ABSTRACT

Breeding of high-erucic acid rapeseed (HEAR) is being focussed on the achievement of erucic acid (22:1) contents substantially higher than 45-55% in the seed oil. Rapid progress is mainly limited due to the lack of 22:1 in sn-2 position of the triacylglycerols (TAG) and the absence of trierucin. Molecular breeding approaches have led to the stepwise improvement of specific biosynthetic pathways in transgenic Brassica napus. Firstly, we have shown that down-regulation of the endogenous lysophosphatidic acid acyltransferase (LPAAT) and expression of the corresponding enzyme from Limnanthes douglasii R. Br. in developing rapeseeds leads to about 50% 22:1 in the sn-2 fatty acid composition and considerable amounts of trierucin. Secondly, with regard to the capacity of very long-chain fatty acid biosynthesis the role of the microsomal condensing enzyme (β-ketoacyl-CoA synthase, KCS) has been investigated by identifying and molecular characterizing more effective alleles and introducing additional KCS genes for 22:1 synthesis. The role of further enzymes putatively responsible for the improvement of 22:1 content is discussed.

INTRODUCTION

In the last years plant breeders and molecular biologists were especially interested in seed oils rich in erucic acid (cis-13-docosenoic acid, 22:1) for the production of basic feedstock being directed to several oleochemical niche markets. Among other cruciferous oilseeds high-erucic acid rapeseed (HEAR) is by far the most economical and readily available source of this very long-chain fatty acid (VLCFA). As Canola cultivars low in 22:1 are widely grown for the edible oil market, HEAR cultivars have been specifically bred for non-food purposes (Lühs and Friedt 1994, Friedt and Lühs 1998). At least two key steps have emerged as the privileged targets for the improvement of 22:1 production in rapeseed (B. napus): the capacity of microsomal fatty acid elongation and the alteration of the LPAAT activity leading to the biosynthesis of trierucin (trierucoylglycerol, 69:3). Though the modification of the triacylglycerol (TAG) biosynthetic pathway has led to a better understanding of biosynthesis and storage of VLCFA, further biochemical constraints are limiting fast success on the way to rapeseed oil with maximum 22:1 content (Frentzen 1998, Lühs et al. 1999).

BIOASSEMBLY OF TRIERUCIN IN RAPESEED OIL

In most members of the Brassicaceae and specifically in natural B. napus, erucic acid and other VLCFA are not incorporated into the central position of the glycerol backbone. This obstacle prevents the biosynthesis of trierucin and restricts the 22:1 content in rapeseed oil to a theoretical maximum of 66.7%. To overcome this biochemical limitation, the strategy is to clone genes which encode heterologous LPAAT enzymes and to insert these genes into appropriate B. napus genotypes conferring rapeseed with the ability to esterify 22:1 into the central position of the TAG (Frentzen 1998). Following molecular breeding procedures, the LPAAT gene of Limnanthes douglasii was cloned and the corresponding pRESS construct genetically engineered into different HEAR, especially the resynthesized rapeseed lines (Weier et al. 1997). Among the pRESS transformants (analyzed in pooled T2 seed oil fractions) those derived from RS lines showed the highest trierucin content (up to 9% 69:3 in ’TR-RS 224’), which can mainly be attributed to the higher 22:1 synthesizing capacity of these lines as compared to conventional HEAR cultivars used before in similar experiments (Lassner et al. 1995, Brough et al. 1996). Though displaying up to 40% sn-2 erucate and considerable amounts of trierucin in the seed oil, none of the pRESS transformants contained total 22:1 proportions higher than the range found in untransformed B. napus material. The same effect with regard to total erucic content was observed in a second generation of rapeseed transformants bearing the pALM1 construct (Münster et al. 1998). However, we were able to show that antisense inhibition of the endogenous rapeseed LPAAT and combined expression of the LdLPAAT gene leads to about 50% 22:1 in the sn-2 fatty acid composition accompanied by a slight increase of trierucin content (Fig. 1).

Keeping in mind that conventional HEAR has less than 1% sn-2 erucate, the genetic engineering procedure has been successful in changing the sn-2 fatty acid composition of rapeseed oil leading to a wide range of 4 to 50% sn-2 erucic acid (summarized in Frentzen 1998, Lühs et al. 1999).

IMPROVEMENT OF FATTY ACID ELONGATION CAPACITY

Once the esterification of the sn-2 position with 22:1 had been accomplished, it has become obvious, that the biosynthetic capacity of VLCFA synthesis is to be considered as a further target for metabolic engineering. VLCFA are synthesized by the acyl-CoA elongase catalyzing the addition of two carbon units from malonyl-CoA to an acyl-CoA similar to the reaction sequence of the plastidial fatty acid synthetase system. Among the constituent enzymes of the microsomal fatty acid elongase the condensing enzyme is regarded to catalyze the first and rate-limiting step determining the acyl chain length of the VLCFA produced (Cassagne et al. 1994, Millar and Kunst 1997). The sequences of FATTY ACID ELONGATION 1 (FAE1) genes encoding putative seed-specific condensing enzymes were firstly characterized in Arabidopsis thaliana (James et al. 1995) and jojoba (Simmondsia chinensis, Lassner et al. 1996). Subsequently a couple of homologous sequences were isolated from B. napus (Clemens and Kunst 1997, Barret et al. 1998, Fourmann et al. 1998, Han et al. 1998) and other Brassica species (cf. Fig. 2). With regard to the improvement of 22:1 content in B. napus, the role of KCS has been investigated by identifying and molecular characterizing more effective 22:1 alleles (Lühs et al. 1999) and introducing additional KCS genes in order to promote VLCFA synthesis (Han et al. 1998).

Figure 2: Phylogram based on the analysis of the amino acid sequences of the condensing enzyme and putative ones of the following plant species: B. napus 4 and 5, B. rapa, B. oleracea (Fourmann et al. 1998), B. napus 3 (Barret et al. 1998), B. napus 1 (Han et al. 1998), B. napus 2 (Clemens and Kunst 1997, Barret et al. 1998), A. thaliana 1 (James et al. 1995), B. juncea (Y11007), A. thaliana 2 (AL023094), A. thaliana 5 (AC004484), A. thaliana 6 (AC005818), Hemerocallis hybrid (AF082033), S. chinensis (Lassner et al. 1996), A. thaliana 4 (AC002411), A. thaliana 3 (AC003105), Lycopersicon esculentum (X83420); * partial cDNA sequence.

Following a genetic engineering approach, as described earlier by Lassner et al. (1996), the effect of specific KCS genes or alleles can be studied in Canola cultivars that show an almost complete abolishment of 22:1 synthesis. As shown in Figure 3, the integration of KCSb5 originating from the winter HEAR cv. ‘Askari‘ into the spring Canola variety ‘Drakkar‘ has led to the restoration of VLCFA biosynthesis in B. napus. In half seeds (T2 seeds) derived from certain transformants (T1 plants), the content of cis-11-eicosenate (20:1) and 22:1 has been increased from about 2% to more than 40% of total fatty acids, which clearly demonstrates that KCS is essential within the microsomal fatty acid elongation process.

CONCLUSION AND PERSPECTIVES

Concerted research efforts in plant breeding and genetic engineering of B. napus have led to the successive genetic alteration of relevant enzymes (e.g. LPAAT, KCS) being involved in the biosynthesis of 22:1-enriched TAG. Though the introduction of the Limnanthes LPAAT genes as well as the down-regulation of the inherent B. napus enzyme proved the feasibility of using acyltransferase genes in modifying the sn-2 composition, these routes did not cause an increase of overall 22:1 content in HEAR oil. Thus, one could assume that the levels of 22:1 in the seed acyl-CoA pool may be too low to support high levels of trierucin synthesis. In this concern, the transfer of cDNAs encoding KCS into Canola varieties was successful in restoring the deficiency of 22:1 synthesis, but this procedure was not sufficient to obtain higher amounts of 22:1 than in conventional HEAR - even not by using resynthesized rapeseed lines as recipient in genetic transformation experiments (data not shown).

This result suggests that the amount of 22:1 in transgenic B. napus could be limited by the level of the activities of the other enzymes of the acyl-CoA elongase complex, viz. β-ketoacyl-CoA reductase, β-hydroxyacyl-CoA dehydrase, and trans-2-enoyl-CoA reductase. In this case, introducing the genes encoding for all the proteins of the microsomal complex would be necessary to obtain a drastic increase in seed oil 22:1 content. Furthermore, VLCFA synthesis from oleic acid (18:1) has been demonstrated to require malonyl-CoA, which is essentially produced by the cytosolic homodimeric acetyl-CoA carboxylase (ACCase). On the other hand, there are strong indications that acyl-CoA synthetase does not play a direct role in supporting fatty acid elongation, and that phosphatidylcholine or another glycerolipid is a more likely donor of acyl primers to the elongase reaction than oleoyl-CoA. Consequently, the flux of 18:1 through distinct intermediate lipid pools before elongation or desaturation may be an additional factor limiting the availability of 18:1 moieties for elongation (Hlousek-Radojcic et al. 1995, 1998, Bao et al. 1998). Although appreciable effects can be achieved by expressing a single transgene, additional genes have to be included in a wholly sufficient approach towards rapeseed oil with a very high 22:1 content.

ACKNOWLEDGEMENTS

The authors are grateful for financial support granted by the Norddeutsche Pflanzenzucht Hans-Georg Lembke KG, Kleinwanzlebener Saatzucht AG, and Deutsche Saatveredelung Lippstadt-Bremen GmbH as well as the Bundesministerium für Bildung, Wissenschaft, Forschung und Technologie, Bonn, Germany (Grants BEO/22/0319412, 0310528, 0311156-311158 and 316601). We wish to thank Dr. K. Sonntag of the Bundesanstalt für Züchtungsforschung (BAZ) in Groß Lüsewitz as well as the Planta GmbH Einbeck for developing transformants derived from cv. ‘Drakkar‘.

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