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Davies, H.M. 1996. Engineering new oilseed crops from rapeseed. p. 299-306. In: J. Janick (ed.), Progress in new crops. ASHS Press, Alexandria, VA.

Engineering New Oilseed Crops from Rapeseed

H. Maelor Davies

  1. LAURICAL™
  2. HIGH-LAURIC OIL
  3. OTHER MEDIUM-CHAIN FATTY ACIDS
  4. ALTERED SATURATION
  5. HIGH-ERUCIC OIL
  6. ALCOHOLS AND WAX ESTERS
  7. LOOKING AHEAD
  8. REFERENCES
  9. Table 1
  10. Fig. 1
  11. Fig. 2
  12. Fig. 3
  13. Fig. 4
  14. Fig. 5
The engineering of new rapeseed oils has made tremendous progress in the last four years. In the II New Crops Conference I reported on the isolation of an unique enzyme involved in the biosynthesis of a valuable 12-carbon fatty acid, laurate (12:0), from the seeds of an undomesticated tree species. We had cloned the corresponding gene and were able to show that its expression in E. coli resulted in the production of large amounts of laurate by the bacterial culture (Davies et al. 1993). At that time there was eager anticipation of the forthcoming transgenic plant data. I can now report that laurate was produced in the transgenic seeds, and also that the high-lauric rapeseed line developed from them is now a new oilseed crop in commercial agriculture. The first commercial production of the resulting specialty oil, Laurical™, which contains 40 mol% laurate, took place last year (1995).

In parallel with this rapid progress in the development of a high-lauric oil, there have been significant advances with other kinds of oil modification. Many important genes in fatty acid and oil biosynthesis have been cloned, and several of them have been introduced into rapeseed. As a result there now exist some very interesting transgenic rapeseed plants which make new and different kinds of seed oils, and which will serve as prototypes for the development of commercial cultivars. Within a few years we can expect a whole series of new rapeseed crops to be developed, each producing a different oil for specific applications, and each representing a new opportunity for rapeseed growers which diversifies and augments, rather than competes with, their existing production. We feel that this will be a very positive development for the grower, oilseed processor, oil user, and for the consumers of products that contain plant oils.

Several recent reviews have presented comprehensive coverage of the biochemistry and molecular biology of plant lipid metabolism (Ohlrogge 1994; Ohlrogge and Browse 1995), as well as recent oilseed engineering work world-wide (Töpfer et al. 1995). In this paper I will briefly review the major progress in the engineering of plant oils at the Oils Division of Calgene Inc. Our vehicle for the production of novel oils is rapeseed (Brassica napus), and I will be referring to the low-erucic type called "canola" unless I indicate otherwise. The chapter organization is based on the new crop applications under development.

LAURICAL™

The 12:0-ACP thioesterase from California bay (Umbellularia californica) interacted successfully with fatty acid biosynthesis in the plastids of rapeseed embryos, with the result that considerable laurate accumulated (Voelker et al. 1992; Voelker et al. 1996). Furthermore this laurate was found almost entirely in the reserve oil, indicating that the "foreign" fatty acid was exported from the plastids and utilized by the enzymes of the Kennedy pathway for the assembly of triglycerides (TAGs). The initial transformants exhibited wide variation in the laurate content of the oil, resulting from the customary variation in expression of the transgene and consequential wide-ranging thioesterase activity. Non-destructive analysis of the oil composition of single seeds enabled high-lauric lines to be selected from the best events, and through subsequent breeding and performance trials a cultivar has been developed. This cultivar is known as 'Laurate Canola'; a considerable area was planted in Georgia in the late-1994 season, ultimately producing a very acceptable yield of an oil in which 40% (by weight) of the fatty acyl groups are laurate (Table 1). Known as Laurical™, this unique oil finds applications in the manufacture of soaps and detergents. The possibility of its use as a food ingredient is also under investigation.

HIGH-LAURIC OIL

The laurate enrichment of the oil in these transgenic plants is correlated with the amount of 12:0-ACP thioesterase activity expressed in the seeds, up to about 60 mol% laurate. Although further increases in thioesterase activity are possible through the introduction of additional gene copies or new genomic locations of those genes, very high activities do not have much additional effect on the laurate content of the oil (Voelker et al. 1996). We are investigating several factors which may contribute to the apparent "ceiling" of 60 mol%. One which has already received considerable attention is the inherent limitation in the use of saturated substrates by the second acyltransferase reaction of the Kennedy pathway. In rapeseed, lysophosphatidic acid acyltransferase (LPAAT) which catalyzes this sn-2 acylation step exhibits a strong preference for 18:1-containing substrates (for example, Oo and Huang 1989). As a result, rapeseed TAGs have an almost exclusively unsaturated acyl composition at the sn-2 position. We found that this was still true of the TAGs in the transgenic plants accumulating laurate. For example, the oil from a transgenic line containing 52 mol% laurate has only 5 mol% laurate at the sn-2 position of the TAGs (Voelker et al. 1996). One can calculate that the average laurate content at the sn-1 and sn-3 positions is therefore 76 mol%. It seems likely that the development of an oil enriched very highly in laurate will require the presence of an additional LPAAT activity able to incorporate saturated, and particularly medium-chain, acyl groups at the sn-2 position.

To locate an LPAAT having such a preference for medium-chain substrates we again turned our attention to seed tissues which accumulate oil rich in laurate. We identified an appropriate activity in the immature endosperm of coconut (Fig. 1), and we have since solubilized this membrane-associated enzyme (Davies et al. 1995). Sufficient purification was undertaken to enable a candidate protein to be identified, and the corresponding gene was then cloned (Knutzon et al. 1995). Expression of this gene in E. coli produced abundant medium-chain LPAAT activity, confirming its identity. Rapeseed plants expressing this LPAAT under the control of the napin promoter have been crossed with a laurate-producing transgenic line. Preliminary analysis of the oil accumulated by the seeds of the F1 progeny plants shows that the introduced coconut LPAAT is incorporating laurate into the sn-2 position (Fig. 2). Laurate enrichments at sn-2 of up to 30 mol% have been seen in individual seed oils averaging 40 mol% laurate content. These results represent the first demonstration of the manipulation of TAG structure, sometimes referred to as the production of "structured triglycerides," by the genetic engineering of an oilseed crop. The F1 seeds from the above crosses were segregating for both the thioesterase and LPAAT transgenes, so we must now examine successive generations of selfed plants to determine whether the introduction of laurate at sn-2 results only in a re-arrangement of the same amount of laurate or whether the total laurate enrichment of the oil is increased.

OTHER MEDIUM-CHAIN FATTY ACIDS

By taking advantage of sequence relatedness amongst acyl-ACP thioesterases, T.A. Voelker, K. Dehesh, J. Kridl, L. Yuan and colleagues at Calgene have cloned many of these genes from a diverse range of species. Some examples are listed in Fig. 3, which shows the sequence relatedness between the respective open reading frames, and summarizes the principal substrate specificities of the corresponding enzyme activities (determined by expression in E. coli). The sequences fall into two distinct classes, "A" and "B," and we believe that representatives of both classes exist in all higher plants (Jones et al. 1995). All examples of class A thioesterases cloned to date appear to be the ubiquitous oleoyl-ACP thioesterase of plant fatty acid biosynthesis. The members of class B all exhibit a preference for saturated acyl-ACP substrates, and it is within this class that the specialized medium-chain-preferring types which are expressed specifically in certain oilseeds are found. Class B also includes thioesterases that exhibit preference for 14:0- and 16:0-substrates, cloned from plants which do not accumulate these fatty acids in their seed oils (e.g. At FatB1 in Fig. 3). The function of these thioesterases, which are quite possibly ubiquitous in higher plants and expressed in many plant tissues, is not yet known. (For a review of these thioesterases and a discussion of their evolutionary origins see Jones et al., 1995).

Several of the class B thioesterases acting on medium-chain substrates have now been expressed in rapeseed. Oils have been obtained containing 8:0, 10:0, and 14:0, and various combinations of these fatty acids in significant amounts. For example, the expression of thioesterase Ch FatB2 from Cuphea hookeriana, a plant which accumulates an oil highly enriched in 8:0 and 10:0, has produced a rapeseed oil containing up to 10 mol% 8:0 and 25 mol% 10:0. Expression of other class B thioesterases has resulted in rapeseed oil containing up to 40 mol% 14:0. Further improvements in the amounts of the medium chains can be anticipated as these prototype lines are developed through breeding and selection. These fatty acids and the respective TAGs have diverse applications in the food ingredients, pharmaceutical, and lubricant industries.

ALTERED SATURATION

There are many food quality and health considerations that encourage the development of oils containing altered ratios of saturated/unsaturated fatty acids. For example, solid glycerides from highly saturated (high-stearic) oils may have novel functionalities, will contain no trans fatty acids, and may be less cholesterolemic than hydrogenated fats. Significant alterations to increase, and decrease, the degree of saturation have been achieved by D.S. Knutzon, G.A. Thompson, J. Kridl, and co-workers via manipulation of the level of stearoyl-ACP desaturase activity (Knutzon et al. 1992, and unpublished work). This enzyme is responsible for the first of the desaturation reactions, by forming oleoyl-ACP from stearoyl-ACP during fatty acid biosynthesis. The introduction of additional activity in canola by transformation with the safflower stearoyl-ACP desaturase gene reduced the saturate content of the oil from c. 2% (by weight) to around 1%. Expressing the rapeseed desaturase gene in the inverse, or "antisense" orientation decreased the native activity and as a result the stearate (18:0) content of the oil increased up to 40%.

Another strategy for elevating the stearate, and hence the saturate, content of the oil is to express an acyl-ACP thioesterase having a higher activity on 18:0-ACP than the incumbent rapeseed enzyme (which shows strong preference for 18:1-ACP). J. Kridl and co-workers recently cloned the gene for such a thioesterase from mangosteen (Garcinia mangostana), whose seeds contain 50% stearate. The expression of this gene in canola raised the stearic content of the oil to 20%. These transgenic lines will now be crossed with those expressing the antisense desaturase construct to combine the traits.

HIGH-ERUCIC OIL

Erucic acid (22:1) for the manufacture of industrial lubricants is currently obtained from high-erucic (HEAR) varieties of rapeseed. These TAGs lack erucoyl residues in the sn-2 "position," and there has been considerable interest in raising the erucate levels further by overcoming this compositional limitation. Such an oil would not only serve as a higher-yielding source of erucoyl residues, but would also provide trierucin for certain clinical applications. Lassner, Metz and colleagues at Calgene have approached this objective in two parallel ways. First they cloned a gene responsible for the initial reaction of the cytoplasmic fatty acyl elongation system, i.e. ketoacyl-CoA synthase (KCS), from the jojoba plant, Simmondsia chinensis. In its native species this enzyme is part of the "elongase" system that produces the C20, C22, and C24 acyl groups that predominate in the stored wax esters. The introduction of this gene into canola resulted in the production of TAGs containing up to 58% of their acyl groups as these very long-chain fatty acids (VLCFAs). Previous work had suggested that a deficiency in the cytoplasmic fatty acyl elongation system was responsible for the near-absence of VLCFAs in canola relative to HEAR (Stumpf and Pollard 1983). This transformation experiment conclusively showed that the metabolic deficiency is a lack of functional KCS. The KCS gene will now be used to isolate the homologous gene from HEAR, in order to over-express it and so to obtain an erucate content higher than the typical HEAR value of 40%-50%.

The second part of the strategy is to correct the limitation at the sn-2 position by the introduction of another specialized LPAAT. Making use of sequence homologies with the coconut LPAAT discussed above, Lassner et al. (1995) have cloned the gene for an appropriately erucate-preferring LPAAT from the meadowfoam plant (Limnanthes alba). The introduction of this gene into a HEAR line resulted in the incorporation of erucoyl residues into the sn-2 position of the TAGs, the oil containing a significant amount of trierucin as a result (Lassner et al. 1995; Fig. 4).

These genes for specialized KCS and LPAAT enzymes are now being combined in a HEAR background with the intention of producing a new, super-erucic rapeseed line.

ALCOHOLS AND WAX ESTERS

All of the above projects are concerned with altering the fatty acyl composition of rapeseed TAGs. A more ambitious plan is the replacement of the TAGs with another kind of reserve lipid, namely a wax ester having industrial applications such as that found in jojoba seeds. This will require the production of significant amounts of very-long-chain fatty acids (VLCFAs), the reduction of part of the VLCFA-CoA pool to form long-chain alcohols, and the condensation of those alcohols with more of the VLCFA-CoAs to form the wax esters. Lassner, Metz, and co-workers have cloned the jojoba acyl-CoA reductase responsible for formation of the alcohol moiety and have expressed that gene in HEAR (Metz et al. 1994). Approximately 4% of the acyl groups in the seed oil were reduced to alcohol groups. Most interestingly, an as-yet uncharacterized rapeseed enzyme activity esterified at least some of this alcohol so that about 8% of the weight of the final seed oil comprised wax esters (Fig. 5). Work is progressing on the cloning of the jojoba "wax synthase" gene to increase the rate of ester formation.

LOOKING AHEAD

Within a few years we can expect the rapeseed grower to be planting a whole series of new rapeseed cultivars that will have sufficiently different applications to represent entirely new crops. These lines will be engineered for the production of diverse oils having well-defined uses in the food, detergent, and lubricants industries. Specialty oils may also be developed with pharmaceutical and chemical feedstock applications in mind. The technology and the prototypes for many of these new crops are here today, and the engineering strategies are already moving from the relatively simple, single-gene transformations of a few years ago to two-gene projects and the introduction of longer biosynthetic pathways. The specialized genes which are required have come from the germplasm of diverse, uncultivated plant species, as well as from crop plants. In the future we can expect non-plant genes to feature as well. Along the way, much has been learnt about the basic mechanisms of the biosynthesis of fatty acids, TAGs etc. and we can continue to look forward to a productive synergy between the applied projects and the underlying basic science. The genetic engineering of oilseed plants promises to be one of the most impactive and impressive sources of new crops for both new and existing applications.

REFERENCES

Table 1. Seed fatty acyl composition of 'Laurate Canola' and canola seed.

Composition (wt %)
Fatty acyl group Laurate canola Control canola
12:0 40.0 0.0
14:0 3.9 0.0
16:0 2.9 6.2
18:0 1.1 1.6
18:1 29.8 60.8
18:2 13.3 9.2
18:3 6.8 9.2
20:0 0.3 0.3
20:1 0.6 0.9

Fig. 1. Substrate specificity of lysophosphatidic acid acyltransferase (LPAAT) from immature coconut endosperm. A crude membrane fraction was prepared from a homogenate of the tissue, the activity was solubilized with detergent and then recombined with phospholipids for assay. The acyl donor substrates were those indicated, and the acyl acceptor substrate was 12:0-lysophosphatidic acid throughout. Very similar results were obtained using 18:1-lysophosphatidic acid as acceptor substrate. For assay (mU = milliunits activity) and other details see Davies et al. (1995).
Fig. 2. Combination of the engineered traits for laurate production (via bay 12:0-ACP thioesterase) and medium-chain LPAAT activity, in rapeseed. Plants expressing the medium-chain-preferring coconut LPAAT activity were crossed with a laurate-producing line in order to examine the effect of the specialized LPAAT on the laurate content of the oil. Each point indicates the result from crossing an independent LPAAT-expressing "event" with the laurate-producing line. From each cross a pool of 20 mature F1 seeds was obtained and analyzed for sn-2 laurate content. Each of these results is plotted against the LPAAT enzyme activity in pooled immature seeds from the corresponding LPAAT parent, measured using 12:0-containing substrates. The F1 progeny are segregating for both the LPAAT activity and the overall laurate content, hence the considerable scatter in the data. Control data, shown by the arrows, were generated by crossing the same laurate-producing line with untransformed canola. Note that all the LPAAT "events" produced F1 seed in which the laurate content at sn-2 was elevated relative that in the control cross, and that three events produced progeny with markedly increased sn-2 laurate contents.

Fig. 3. Examples of homologous, higher plant, acyl-ACP thioesterases. The left-hand side of the figure shows a phylogenetic tree of some of the acyl-ACP thioesterases cloned to date, constructed from an alignment of amino acid sequences. [For a more complete listing, and for additional details and references, see Jones et al. (1995). For details of the thioesterase At FatB1 see Dörman et al. (1995)] The right-hand diagram summarizes the substrate specificities of the corresponding enzyme activities, semi-quantitatively. The activity on each acyl-ACP substrate is represented by a dot below the corresponding substrate acyl group (8:0 through to 18:1), and the diameter of the dot is an approximate indication of relative activity. The sources of these thioesterases are as follows [for terminology see Jones et al. (1995)]: Ap, Allium porrum (leek); At, Arabidopsis thaliana; Bn, Brassica napus (rapeseed); Cc, Cinnamomum camphora (camphor); Ch, Cuphea hookeriana; Cp, Cuphea palustris; Ct, Carthamus tinctorius (safflower); Mi, Mangifera indica (Mango); Ua, Ulmus americana (elm); Uc, Umbellularia californica (California bay).

Fig. 4. Resolution of triglycerides from rapeseed (HEAR) oil of a control plant and a transgenic plant containing the meadowfoam LPAAT gene. Triglycerides were separated by silver-phase HPLC [for details see Lassner et al. (1995)] according to their degrees of unsaturation. Portions of the chromatograms occupied by trienoic and tetraenoic TAGs are shown, and peaks of interest are labeled with the total carbon content of the corresponding TAGs. Trierucin has a total carbon content of 69. The introduced LPAAT also enabled the formation of 22:1/20:1/22:1 TAG (C67).

Fig. 5. Resolution of triglycerides and wax esters from rapeseed (HEAR) oil of a control plant and a transgenic plant containing the jojoba acyl-CoA reductase gene. Triglycerides and wax esters were separated by high-temperature GLC [for details see Metz et al. (1994)]. The wax esters contain alcohol and acyl moieties derived from 20:1 and 22:1 fatty acyl groups as indicated (e.g. 40:2 contains 20:1 alcohol and 20:1 acyl moieties). The 35:1 peak is from an internal standard wax ester.

Last update August 19, 1997 aw