EPOXYGENASE EXPRESSION IN SOYBEAN AND BIOLOGICAL EFFECTS OF EPOXY FATTY ACIDS

Epoxy fatty acids (EXA) are valuable to industry as they are used in synthesizing plasticizers such as of poly vinyl chloride, resins, adhesives, coating materials such as paint, lubricant, lubricant additives, insecticides, insect repellants, crop oil concentrates and formulations of carriers for slow release pesticides and herbicides. There is interest in developing commercial oilseeds accumulating epoxy fatty acids to at least 50% of the seed oil. Soybeans are the most widely cultivated oilseed and its oil has high levels of linoleic acid which can be a substrate for epoxygenase enzymes. Cahoon et al., expressed a cytochrome P450 enzyme (CYP726A1) from Euphorbia lagascae in soybean somatic embryos and found that the epoxy fatty acid, vernolic acid, reached ~8% of the total fatty acids in transgenic somatic embryos. Rabbit Livers possess a cytochrome P450, CYP2C2, which catalyzes the same epoxidation reaction as the E. lagascae enzyme but might be less likely to be influenced by regulatory machinery in plant cells. This CYP2C2 gene was placed in a plant expression vector under a seed-specific promoter and used to transform soybean, Glycine max, somatic embryos. The ten putative transgenic clones observed after 4-5 weeks were separated and proliferated under selection. glucuronidase (GUS) assays and PCR analyses performed on selected clones were positive. However vernolic acid in total lipids and specific lipid classes was not detected as analyzed by GC. In vitro enzyme assay performed on microsomes isolated from mature somatic embryos at three weeks of maturation using [14C] 18:2 PC as substrate showed presence of [14C] methyl vernoleate. Preliminary analyses on toxicity of epoxy fatty acids and corresponding diols in bacteria, yeast and caco-2 cells showed that leukotoxin diol (LD) most toxic