Metabolic Engineering of Lipid Metabolism

A plant cell contains a plethora of lipid species, which are mainly represented by free fatty acids, glycolipids, phospholipids, waxes and neutral glycerolipids. Vegetable oil for human consumption almost exclusively consists of triacylglycerols (TAGs), which are composed of three fatty acids esterified to glycerol. TAGs dominate the storage lipid pool in oilseeds from which most plant oils are isolated. There are five fatty acids that are commonly esterified to triglycerides in the predominant oilseed crops (Dyer et al. 2008; see also Chap. 21), namely palmitic acid (16:0), stearic acid (18:0), oleic acid (18:1A9), linoleic acid (18:2A9,12) and a-linolenic acid (18:3A9,12,15). In order to obtain plant oil with improved technological or nutritional value by metabolic engineering, either unusual fatty acids that are highly abundant in exotic non-crops or novel fatty acids are produced in oilseeds, which can be cultivated on a large scale in contrast to the species from which these unusual fatty acids originate. As a prerequisite for efficient retrieval of the engineered fatty acids, these need to be targeted into storage lipids of seed oil.

Before we discuss potential applications of plants producing unusual fatty acids and the limitations and bottlenecks that were encountered upon engineering of glycerolipids in transgenic oilseed crops, we will briefly outline the route of triacylglycerol biosynthesis.

11.3.2.1 Biosynthesis of Storage Lipids

The biosynthesis of glycerolipids (triacylglycerols; TAGs) is a complex, non-linear pathway that involves three subcellular compartments, the chloroplast, the cytosol and the endoplasmatic reticulum (ER). Utilizing the photochemically generated reducing power in the chloroplast stroma, the de novo biosynthesis of palmitic acid (16:0), stearic acid (18:0) and oleic acid (18:1A9) from activated Acetyl-CoA building blocks takes place while the nascent acyl chain is covalently bound to acyl carrier protein (ACP) complex. Acyl-ACP can undergo two different fates:

  1. For the biosynthesis of phospholipids and galactolipids at the chloroplast envelope, the acyl chains can be directly transferred from acyl-ACPs to glycerol-3-phosphate and subsequently to lysophosphatidic acid (LPA). The product, phosphatidic acid (PA), represents the substrate for the production of phospho-lipids via the transfer of choline, ethanolamine or serine. However, phospholipid synthesis via the prokaryotic plastidic pathway has a very minor impact on seed oil production and is not discussed further.
  2. More importantly, fatty acids can be liberated from the plastidic acyl-ACP pool and transferred to the cytosol (Fig. 11.2), where they are: (i) re-esterified to coenzyme A and (ii) subsequently incorporated into the phospholipid pool at the ER. While acyl-CoAs are the substrates for elongases at the cytosolic leaflet of the ER, the cytochrome b5 containing desaturases FAD2 and FAD3 utilize the phospholipid-bound fatty acid pool (Ohlrogge and Browse 1995).

Precursors and intermediates for TAG biosynthesis at the ER derive from both free acyl-CoA thioesters and phospholipids. The route of TAG synthesis via glycerol-3-P and free acyl-CoA is known as the Kennedy pathway (Fig. 11.2) and involves glycerol-3-phosphate acyltransferase (GPAT), lysophosphatidic acid acyl-transferase (LPAAT), phosphatidic acid phosphatase (PAP) and diacylglycerol acyltransferase (DGAT). In each of the three acyl transferase steps (GPAT, LPAAT, DGAT), one more acyl chain is esterified to the glycerol backbone. Alternatively (see Fig. 11.2), fatty acids can be directly transferred from the phospholipid pool into TAG by phospholipid:diacylglycerol acyltransferase (PDAT) or enter the diacylglycerol (DAG) pool by the reversible removal of the phospholipid head group via choline phosphotransferase (CPT). DAG can then be utilized by DGAT or PDAT to yield TAG.

In the production of novel plant oils, both DGAT (Jako et al. 2001; Yu et al. 2006) and PDAT (Dahlqvist et al. 2000) were found to represent rate limiting steps for the entry of heterologously produced fatty acids into the TAG pool in metabolically engineered oilseeds (Bates et al. 2007), identifying the ultimate step

Fig. 11.2 Routes for triacylglycerol biosynthesis in oilseeds. Triacylglycerols (TAGs) can be synthesized from the glycerol-3-phosphate and the acyl-CoA pool via the Kennedy pathway by subsequent acylation of the triose backbone. Alternatively, the penultimate intermediate, diacylglycerol (DAG) and the TAG end-product can be generated via acyl transfer from the phospholipid pool. Please see the text for more detailed explanation (modified after Napier 2007 and Dyer et al. 2008). Acetyl-CoA Acetyl coenzyme A, Acyl-ACP acylated acyl carrier protein, CPT choline phosphotransferase, DGAT diacylglycerol acyltransferase, GPAT glycerol-3-phosphate acyltransferase, LPAAT lysophosphatidic acid acyltransferase, LPCAT lysophosphatidylcholine acyltransferase, Malonyl-CoA malonyl coenzyme A, PAP phosphatidic acid phosphatase, PDAT phospholipids:diacylglycerol acyltransferase

Glycerol Phosphate Pathway Dgat

Fig. 11.2 Routes for triacylglycerol biosynthesis in oilseeds. Triacylglycerols (TAGs) can be synthesized from the glycerol-3-phosphate and the acyl-CoA pool via the Kennedy pathway by subsequent acylation of the triose backbone. Alternatively, the penultimate intermediate, diacylglycerol (DAG) and the TAG end-product can be generated via acyl transfer from the phospholipid pool. Please see the text for more detailed explanation (modified after Napier 2007 and Dyer et al. 2008). Acetyl-CoA Acetyl coenzyme A, Acyl-ACP acylated acyl carrier protein, CPT choline phosphotransferase, DGAT diacylglycerol acyltransferase, GPAT glycerol-3-phosphate acyltransferase, LPAAT lysophosphatidic acid acyltransferase, LPCAT lysophosphatidylcholine acyltransferase, Malonyl-CoA malonyl coenzyme A, PAP phosphatidic acid phosphatase, PDAT phospholipids:diacylglycerol acyltransferase

Lysophosphatidyl choline (LPC)

Lysophosphatidyl choline (LPC)

of TAG biosynthesis as a committed entry site for fatty acids. However additional bottlenecks for the flux of novel fatty acids into TAG were identified, which are discussed later.

11.3.2.2 Genetic Engineering of Plant Lipid Metabolism

The manipulation of lipid metabolism in genetically engineered plants provides an enormous economic potential. The world annual production of vegetable oils amounts to 128.2x106 t in 2007, which is only 30x lower than the annual production of crude mineral oil of 4100x 106 t/year. In contrast to mineral oil, plant oils represent both a renewable resource and a versatile commodity for food, feed and industrial applications. About 14% of the annual plant oil production is being used for industrial processing, 5% are used as feed and for biodiesel production, respectively, while the rest is consumed as human food (Metzger and Bornscheuer 2006; Durrett et al. 2008).

Soybean, oil palm, rapeseed and sunflower are the predominant oil crops in the world (Dyer et al. 2008; also covered in Chaps. 21, 24). Other important oil crops are peanut, cottonseed, palm kernel, coconut and olives. However, more than 60% of the annual vegetable oil production is derived from soybean and palm oil. As summarized by Dyer et al. (2008), seed oil from these major oilseeds is mainly composed of the five major fatty acids palmitic acid (16:0), stearic acid (18:0), oleic acid (18:1A9), linoleic acid (18:2A9,12) and a-linolenic acid (18:3A9,12,15). Vegetable oil enriched for fatty acids uncommon to these major oilseed crops and fatty acids with additional functions provide a huge potential as chemical feedstock for the industrial production of detergents, cosmetics, drying oil, paint, ink, specialized lubricants or plastics providing a much higher versatility than mineral oil (Metzger and Bornscheuer 2006; Dyer et al. 2008). Consequently, engineering the lipid composition of seed oil has mainly followed three objectives: (i) to produce unusual fatty acids in oil crops that are of special value as chemical feedstock, (ii) to generate a fatty acid composition optimized for chemical processing and (iii) to introduce fatty acids with a special nutritional value like very long polyunsaturated fatty acids (VL-PUFAs).

In the following, we discuss the current advance in the production of: (i) unusual, short chain fatty acids like lauric acid (12:0), caprylic acid (8:0) and capric acid (10:0), (ii) long-chain fatty acids like erucic acid (22:1) and very-long-chain polyunsaturated fatty acids (VL-PUFAs) like arachidonic acid (AA; 22:4), eicosapenta-noic acid (EPA; 20:5) and docosahexaenoic acid (DHA; 22:6) as well as (iii) various fatty acids with additional functional groups in transgenic oilseed crops.

Unusual Medium-Chain Fatty Acids

Glycerolipids (TAGs) containing medium-chain acyl residues are of outstanding interest for the use as biofuel. Medium-chain TAGs are devoid of two major disadvantages intrinsic to conventional biodiesel consisting of TAG containing the five major fatty acids (Durrett et al. 2008). First, complications caused by biodiesel viscosity are alleviated when medium-chain TAGs are used, as TAG viscosity decreases with the chain length of the esterified fatty acids. The viscosity of regular biodiesel is tenfold higher compared to fossil fuel and is commonly prevented by utilizing fatty acid methyl esters (FAMEs) after the trans-esterification of the TAG fatty acids to methanol. Second, the coking index of medium-chain containing TAGs is lower, compared to other fuels.

The most distinguished example for metabolic engineering of medium-chain fatty acids in oilseeds is the generation of transgenic high-lauric acid (12:0) rap-eseed, which is currently approved for commercial use. In the initial approaches, the overexpression of a laureate-specific ACP from the California bay tree (Umbellularia californica) in Arabidopsis and Brassica napus (rapeseed) led to an accumulation of more than 50% of lauric acid in seed TAGs (Voelker et al. 1992; Wiberg et al. 2000). However, the sn-2 position of glycerol barely contained lauroyl residues in these transgenics. Additional overexpression of a LPAAT from coconut with high specificity for lauroyl-CoA increased the yield of laureate in rapeseed TAG to 67%, indicating that a limitation in the Kennedy pathway (see Fig. 11.2) restricted the accumulation of lauric acid in seed oil of the transgenics (Knutzon et al. 1999). Likewise, Cuphea lanceolata, which accumulates more than 80% of capric acid (10:0) in seed TAG was found to contain one set of GPAT and LPAAT specific for medium-chain acyl-CoAs and an DGAT that preferentially funnels di-medium-chain DAGs into TAG (Dehesh 2001). The specificities of these three Kennedy pathway enzymes obviously leads to an effective channelling of medium-chain fatty acids into Cuphea TAG.

Attempts to introduce valuable medium-chain fatty acids like capric acid (10:0) or caprylic acid (8:0) into rapeseed TAG were less successful, leading to 8% and 30% medium-chain acyls residues in rapeseed oil (Wiberg et al. 2000). However, the acyl-CoA pool in the transgenic seeds was dominated by the introduced medium-chain fatty acids, again indicating that the incorporation into glycerolipids via the Kennedy pathway was the limiting step preventing a high yield of caprylic and capric acid in the TAG pool (Larson et al. 2002).

Unusual Long-Chain Fatty Acids

Coriander and Thunbergia alata seed oil contain more than 80% of the unsusual monoenoic fatty acids petroselinic acid (18:1A6) and 16:1A6, respectively, both of which are valuable precursors for the production of various plastic polymers and cyclic hydrocarbon skeletons. Interestingly, both unusual fatty acids are synthesized by plastidic acyl-ACP desaturases. Palmitoyl-ACP (16:0-ACP) is utilized as a substrate for desaturation at the A4 and A6 position, respectively and the monoenoic fatty acid products are targeted to seed TAG via the phospholipid pool at the ER (Cahoon and Ohlrogge 1994; Schultz et al. 2000).

The 16:1 A4 product of the coriander desaturase is then subsequently elongated to yield petroselinic acid, while the 16:1A6 fatty acid is a direct product of the Thunbergia desaturase. The accumulation of these two unusual monoenoic fatty acids in transgenic Arabidopsis overexpressing the coriander and Thunbergia ACP-desaturases amounted to less than 15% of total seed TAG (Suh et al. 2002). In coriander, specific ACP, 3-ketoacyl-ACP synthase and thiesterase are present for the synthesis of petroselinic acid in plastids (Suh et al. 2002), suggesting that an inefficient substrate channelling between the prokaryotic pathway enzymes in Arabidopsis and the heterologously expressed desaturase may be the cause to the relatively low abundance of petroselinic acid in seed oil of the transgenics.

In contrast to petroselinic acid, erucic acid (22:1 A13) is produced in high amount in oilseed rape and other Brassicaceae. However, erucic acid is largely restricted to the sn-1 and sn-3 positions of TAG. Again, the specificity of the endogenous LPAAT seems to prevent the incorporation of erucic acid at the sn-2 position, identifying the same bottleneck that limited lauric acid accumulation in TAG of transgenic rapeseed. When an LPAAT from Limanthes specific for erucic acid and the endogenous FAE1 elongase were overexpressed in parallel, the TAG pool of the resulting transgenic rapeseed contained more than 70% erucic acid (Nath et al. 2006).

The production of the very-long-chain polyunsaturated fatty acids (VL-PUFAs) AA (an o6-fatty acid), EPA (an o3-fatty acid) and DHA (an o3-fatty acid) has drawn considerable attention due to their importance for human nutrition. Furthermore, the application of VL-PUFAs isolated from transgenic oilseed crops as a feed supplement to enable more sustainable salmon farming was supposed (Cahoon et al. 2007). Nevertheless, the production of VL-PUFAs in transgenic plants is complicated as it involves several cycles of desaturation and chain elongation of the endogenous precursors linoleic acid (18:2) and a-linolenic acid (18:3). As outlined in the previous section, the substrates for fatty acid desaturases are PC bound fatty acids, while elongases use free acyl-CoAs as their substrates, necessitating a substrate shuttling between the phospholipid and the acyl-CoA pool. Metabolic engineering of transgenic plants for VL-PUFA production has been accomplished by the introduction of several desaturases and elongases in Arabidopsis and Brassica juncea, totalling to up to nine transgenes (Wu et al. 2005). However, various routes can be chosen for VL-PUFA production. Apart from the A6 desaturase pathway, on which most attention has been focused to date, as it allows for the simultaneous biosynthesis of AA, EPA and DHA, the A8 desaturase pathway has proven an interesting alternative for the production of AA and EPA (Qi et al. 2004) Commonly, the maximum yield of VL-PUFAs in TAG of transgenic Arabidopsis, Brassica juncea and soybean obtained to date is low and ranges between 3% for DHA (Wu et al. 2005; Kinney 2006) and 8% for EPA (Qi et al. 2004; Wu et al. 2005). Recently, desaturases that act on acyl-CoAs have been identified from microalgae and higher plants, possibly making trans-esterification between acyl lipids and the acyl CoA pool dispensable in the future, which could also improve the yield of VL-PUFAs (Sayanova et al. 2007; Hoffmann et al. 2008).

Fatty Acids with Additional Functional Groups

Fatty acids with additional functional groups and their chemical derivatives represent an emerging valuable resource as industrial feedstocks for the production of cometics, lubricants, nylon, resins, polyvinylchloride (PVC), polyurethane and drying oils in paint and ink (Metzger and Bornscheuer 2006). Here, we briefly discuss unusual fatty acids that contain hydroxyl, epoxy and stereochemically unusually conjugated hexatriene groups, which have in common that they all are synthesized by divergent forms of the ER A12-oleic acid desaturase FAD2 (van de Loo et al. 1995; Lee et al. 1998; Dyer et al. 2002).

Ricinoleic acid is produced by a A12-hydroxylase and represents almost 90% of the castor bean (Ricinus communis) seed oil pool. Ricinoleic acid carries a hydroxyl group at the C-12 position in addition to a cis-double bond at the C-9 position, which renders it to a versatile substrate for various organic syntheses (Metzger and Bornscheuer 2006). Vernolic acid is synthesized by a A12-epox-genase and is abundant in the seed oil of, e.g. Vernonia galamensis, Crepis palaestina and Euphorbia lagascae. It contains an epoxy group at position C-12 in addition to the C-9 double bond and can be used as a binder in coatings and for the synthesis of enantiomerically pure products. Calendulic acid (18:3A8trans,10trans,12cis) and a-eleostearic acid (18:3A9cis,11trans,13trans), which are abundant in the seed TAG pool of marigold (Calendula officinalis) and the Chinese tung tree (Vernicia fordii), respectively, are used as drying oils in paints, inks and coatings. The conjugated hexatrienic double bonds of calendulic and a-eleostearic acid are synthesized from linoleic acid by a FAD2 conjugase (Cahoon et al. 1999).

Intriguingly, the overexpression of these three divergent FAD2 genes in Arabidopsis and soybean lead to less than 20% accumulation of ricinoleic, vernolic, calendulic and a-eleostearic acid in seed TAG as compared to 60% to 90% in the native species (Broun and Somerville 1997; Lee et al. 1998; Cahoon et al. 1999). Instead, oleic acids contents were increased in all these transgenics and the unusual fatty acids accumulated in the PC pool (Thomaeus et al. 2001; Cahoon et al. 2006), indicating that: (i) the conversion from oleic to linoleic acid by the endogenouse FAD2 desaturase is disturbed by the transgene expression and (ii) the channelling of the unusual fatty acids into the TAG pool is inefficient in the transgenics. In Vernonia galamensis, castor bean and tung tree, the respective DGAT2 isoforms were identified to specifically confer the transfer of vernolic, ricinoleic and a-eleostearic acid into seed oil, respectively (Cahoon et al. 2006; Kroon et al. 2006; Shockey et al. 2006), identifying DGAT as the potential bottleneck for the accumulation of these fatty acids in the TAG pool.

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