Epoxy Fatty Acid Accumulation in Seed Oil

The use of plants as chemical factories such as the production of epoxy compounds proposed here promises to greatly enhance the variety and quality of products available in the future. This requires no fossil fuel for the actual biosynthesis reactions instead using sunlight, H2O and atmospheric CO2 for the hydrocarbon syntheses. They also can directly and indirectly provide domestically produced renewable fuels. This is considered to be part of the new "green" industrial revolution. The aim of this research is to expand or provide additional renewable resources derived from crop plants that are industrially valuable. This could also provide the basis of gTeatly increased value of a byproduct of production of ethanol and other fuels from plant materials such as cornstarch. This in turn will make production of such crop-derived fuels more economically competitive with petroleum-derived fuels. Engineering oilseeds for high epoxy fatty acid accumulation in triglyceride is the specific goal. This would greatly increase the value of such modified seeds and reduce epoxide production costs. Safety concerns associated with industrial epoxidation of oils would also be eliminated. A considerable market currently exists for epoxy fatty acids particularly for epoxy coatings and plasticizers. Presently most of these are derived from petroleum. Soybean and linseed oil are currently utilized to some extent to produce epoxidized oil by introducing an epoxy group across the double bonds of polyunsaturated fatty acids. This is a costly process and it would likely be more economical if the biosynthetic reactions in major oilseed crops were altered such that the seeds themselves converted the polyunsaturated fatty acids into epoxy fatty acids. There is no known way to produce a commercial oilseed that accumulates epoxy fatty acids by conventional breeding and genetics. Certain genotypes of several plant species, however, accumulate high levels of epoxy fatty acids in the seed oil. The best examples of this are Vernonia galamensis, Stokesia laevis and Euphorbia lagascae. There are three major proposed objectives: 1) Further investigation of the mechanism of high-oxygenated fatty acid accumulation in triacylglycerol. 2) Clone genes responsible for high epoxy fatty acid accumulation in source plants and test these genes in soybean embryos. 3) Clone additional genes for unique epoxy fatty acid biosynthesis. V. galamensis, S. laevis or E. lagascae cannot readily be grown to produce seed on an industrial scale limiting their current potential to replace much fossil fuel use as sources of epoxy compounds. Epoxygenase genes have been cloned from several accumulators of vernolic acid and high expression in developing Arabidopsis or soybean embryos can result in up to -10% vernoleate in the seed oil but this is insufficient for commercial production of epoxides in oilseeds. The effective channeling or selective accumulation of epoxy fatty acids into triacylglyceride appears to be the key to the effective commercialization of this process. Diacylglycerol acyltransferase (DGAT) and/or phospholipid:diacylglycerol acyltransferase (PDAT) is likely to be of major importance to the accumulation of epoxy fatty acids in seed oil triacylglycerols. We have already cloned a DGAT from V.galamensis for use in the proposed studies. Genes encoding other enzymes identified to be important in the selective accumulation of oxygenated fatty acids in triacylglycerols in addition to or instead of DGAT will be cloned. . In order to better understand the selective ~c.cumula~ionof epoxy ~d r~late~ o~lgenated .fatty aCIds into triacylglycerols, we will do competItIve feedmg studIes of C lInoleIc, C vernolIc CoAs and 14Cvernolic acid containing diacylglycerols with cultured developing seeds of soybeans, Vernonia, and some analogous studies with Euphorbia and Stokesia. The fa~eof epoxide produced in vivo in soybean and Arabidopsis oil accumulating seed tissue wIll also be evaluated.