been demonstrated to encode a plastid two-carbon extension of the acyl chain per cycle. The envelope enzyme (Schnurr et al., 2002). However, loss of chain lengths of aliphatic wax components are typically in function of LACS9 does not result in reduced export of acyl the range of 20–34 carbons, thus multiple elongation groups from the chloroplast, or a wax-deficient phenotype cycles are needed to extend the acyl chain to its final (Schnurr et al., 2002), suggesting that the LACS isozyme length. The differential effects of inhibitors on incorpora- primarily responsible for CoA esterification of fatty acids en tion of radiolabelled precursors into wax components of route to wax biosynthesis has yet to be identified. Move- various chain lengths, and analyses of mutants with ment of the fatty acyl group from the thioesterase to LACS defects in fatty acid elongation, demonstrated that has been proposed to occur by some type of facilitated sequential acyl chain extensions are carried out by several diffusion (Koo et al., 2004), but the exact mechanism of distinct FAEs with unique substrate chain-length specific- transfer is not known. An alternative model for fatty acid ities (von Wettstein-Knowles, 1993). Specificity of each export from the plastid was recently suggested by Bates elongation reaction resides in the condensing enzyme of et al. (2007). Their radiolabelling studies revealed that 16:0 the FAE complex (Lassner et al., 1996; Millar and Kunst, and 18:1 fatty acids synthesized de novo in the plastid can be 1997). Consistent with the requirement for fatty acyl incorporated into phosphatidylcholine (PC), perhaps by precursors of diverse chain lengths for the synthesis of direct acylation of lyso-PC. The acyl groups removed from cuticular waxes, a family of 21 FAE condensing enzyme- PC by acyl editing may then be fed into the acyl CoA like sequences has been identified in the A. thaliana pool. However, mechanistic details and the relevance of genome (Dunn et al., 2004). An unrelated ELO-like gene this process for epidermal wax formation have not been family of putative condensing enzymes, related to the established. Saccharomyces cerevisiae condensing enzymes ELO1, Translocation of fatty acids to the ER, where additional ELO2 and ELO3, has also been annotated (Dunn et al., acyl chain elongation and modification of VLCFAs to diverse 2004). It is not known how many of these putative aliphatic wax components take place, appears to involve condensing enzymes participate in wax production and plastid-associated membranes (PLAMs; Andersson et al., how many different condensing enzymes are needed

2007). Physical manipulation of GFP-labelled ER strands, for the elongation of a C18 to a C34 fatty acyl CoA, as using laser scalpels and optical tweezers, experimentally single condensing enzymes may catalyse multiple elon- veriﬁed the intimate connection between the plastid and the gation steps. The only wax-speciﬁc condensing enzyme ER of Arabidopsis leaf protoplasts. Therefore, PLAMs have characterized to date is CER6 (Fiebig et al., 2000; Hooker been proposed to be major routes for lipid transfer between et al., 2002; Millar et al., 1999), which is involved in the the two organelles. elongation of fatty acyl CoAs longer than C22.

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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.

Unlike the condensing enzymes, the other three enzyme demonstrating that it encodes a functional ECR. The A. tha- activities of the FAE complex, the b-ketoacyl reductase, liana ECR gene is ubiquitously expressed, and the protein b-hydroxyacyl dehydratase and enoyl reductase, are shared physically interacts with the Elo2p and Elo3p condensing by all VLCFA elongase complexes. Thus, these three enzymes when expressed in yeast (Gable et al., 2004). The enzymes have broad substrate specificities and generate a A. thaliana ECR was shown to be identical to CER10 (Zheng variety of acyl products used to make different classes of et al., 2005), the protein defective in one of the original lipids (Millar and Kunst, 1997). Because genetic screens in A. thaliana eceriferum mutants isolated by Koornneef et al. Arabidopsis did not result in isolation of mutants defective (1989). These eceriferum (literally ‘not bearing wax’) in the reductases or the dehydratase, suggesting that these mutants lack epicuticular wax crystals and therefore have enzymes are essential and/or functionally redundant (Millar glossy green inflorescence stems that can easily be recog- and Kunst, 1997), genes encoding the b-ketoacyl reductase nized in visual screens. Biochemical analysis of the cer10 and enoyl reductase were cloned by homology to the mutant demonstrated that the ECR gene product is involved corresponding sequences from Saccharomyces cerevisiae in the VLCFA elongation that is required for synthesis of all (Beaudoin et al., 2002; Kohlwein et al., 2001). Two b-ketoacyl the VLCFA-containing lipids, including cuticular waxes, seed reductase (KCR) genes are present in both the A. thaliana triacylglycerides and sphingolipids (Zheng et al., 2005). and maize (Zea mays) genomes. The maize genes, named Although the plant dehydratase remains unknown, recent GL8A and GL8B (Dietrich et al., 2005; Perera et al., 2003; Xu identification of the yeast b-hydroxyacyl dehydratase PHS1 et al., 2002), are not only expressed in the epidermis, but (Denic and Weissman, 2007) should permit cloning and also in internal tissues. Attempts to generate double characterization of this enzyme from plants. mutants by crossing gl8a · gl8b failed because embryos carrying both mutations were not viable. Thus, the KCR has Compound classes an essential function in plants, most likely in the production of sphingolipids (Dietrich et al., 2005). In addition to variations in the chain-length distributions, An A. thaliana single-copy gene was identified as an enoyl cuticular wax mixtures from diverse plants and plant reductase (ECR) candidate. Heterologous expression of the organs also contain various constituent compound classes. putative plant ECR gene rescued the temperature-sensitive These compounds vary in the nature and position of the lethality of yeast tsc13-1elo2D cells (Gable et al., 2004), (typically oxygen-containing) functional groups, with the

ª 2008 The Authors Journal compilation ª 2008 Blackwell Publishing Ltd, The Plant Journal, (2008), 54, 670–683 674 Reinhard Jetter and Ljerka Kunst extreme case of hydrocarbons that are devoid of functional including the green alga Euglena gracilis (Kolattukudy, groups (Jetter et al., 2006). Five or more parallel reactions 1970) as well as the angiosperms jojoba (Simmondsia (or pathways), all competing for the VLCFA CoA precursors, chinensis; Pollard et al., 1979), pea (Pisum sativum; Vioque can be envisioned leading to these ubiquitous wax com- and Kolattukudy, 1997) and A. thaliana (Rowland et al., ponents: (i) acyl reduction, (ii) esterification with an alkyl 2006). For example, functional expression of genes speci- alcohol, (iii) hydrolysis, (iv) aldehyde formation and (v) fying alcohol-forming FARs from jojoba (Metz et al., 2000) alkane formation (Figure 2). Knowledge of all the wax and A. thaliana (Rowland et al., 2006) in heterologous biosynthetic reactions will assist in their exploitation for systems demonstrated that alcohol biosynthesis from biotechnological production of individual compounds VLCFAs in these species is carried out by a single alcohol- and/or mixtures of compounds with specific combinations forming FAR. In contrast, biochemical feeding experiments of functional groups. that allowed isolation of an aldehyde intermediate suggest In virtually all vascular plants, wax compound classes that the two-step process of alcohol formation operates in with predominantly even numbers of carbons are produced Brassica oleracea (Kolattukudy, 1971). However, similar by the so-called acyl reduction pathway (Figure 2; Kunst biochemical evidence from other species and molecular et al., 2006). The most important of these compounds are information supporting the two-step process in any system primary alcohols and alkyl esters. The latter are essentially is currently lacking. dimeric compounds, in which the primary alcohols are It is generally assumed that primary alcohols serve as bonded to acyl groups, most commonly C16,C18 or VLCFAs precursors for ester biosynthesis. However, detailed analy- (>C20; Figure 3). Primary alcohols are thus central metabo- ses of esterified and free alcohols of various mutants of lites of wax biosynthesis, and their formation from VLCFA A. thaliana only recently demonstrated a clear correlation of CoA esters has been studied extensively. alcohol chain lengths in both types of compounds, indicat- Two reduction steps are required to transform acyl ing that the free alcohols are indeed incorporated into the precursors into primary alcohols, and aldehydes must wax esters (Lai et al., 2007). In addition, this study revealed occur as intermediates of the reaction sequence. It has that the levels of free alcohols are limiting for ester forma- been much debated whether both reduction steps are tion. Thus, a pool of primary alcohols, generated in the catalysed by one fatty acyl reductase (FAR), or whether two A. thaliana epidermal cells, is available either for export separate enzymes are necessary for alcohol formation. towards the cuticle or for esterification with an acyl CoA. There is currently substantial evidence for the existence of Other plant species exhibit large variations in compositions a one-enzyme system in a number of plant species, of cuticular wax esters, characterized in some cases by broad

Figure 4. Diversity of acyl and alkyl compositions 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.

ª 2008 The Authors Journal compilation ª 2008 Blackwell Publishing Ltd, The Plant Journal, (2008), 54, 670–683 Metabolic engineering of waxes and hydrocarbon biofuels 675 distributions of acyl and/or alkyl moieties and in other cases into alkanes and a second series of reactions modifying by relatively high preferences for certain isomers (Figure 4). them into secondary alcohols and ketones (Kolattukudy, In higher plants, mammals and bacteria, ester biosynthe- 1965). Subsequent experiments conﬁrmed the central role of sis is catalysed by one of three classes of wax synthase (WS) alkanes in this pathway (Kolattukudy, 1968; Kolattukudy and enzymes: jojoba-type WS, mammalian WS, and WS/DGAT Brown, 1974; Kolattukudy et al., 1974), either as intermedi- bifunctional enzymes. Jojoba-type WS uses a wide range of ates en route to mid-chain functionalized compounds or as saturated and unsaturated acyl CoAs ranging from C14 to end products if the downstream reactions are missing.

C24, with 20:1 as the preferred acyl and 18:1 as the preferred Overall, the second stage of the pathway is relatively alcohol substrate (Lardizabal et al., 2000). In A. thaliana, well characterized, whereas the first part remains poorly there are 12 wax synthases with high homology to the jojoba understood. WS, but none have yet been characterized. Mammalian WS Although conversion of VLCFA precursors into alkanes enzymes do not have homologues in plants, and have could proceed directly in one reaction, the net acyl decar- highest activities with C12–C16 acyl CoAs and alcohols boxylation is apparently brought about by a sequence of shorter than C20 (Cheng and Russell, 2004a,b). A bifunctional transformations. This multistep pathway is supported by the WS/DGAT enzyme from Acinetobacter calcoaceticus has a fact that a number of different A. thaliana mutants with preference for C14 and C16 acyl CoA together with C14–C18 alkane-deficient cuticular wax mixtures have been described alcohols (Sto¨ veken et al., 2005). Nearly a hundred WS/DGAT (Hannoufa et al., 1993; Jenks et al., 1995; Rashotte et al., homologues have been identified from over 20 other micro- 2001, 2004). Cloning of several of these mutated genes organisms so far (Wa¨ ltermann et al., 2007), and ten (CER1, CER2 and CER3/WAX2) revealed that the proteins sequences in the A. thaliana genome have also been anno- they encode contain motifs similar to known biosynthetic tated as WS/DGATs. One of these enzymes, WSD1, has been enzymes (Aarts et al., 1995; Ariizumi et al., 2003; Chen et al., characterized and shown to be responsible for the formation 2003; Kurata et al., 2003; Negruk et al., 1996; Rowland et al., of cuticular wax esters in A. thaliana stems (R.J., L.K., F. Li, 2007; Xia et al., 1996). While this suggests a potential X. Wu and A.L. Samuels, University of British Columbia, enzymatic role for these proteins, their exact function Canada, unpublished results). The enzyme utilizes mostly remains unknown. Due to this lack of molecular information, saturated C16 acyl CoA precursors, showing that this it is currently not possible to predict the exact number of upstream precursor of wax production must be co-localized reaction steps involved in the conversion of acyl precursors in the cell with the primary alcohols, which are synthesized into alkanes, the nature of these steps or the resulting far downstream in the wax biosynthetic pathway. intermediates. Two additional compound classes with predominantly Two alternative pathways have been proposed for the even-numbered chain lengths, aldehydes and free fatty conversion of acyl compounds into alkanes, which vary in acids, are also found in the wax mixture of most plant the central reaction in which a C1 unit is cleaved off (Bianchi, species, albeit usually at relatively low concentrations. 1995; Bognar et al., 1984; Chibnall and Piper, 1934). The Currently, our knowledge on their biosynthesis is very difference lies in the nature of the immediate precursor from limited. Formation of free fatty acids must involve hydrolysis which cleavage occurs and whether the C1 unit is CO or CO2 of the elongated acyl CoA precursors (Figure 2). However, it (decarbonylation versus decarboxylation). Only one model, is not clear whether this reaction occurs spontaneously or which describes alkane formation as the decarbonylation of whether it is enzyme-catalysed. Aldehyde formation an aldehyde intermediate, has been tested experimentally to requires reduction of acyl CoA precursors, and may occur some extent (Cheesbrough and Kolattukudy, 1984). How- as an intermediate step during alcohol formation (see ever, conclusive molecular genetic and biochemical evi- above), during alkane formation (see below), or indepen- dence for either model is lacking, leaving alkane formation dently of either of these pathways (Figure 2). Only a single as the least understood part of wax biosynthesis. wax aldehyde-forming reductase enzyme has been partially In A. thaliana leaves, alkanes are the major odd-num- purified to date, and the gene encoding this enzyme has not bered product, while a high level of secondary alcohols and been identified (Vioque and Kolattukudy, 1997). ketones accompanies alkanes in the stem wax, as well as in A separate set of wax biosynthetic reactions is responsible wax from B. oleracea leaves (Baker, 1974; Jenks et al., 1995). for the formation of compounds with predominantly odd In these instances, a second stage of the pathway is numbers of carbons (Figure 2). Examples of such compound additionally involved, transforming alkanes first into sec- classes include the alkanes, secondary alcohols and ketones ondary alcohols and then into ketones (Figure 2). This that occur together in many wax mixtures and typically reaction sequence is well-supported by chemical evidence share similar chain-length distributions (Jetter et al., 2006). correlating chain-length and isomer compositions of all Early biochemical experiments led to a model describing the three compound classes (Jenks et al., 1995), and by bio- biosynthesis of these compounds as a two-stage process, chemical evidence provided by feeding experiments and with a first set of reactions transforming VLCFA precursors detailed studies of label positions in resulting products

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(Kolattukudy and Liu, 1970; Kolattukudy et al., 1971, 1973). pathway in A. thaliana stems, is also confined to the Recently, a reverse genetic approach led to the discovery of ER (Greer et al., 2007). These two downstream pathway a cytochrome P450 enzyme that is involved in secondary enzymes are thus co-localized with the VLCFA-generating alcohol and ketone formation in A. thaliana (Greer et al., FAEs (Kunst and Samuels, 2003; Xu et al., 2002; Zheng et al., 2007). The protein is a mid-chain alkane hydroxylase (MAH1) 2005), and it is very likely that the entire wax biosynthesis catalysing two consecutive reactions by first hydroxylating process occurs in a single subcellular compartment. All the the central CH2 group of alkanes, and then probably wax biosynthetic enzymes and precursors are therefore re-binding the resulting secondary alcohol for a second expected to be present in the ER of epidermal cells, leading hydroxylation of the same carbon. Overall, this confirms the to accumulation of all intermediates and products in the original hypothesis that the pathway involves alkanes as extensive membrane system of this organelle. central intermediates that may be further oxidized depending on plant species and organ. Current applications and commercial sources of waxes Waxes from certain taxa and/or organs can also contain other compound classes (Figure 1), most prominently aro- Wax applications matic esters and compounds with two hydroxyl or carbonyl functions (diols, ketols, ketoaldehydes and diketones; Jetter From the applied perspective, waxes are defined as mixtures et al., 2006). These wax constituents can be regarded as of lipophilic compounds that are solid at room temperature, downstream or side products of the ubiquitous biosynthetic range from transparent to opaque, and are ductile and easy reactions forming the common product classes as described to polish (Illmann et al., 1983; Warth, 1956). Physical above. This implies that additional enzymes, expressed at parameters used to characterize waxes include hardness, high levels in certain plant species, can intercept intermedi- cure speed, melting point or range, pour point, viscosity, ates and/or final products of the ubiquitous pathways before (low) surface tension, adhesive strength, optical transpar- they are exported to the cuticle. As these enzymes can ency and durability, and thermal expansion coefficient apparently handle the pre-formed wax compounds, they (Anwar et al., 1999; Imai et al., 2001; Kim and Mahlberg, could be added in a modular fashion to the standard 1995; Kobayashi et al., 2005; McMillan and Darvell, 2000). pathways in heterologous expression systems. This would Due to their special properties, waxes are used as allow stepwise addition and modification of secondary lubricants, adhesives, coatings, sealants, impregnation functional group(s), and substantially increase the chemical materials and adjuvants in formulations of (bio)active com- diversity of biotechnologically produced wax mixtures. The pounds. A wide range of commercially important final necessary biochemical and molecular genetic information products rely on waxes, including automobiles, textiles, on the biosynthesis of these compound classes is currently papers and specialty inks, pesticides, candles, plastics not available. However, cloning and characterization of the and wood–plastic composites, furniture and shoe polish, genes involved may become possible in the near future, household cleaners, cosmetics, dental treatment products, once the standard wax biosynthetic pathways are better drugs (lozenge coating) and food (chewing gum, cheese understood in A. thaliana, so that the rapidly growing packaging, confectionery coating). genomic information from other species (Pennisi, 2007) can be further exploited. Wax sources With the isolation and characterization of a number of key genes involved in modification of VLCFA precursors into the To meet the demand for material applications, waxes are diverse wax compound classes, important information on currently generated by chemical syntheses, obtained from the intracellular localization of wax biosynthetic pathways geological deposits originating from past organisms (fossil has emerged. The site of primary alcohol formation appears waxes) or obtained from living organisms (recent waxes) to be the ER, as shown by localization of the alcohol-forming (Illmann et al., 1983). The vast majority of these waxes are Arabidopsis enzyme CER4 after expression in yeast (Row- based either on alkane or ester structures: synthetic waxes land et al., 2006). This is in contrast with mammalian FARs, are mainly generated by the Fischer–Tropsch process which are associated with peroxisomes (Burdett et al., 1991; (CO + H2) and olefin (ethylene, propylene) polymerization, Cheng and Russell, 2004a,b), and therefore the localization giving rise to mixtures of normal and branched alkanes (Ill- of the CER4 FAR will have to be verified in planta. mann et al., 1983; Schulz, 1999; Warth, 1956). Fossil waxes, Meanwhile, the subsequent enzyme in the wax biosynthetic on the other hand, are extracted from crude oil and coal pathway, the wax ester synthase WSD1, has been localized deposits, yielding alkanes and alkyl ester mixtures (together to the ER, (R.J., L.K., F. Li, X. Wu and A.L. Samuels, with the corresponding free acids and alcohols), respectively University of British Columbia, Canada, unpublished (Illmann et al., 1983; Warth, 1956). results). Similarly, the mid-chain alkane hydroxylase MAH1 Beeswax and wool wax are the prime commodities of (CYP96A15), which catalyses the last two steps of the alkane- recent waxes from animal sources (Tulloch, 1971; Warth,

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1956), whereas the most important plant sources for com- lubrication alone (Carlsson, 2006). However, these special mercial production of waxes are carnauba (Copernicia wax commodities can currently be commercially extracted cerifera), candelilla (Euphorbia cerifera and E. antisyphiliti- only from jojoba seeds, i.e. their production relies on the ca), ouricouri (Syagros coronata), sugar cane (Saccharum agriculture of a single plant species (Carlsson, 2006; Purcell sp.) and jojoba (Simmondsia chinensis). All these recent et al., 2000; Yermanos, 1975). Jojoba cultivation is limited by waxes are relatively rich in aliphatic esters, with varying special growth conditions and low yields with respect to overall chain lengths of both acyl and alkyl groups, and time and agricultural area, resulting in the high cost of jojoba contain characteristic admixtures of cinnamates, hydroxy- oil and its almost exclusive use for high-value products such esters and lactones, steryl esters, estolides and alkanes as cosmetics and specialty lubricants. Genetic engineering (Basson and Reynhardt, 1988; Holloway, 1984; Illmann et al., of the jojoba-type wax biosynthetic pathway in a conven- 1983; Lamberton and Redcliffe, 1960; Regert et al., 2005; tional oilseed crop would result in a new cost-effective Vandenburg and Wilder, 1967; Warth, 1956). supply of these wax esters and enable their extensive use.

Chemical diversity of commercial waxes Exploitation of plant cuticular waxes The chemical diversity that is currently available is relatively With the exception of the esters produced in jojoba seeds, all broad for the wax alkanes, which include a wide variety of other commercial plant waxes are harvested more or less isomeric branching patterns and chain lengths. For example, directly from plant surfaces, where they are deposited by the alkane isomers with various patterns of methyl branches can epidermal cells. Cuticular wax biosynthesis is largely con- be synthesized through polymerization of propylene and/or trolled by developmental genetic programs, resulting in co-polymerization of propylene and ethylene (Illmann et al., fairly constant, specific compositions for each plant species 1983; Warth, 1956). Furthermore, broad mixtures of (syn- and organ. Plant cuticular waxes are therefore chemically thetic and fossil) alkanes with diverse chain lengths can be much more diverse than all the other wax sources, and this distilled into mixtures with desired chain-length ranges and greater chemical diversity goes hand in hand with the vari- average carbon numbers, or even purified into single chain ations in wax physical properties that are desirable for lengths (Illmann et al., 1983; Warth, 1956). industrial applications. At present, however, the chemical The chemical nature of wax esters allows a similar diversity of plant cuticular waxes is not being exploited diversity of isomers and chain lengths through variation of because waxes are commercially harvested from only a acyl and/or alcohol carbon numbers (Figure 3). However, small number of plant species. For example, the carnauba this diversity is presently not commercially exploited, palm (Copernicia cerifera) is grown exclusively for cuticular because the natural wax sources are characterized by ester wax production. Its large leaves are covered by an excep- mixtures with relatively narrow ranges of chain lengths in tionally thick layer of wax reaching a coverage of their acyl and alcohol moieties, and therefore also of overall 300–1000 lgcm)2 of plant surface (Tulloch, 1976). This ester chain lengths (Illmann et al., 1983). To increase the wax greatly facilitates mechanical wax harvest, but yields only ester diversity and expand the array of ester applications, 10–100 kg per hectare (Da Silva et al., 1999; Johnson and chemical variations beyond those available in the current Nair, 1985). The vast majority of other plant species have leaf sources will have to be explored. Examples of highly wax coverages in the range of 1–100 lgcm)2 (Jetter et al., desirable modifications in wax ester structures include 2006), but these low wax amounts can be offset by large in-chain and x-terminal functional groups on the alkyl surface areas reached in crop fields (Gower et al., 1999). For and/or on the acyl chains, as well as further variations in example, wheat fields are estimated to contain approxi- average chain lengths and chain-length distributions. The mately 10–200 kg of wax per hectare (Austin et al., 1986; target wax esters will also have to accumulate to high levels Bianchi and Corbellini, 1977). However, substantial invest- in waxes of the source plants to make them viable industrial ments would be necessary to make harvesting this wax raw materials. These goals can only be accomplished by source commercially viable. Sugar cane (Saccharum sp.) is genetic engineering of wax biosynthetic pathways in oilseed the only crop species from which cuticular wax is currently crops as described below. exploited as a side-commodity, as wax is easily accessible Because of their special chain-length composition, some by extraction of the filter cakes from sugar production. of the wax ester mixtures from natural sources have proven Approximately 40–240 kg of wax can be produced per to be important commercial commodities. For example, wax hectare of sugar cane, assuming average crop yields of esters consisting of 20:1 fatty acid bonded to 20:1 and 22:1 50 000 kg ha)1, with filter cakes amounting to 4% of the alcohols are known to have outstanding lubrication proper- mass and waxes to 2–12% of the filter cake (US patent ties combined with high resistance to hydrolysis and 3931258; Paturau, 1982; Azzam, 2006; FAOSTAT, 2008). oxidation (Carlsson, 2006). It has been estimated that there In addition to the modest wax coverages and the lack of is a market for millions of tonnes of these esters in structural diversity in the wax mixtures, wax utilization from

ª 2008 The Authors Journal compilation ª 2008 Blackwell Publishing Ltd, The Plant Journal, (2008), 54, 670–683 678 Reinhard Jetter and Ljerka Kunst plant surfaces is also limited by the poor agronomic formation of sphingolipids (Dietrich et al., 2005; Zheng et al., properties and special growth conditions of plant species 2005), which are essential membrane components of all currently used for wax production. In order to make the cells, it is likely that sufficient quantities of VLCFA precursors surface wax a lucrative side-commodity in the future, the for wax ester production will be available in all target species wax coverage and composition of temperate plant species and tissues. (ii) Heterologous overexpression of a fatty acyl would have to be genetically manipulated in a controlled reductase (FAR) together with a wax ester synthase (WS) way. This is currently not feasible, however, because neither should therefore lead to wax ester formation. (iii) Wax ester the regulation of cuticle-forming genes nor the effect of formation can be manipulated to enhance flux or generate changed wax composition on the critical cuticle functions novel products. For example, downregulation of triacyl- have been investigated in any detail. glycerol biosynthesis competing for fatty acid percursors should increase wax ester production. On the other hand, up-regulation of steroid biosynthesis should increase the Potential for plant wax production in seeds levels of steryl alcohols and result in greater production of The apparent shortcomings of wax production on plant steryl esters and/or mixtures of wax and steryl esters. (iv) To surfaces can be circumvented by genetic engineering further increase the wax ester diversity, additional enzymes approaches using established high-yielding oil crops as a may be co-expressed that would lead to the hydroxylation, platform. By introducing wax biosynthetic pathways into desaturation or other modifications of the hydrocarbon oilseeds, waxes with optimal chemical compositions for chains of either the acyl or alkyl moieties. various specialty markets could be produced, including Proof of concept exists that jojoba-type wax esters (C38– high-value lubricants, cosmetics and pharmaceuticals as C44) can be produced at high levels by engineering of well as high-energy fuels. Potential wax yields from oilseed oilseeds (Lardizabal et al., 2000). A recent study concluded engineering can be estimated based on the current yields for that production of wax esters by introduction of a three- major plant oil commodities. For example, Brassica seed oil enzyme biosynthetic pathway in the crucifer Crambe abyss- yields are in the range of 500–4000 kg ha)1, with typical inica is a viable enterprise for the EU (Carlsson, 2006). This values for Canadian canola between 1500 and 1800 kg ha)1 may lead to high volume production of wax esters at (FAOSTAT, 2008). Even though wax yields from engineered substantially reduced cost, and to their use for general oilseed crops will probably be lower than the current oil automotive lubrication applications, for example, as amounts, this would represent a several-fold increase over transmission and hydraulic fluids. current surface wax yields. That this estimated potential is Wax esters with a vast array of compositions of constit- realistic can be seen from comparison with jojoba, the only uent fatty acids and alcohols are present in various plant species known to accumulate wax in its seeds, which yields species (Figures 3 and 4). Unfortunately, it is currently not 75–750 kg of wax per hectare (Botti et al., 1998). 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 Metabolic engineering for wax ester production availability. Chemical evidence for A. thaliana stem wax Metabolic engineering of high-yielding oilseed species is a showed that epidermal ester biosynthesis was limited by rapid, cost-effective and rational approach for mitigating wax alcohol pools, but the study did not address enzyme current limitations in the chemical diversity and yield of specificity (Lai et al., 2007). Biochemical characterization of surface wax crops. Success will require the following indi- various wax ester synthases is currently under way. Once vidual steps to be accomplished: (i) elucidation of wax bio- the substrate specificities of these enzymes are known, it will synthetic pathways, (ii) reconstitution of selected pathways, be possible to increase the chemical diversity of wax esters one at a time, in transgenic systems, (iii) modification of through introduction of the desired enzymes in transgenic other lipid biosynthetic pathways by up- or down-regulation crops. The various types of wax esters will have unique of certain enzymes, to control flux and to generate appro- properties and will serve as substrates for the production of priate product mixtures, and (iv) integration of additional high-value specialty lubricants, cosmetics and pharmaceu- unique modification steps into pathways. The first step in ticals. Additional wax ester diversity can be generated by this process is nearing completion, and the second step is engineering of artificial enzymatic steps into pathways that currently being attempted. The remaining steps can be do not normally occur in nature in a single species, to tackled in the near future. The following example illustrates introduce novel functional groups in either the acid or the whole process. (i) Wax ester biosynthesis is relatively alcohol moieties of the esters. well understood, with genes cloned and characterized from Examples of novel wax products with broad industrial at least one plant species for each enzymatic step involved applications include esters containing acyl and/or alkyl (Lardizabal et al., 2000; Metz et al., 2000; Rowland et al., moieties with C=C double bonds, cyclopropane rings and 2006). As the early FAE steps of this pathway are shared with methyl branches. Interestingly, a recent study on wax

ª 2008 The Authors Journal compilation ª 2008 Blackwell Publishing Ltd, The Plant Journal, (2008), 54, 670–683 Metabolic engineering of waxes and hydrocarbon biofuels 679 hydrocarbons in barley spikes showed that all three struc- erences (see description of ester biosynthesis above). Cor- tural features are biosynthetically related (von Wettstein- responding genes from birds, fungi and other organisms, Knowles, 2007), implying that a small set of enzymes can once they are identiﬁed and characterized, will help to convert elongated wax precursors into these compounds. It further broaden the repertoire of designer wax esters, for was hypothesized that a desaturase introduces a double example with methyl branched moieties. Similarly, genes bond with high positional speciﬁcity, and then a cyclopro- involved in the formation of cuticular alkanes of insects pane synthase and/or a methyl transferase generate(s) (Howard and Blomquist, 2005) should be explored as branched structure(s). Although the exact nature of the candidates for engineering hydrocarbons (see below). involved enzymes remains to be determined, their potential The biosynthetic pathways for the production of novel biochemical function makes them very important targets commercial wax esters can be engineered in oilseed crops for cloning and future wax engineering studies. that are well suited for their synthesis and utilization. At

Esters consisting of C20 and C22 alcohols and a hydroxy present, two crop species are being considered as platforms fatty acid, for example ricinoleic acid (18:1 ) OH), are for ester production, Crambe abyssinica and Brassica cari- another type of novel wax product. The presence of hydroxy nata (Carlsson, 2006). These crops are not intended for food fatty acids disrupts the packing of hydrocarbon chains, production and will grow wherever other Brassica oilseed thereby reducing the melting temperature of the wax esters crops are cultivated. C. abyssinica has an advantage over and improving their lubrication properties at low tempera- B. carinata in that it is a high-yielding crop, and does not out- tures (Carlsson, 2006). cross with any other agricultural species (Carlsson, 2006). Wax ester fatty acid and alcohol components are also However, despite its inferior seed yield and some valuable industrial raw materials, and wax esters containing out-crossing with other Brassica crops, B. carinata may ricinoleic acid would be of particular interest due to high have preference because it can be easily and efficiently demand for this chemical as an additive to base oils in transformed. lubricant formulations, and as a feedstock for the manufac- Possible bottlenecks for wax production in oilseeds will ture of nylon, surfactants, paints, cosmetics and biodegrad- also have to be addressed for each species, including the able polymers for medical applications (Ogunniy, 2006). low germination rates of transgenic lines and the intracel- Castor oil, in which nearly 90% of acyl residues are ricinoleic lular autotoxicity of waxes accumulating in seed embryo acid, is currently the only commercial source of hydroxy cells, whereas they would be exported to the plant surface fatty acids (Atsmon, 1989; Hogge et al., 1991). However, when produced in epidermal cells (Bird et al., 2007; Panik- castor beans are far from an ideal source, as they require ashvili et al., 2007; Pighin et al., 2004). In addition, the manual harvesting and contain complex allergens together physical behaviour of VLCFA derivatives in seeds will have with the potent toxin ricin. Castor is also not a temperate to be tested in these transgenic crops. It has to be noted that climate crop, making it necessary to import castor oil into jojoba, the only currently available model for seed wax many countries from India and China, with irregular supplies accumulation, has wax esters with relatively short chain and fluctuating prices. Engineering of an existing oilseed lengths and a substantial amount of unsaturated acyl and crop to replace castor bean as a major source of hydroxy alcohol moieties. The resulting low melting points make fatty acids is therefore highly desirable. To date, attempts to jojoba wax esters liquid at ambient temperatures. In con- produce oils rich in ricinoleic acid have not been successful trast, longer-chain fully saturated esters are solid at room due to a general lack of understanding of the mechanisms temperature, and it is not clear whether this might affect involved in channelling this unusual fatty acid from PC, their accumulation in transgenic seeds. where it is synthesized, to storage triacylglycerols (Jaworski and Cahoon, 2003). In contrast to triacylglycerol synthesis, Potential for biotechnological alkane production the enzymology of wax ester production is not as complex, so engineering of crop plants that efficiently incorporate Alkanes, the other large group of currently used very-long- ricinoleic acid into wax esters may be a viable alternative. chain wax compounds, can be generated by chemical syn- To further increase the structural diversity of genetically thesis and extracted from fossil sources with sufficient engineered wax esters, biosynthetic genes from organisms chemical diversity and at very low cost (Schulz, 1999; Warth, other than plants should be considered. Wax esters occur in 1956). It is therefore not commercially attractive to produce a wide variety of organisms (Kolattukudy, 1976), including alkane-rich waxes using biotechnological approaches. mammals (Jakobsson et al., 2006; Yen et al., 2005), birds Nevertheless, biosynthesis of cuticular alkanes has great (Dekker et al., 2000; Haribal et al., 2005; Sweeney et al., potential for application in commodities other than waxes. 2004), fungi (Cooper et al., 2000) and bacteria (Ishige et al., One important future market for these hydrocarbons is in the 2002, 2003). Mammalian and bacterial ester formation is fuel sector, where gasoline and diesel are currently provided fairly well understood, providing gene candidates for ester by crude fossil oil consisting of various hydrocarbons. Much synthases with various substrate/product chain-length pref- of our transportation system relies on these hydrocarbons,

ª 2008 The Authors Journal compilation ª 2008 Blackwell Publishing Ltd, The Plant Journal, (2008), 54, 670–683 680 Reinhard Jetter and Ljerka Kunst because the highly reduced carbon contained in them has dedicated to industrial use to avoid threat to the existing maximum chemical energy. Hydrocarbons can be replaced food and feed systems, and will result in production of to some extent by other compounds including ethanol and renewable, high-value waxes that will be able to compete biodiesel (Doran-Peterson et al., 2008; Dyer et al., 2008; with petroleum-based products, thus reducing our depen- Pauly and Keegstra, 2008), but for some applications (e.g. dency on fossil oils. In addition, a better understanding of aircraft), high-energy hydrocarbon fuels will remain essen- cuticular alkane biosynthesis might provide renewable tial. Therefore, it is highly desirable to develop renewable sources for high-energy hydrocarbons and lead to applica- hydrocarbon sources. tions that do not rely on the physical properties of waxes. Production of hydrocarbon-rich biofuels can be accomplished by biotechnological approaches harnessing photosynthetic organisms. To that end, a number of enzymes have Acknowledgements to be combined so that plant compounds, most importantly This work has been supported by the Natural Sciences and Engi- fatty acids, can be utilized and transformed into the desired neering Research Council (Canada), the Canada Research Chairs products. While the enzymes necessary for the hydrocarbon Program, and the Canadian Foundation for Innovation. 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 References the hydrocarbons that accumulate in the cuticular wax Aarts, M.G.M., Keijzer, C.J., Stiekema, W.J. and Pereira, A. (1995) deposited on the plant surface (see above). The cuticular Molecular characterization of the CER1 gene of Arabidopsis hydrocarbons are synthesized at too low a level for direct involved in epicuticular wax biosynthesis and pollen fertility. industrial use. However, this system serves as an ideal Plant Cell, 7, 2115–2127. Andersson, M.X., Goksor, M. and Sandelius, A.S. 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ª 2008 The Authors Journal compilation ª 2008 Blackwell Publishing Ltd, The Plant Journal, (2008), 54, 670–683 682 Reinhard Jetter and Ljerka Kunst

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