New very long chain fatty acid seed oils produced through introduction of strategic genes into Brassica carinata

October 2010

Editor's note: The following article is the second in a two-part series. The first part-"Surfactants based on monounsaturated fatty acids for enhanced oil recovery" by Paul Berger-appeared in the September 2010 issue of inform.

For the emerging global bioeconomy, crop development and enhancement of species diversity are essential. Equally important is maximizing crop value through total crop utilization. We are developing Ethiopian mustard (Brassica carinata) as a biorefinery and bioindustrial oils crop platform using traditional and molecular breeding techniques and tools (see review by Taylor et al., 2010).

Very long chain fatty acids (VLCFA) containing more than 18 carbon atoms are common components of seed oils and plant waxes in a number of plant families including the Cruciferaceae, Limnanthaceae, Simmondsiaceae, and Tropaeolaceae.

Erucic acid (cis-docosa-13-enoic acid, 22:1 ∆13) is the major VLCFA in the seed oil from HEAR (high erucic acid rapeseed) B. napus cultivars, accounting for 45-55% of the total fatty acids. HEAR cultivars are of high interest since 22:1 is a valuable feedstock with more than 1,000 patented industrial applications. Currently the major derivative of erucic acid is erucamide, which is used as a surface-active additive in coatings and in the production of plastic films as an anti-block or slip-promoting agent. Many other applications are foreseen for erucic acid and its hydrogenated derivative behenic acid, for example, in lubricants, detergents, photographic film-processing agents, coatings, cosmetics, and pharmaceuticals.

Studies have confirmed that HEAR oil and its derivatives have a higher energy potential than low erucic acid oil. HEAR oils are more suitable for biodiesel production than low erucic Brassica oils, because the iodine value is lower in the former (i.e., lower proportion of polyunsaturated fatty acids in the oil) and within the European Union specifications. US industry uses 18 million kg of HEAR oil annually, mostly from imports, but supplies are historically limited. Therefore, a large overall market potential exists for expansion and development of new, annually renewable domestic sources of erucic acid oil.

Nervonic acid (cis-tetracosa-15-enoic acid; 24:1 ∆15) is another strategic VLCFA. It exists in nature as an elongation product of oleic acid (18:1 ∆9), its immediate precursor being erucic acid. Nervonic acid has been identified in the triacylglycerols in the seeds of only a few plants: Lunaria spp. (money plant), borage, hemp, Acer truncatum (purple-blow maple), Tropaeolum speciosum (flame flower), and Cardamine graeca (bittercress). Nervonic acid is particularly abundant in the white matter of animal brains and in peripheral nervous tissue where nervonyl sphingolipids are enriched in the myelin sheath of nerve fibers.

Interest in dietary therapy with nervonic acid-containing fats and oils developed when a hypothesis was put forward that dietary nervonic acid could support the normal synthesis and functionality of myelin in brain and nerve tissues. Dietary supplementation with nervonic acid might be beneficial, for neurological development/function, in: (i) individuals with genetic disorders of lipid metabolism specifically associated with peroxisomes (adrenoleukodystrophy, Zellweger's syndrome, others); (ii) individuals with multiple sclerosis and other nervous disorders such as Parkinson's disease; and (iii) infants, particularly prematurely born infants, receiving formula as a source of nutrition. Realization of these potential nutritional applications is limited by the lack of available sources of a nervonic acid-rich oil that has minimal amounts of erucic acid. Provided the erucic acid content is very low, dietary intake of oil with high proportions of nervonic acid is predicted to be nontoxic to humans and animals. Nervonic acid is therefore a strong candidate for further evaluation as a bioactive lipid supplement, similar to arachidonic acid, docosahexaenoic acid, and conjugated linoleic acids, for the promotion of human and animal health (see review by Taylor et al., 2009). Surprisingly, nervonic acid also has applications as an industrial feedstock, in a manner similar to erucic acid.

A strategic goal of our research is to modify seed oil composition in members of the Brassicaceae to increase the proportion of VLCFA. Specific genetic modifications have been made to produce B. carinata prototype lines delivering oils highly enriched in VLCFA; in addition we have used transgene technology to enhance B. carinata seed oil content, which is 5-7% lower than B. napus (canola and HEAR cultivars). While we have HEAR cultivars seeded in May and harvested in August-September in western Canada and European winter varieties, seeded in the fall and harvested in late spring, we are advocating that B. carinata be developed as an alternative crop platform for industrial oil production and high VLCFA oils, in particular on the Canadian prairies. Brassica carinata is genetically transformed at a very high efficiency; the crop is highly disease resistant (e.g., to the fungus blackleg), is drought-tolerant, and is amenable to growth in hotter, drier regions.

Total crop utilization can be illustrated with two examples: High allyl glucosinolate meal from B. carinata can be used directly as a biopesticide; alternatively, the meal can be processed with a solvent wash to remove the glucosinolates so that the remaining low-fiber meal can be used as fish feed. We are currently working closely with B. carinata breeders at Agriculture and Agri-Food Canada, Saskatoon, to make sure that we can cross many of our oil-quality enhanced traits into their elite breeding lines.

Some of the many industrial and health-dietary supplement-related uses of VLCFA oils are shown in Table 1 and Tables 2 and 3, respectively. Specifically, the high-VLCFA feedstocks are essential components of new products developed by Paul Berger and associates at Oil Chem Technologies, Inc. (Sugar Land, Texas, USA), which include viscoelastic and high molecular-weight anionic surfactants. Some of these products show great potential in enhanced oil recovery (EOR) strategies as described recently in inform (21:542-543, 592, 2010).

Most transformation systems for introducing new genes into B. carinata rely on Agrobacterium-mediated infection of cotyledon (first leaf) petiole tissue with a genetic cassette containing the gene or genes of interest, promoter and terminator elements, and a selectable marker-usually a gene conferring resistance to a specific antibiotic or herbicide. The latter makes it possible to "screen" the putative transgenic petiole pieces for positive incorporation and integration of the cassette into the target plant tissue. Then the positive transgenic tissues are regenerated into whole transgenic plants by growth on root- then shoot-promoting medium and transferred to soil. These were the methods we used to introduce the transgenes into B. carinata as described next.

Target transgenes and bioproducts

To enhance the 22:1 or 24:1 acid content of B. carinata oil, we selected two nontraditional sources of genes encoding ketoacyl-CoA synthases (KCS), the KCS being the first of four enzymes in an elongase complex that ultimately converts 18:1 ∆9 (oleoyl)-CoA to erucoyl-CoA and then nervonoyl-CoA by successive cycling of the complex, with malonyl-CoA providing the two carbons needed for each extension cycle. These gene sources were Crambe abyssinica and Cardamine graeca (Fig. 1).

When introduced into B. carinata, Crambe KCS and Cardamine KCS genes resulted in seed oils highly enriched in erucic acid and nervonic acid (Figs. 2 and 3, respectively).

Outlook for B. carinata as a platform crop for delivery of bioproducts

When considering potential platform crops for the delivery of bio-oils and industrial feedstocks, seed yield and oil and protein content are major considerations. Consequently, the plant must be more efficient in resource utilization than the usual cultivars while maximizing yield. Brassica carinata delivers high yields among the Brassicaceae (up to ca. 2,500 kg/ha). A key component of any new crop development is the question of whether it contributes positively to sustainable agriculture. This fledgling crop platform can meet or exceed many of the targets for sustainable agriculture. Specifically:

• Plant-produced VLCFA oils provide renewable, biodegradable, non-fossil fuel feedstocks for the production of polymers, plastics, waxes, pharmaceutical and nutraceutical oils.
• For example, high nervonic acid oil from B. carinata can find direct applications equally in polymers, paving substances, and surfactants for oil recovery/reclamation products, as well as potential new products for enhancing infant nutrition and fighting the symptoms of neurodegenerative diseases.
• Brassica carinata is well suited for growing in the drier southern regions of western Canada.
• Creation of a new crop platform adds genetic diversity. It creates a new delivery system for bioindustrial and pharmaceutical oils that do not impact/compete with the food sector, specifically canola.
• Growing B. carinata in areas not suitable for canola means that one is adding a new oilseed to crop rotations in those areas.
• Brassica carinata requires fewer inputs owing to its natural resistance to drought and blackleg; more robust architecture means crop stands are able to better compete with weeds.
• Brassica carinata provides the grower with enhanced yield (kg/ha) compared with other Brassicas (canola) and is therefore an attractive incentive for farmers as it could result in increased returns at the farmgate.
• The unique characteristics of B. carinata meal provide new opportunities as feedstocks for plastics and antigen delivery systems; the utility of both oil and meal is essential for complete utilization of the seed products, providing greater sustainability.

In conclusion, as indicated by the two case studies discussed here, B. carinata is well suited for genetic engineering, and the generation of transgenics will play a major role in designing this crop for the delivery of VLCFA-enhanced oil feedstocks for bio-products.

David Taylor has been active for 22 years in the field of oilseed biochemistry, biotechnology, and oil modification at the National Research Council of Canada (NRCC) Plant Biotechnology Institute in Saskatoon, Saskatchewan. His research focuses on the creation of value-added oils as industrial feedstocks or for human consumption using biotechnology. He is also actively involved in research to improve the oil content of a number of oilseed crops including canola, Brassica carinata, and flax. He is a strong advocate for adoption of B. carinata as a new platform crop for delivery of customized bioindustrial oils. Email him at David.Taylor@nrc-cnrc.gc.ca. This is NRCC publication no. 50172.

information

For further reading:

Taylor, D.C., Y. Guo, V. Katavic, E. Mietkiewska, T. Francis, and W. Bettger, New seed oils for improved human and animal health and as industrial feedstocks: Genetic manipulation of the Brassicaceae to produce oils enriched in nervonic acid, in Modification of Seed Composition to Promote Health and Nutrition, edited by A.B. Krishnan, ASA-CSSA-SSSA Publishing, Madison, Wisconsin, USA, 2009, pp. 219-233.

Taylor, D.C., K.C. Falk, C.D. Palmer, J. Hammerlindl, V. Babic, E. Mietkiewska, A. Jadhav, E.-F. Marillia, T. Francis, T. Hoffman, E.M. Giblin, V. Katavic, and W.A. Keller, Brassica carinata-a new molecular farming platform for delivering bio-industrial oil feedstocks: Case studies of genetic modifications to improve very long-chain fatty acid and oil content in seeds, Biofuels, Bioproducts and Biorefining 4:538-561, 2010.