Final Report: Biological Effects of Epoxy Fatty Acids

EPA Grant Number: R829479C003 aka R829479C011
Subproject: this is subproject number 003 aka R829479C011 , established and managed by the Center Director under grant R829479
(EPA does not fund or establish subprojects; EPA awards and manages the overall grant for this center).

Center: The Consortium for Plant Biotechnology Research, Inc., Environmental Research and Technology Transfer Program
Center Director: Schumacher, Dorin
Title: Biological Effects of Epoxy Fatty Acids
Investigators: Hildebrand, David
Institution: University of Kentucky
EPA Project Officer: Lasat, Mitch
Project Period: August 7, 2003 through September 30, 2003
RFA: The Consortium for Plant Biotechnology Research, Inc., Environmental Research and Technology Transfer Program (2001) RFA Text |  Recipients Lists
Research Category: Targeted Research , Hazardous Waste/Remediation


Epoxy fatty acids are valuable industrial feedstocks for a variety of applications. Several plants are known to accumulate high levels of epoxy fatty acids in their seed oil. It is thought that plants have evolved this characteristic as a chemical defense. There is interest in producing various epoxy fatty acids in bioreactors or engineered plants. The overall objective of this research project was to determine the impact of epoxy fatty acids on seed pests as well as on microorganisms, including bacteria and yeast.

The specific objectives of this research project were to:

  1. isolate epoxy fatty acids from suitable natural sources that are models for oilseed improvement such as Vernonia seeds;
  2. produce other epoxy fatty acids using in vitro enzymes, microorganisms, and engineered plant tissues;
  3. test the effects of these epoxy fatty acids on stored insect pests and model bacteria;
  4. and test some of these epoxy compounds in the field as a new natural pest control approach.

Dr. Hildebrand’s laboratory at the University of Kentucky is primarily responsible for carrying out the research for Objectives 1 and 2 and the later part of Objective 3 as well as some epoxy fatty acid biosynthesis studies. John Sedlacek's laboratory at Kentucky State University (KSU) leads the work on stored insects in Objective 3 as well as Objective 4 (see Annual Summary report for subproject R829479C011).

Summary/Accomplishments (Outputs/Outcomes):

For Objective 1, we obtained soybean oil, Vernonia oil, and castor oil and prepared vernolic acid from Vernonia oil and methyl-vernolate for gram quantities. Vernonia oil has about 70-80 percent of a (12S,13R)-epoxy fatty acid, vernolic acid. Castor oil contains 90 percent of another oxygenated (12-hydroxy) fatty acid, ricinoleic acid. It is hypothesized that these oxygenated fatty acids accumulate in Vernonia and castor seeds as part of a pest-defense mechanism, but the effects of these compounds have not been investigated yet. The compounds were added to corn at ppm levels and tested against corn insect pests by Dr. Sedlacek and a student at KSU. The levels were chosen to approximate levels of insecticides that might be used. At such relatively low levels, there was little or no impact on the feeding insects; therefore, in discussions with Dr. Sedlacek, we decided to greatly increase the levels of these materials in the feeding diets to approach levels that these high accumulating seeds can reach. For this we needed to prepare each testing compound in excess of 100 g for KSU’s insect feeding studies. More castor oil was purchased from a local drug store ("castor oil USP"). Hydrolyzed castor oil was made by alkaline hydrolysis. The Vernonia oil was obtained from Ver-Tech International, Inc. (Refined Vernonia oil VO-100, M3-1-02). The material used to make vernolic acid and methyl vernolate was trivernolin, which was purified from the Vernonia oil by recrystallization 11 times. The vernolic acid content in the prepared trivernolin is about 98 percent of the total fatty acids based on gas chromatography-mass spectrometry analysis. The vernolic acid was made by alkaline hydrolysis of trivernolin followed by ethyl acetate extraction with acetic acid. There was a very small amount of an unknown compound seen in thin layer chromatography analysis that we believe to be a diol of vernolic acid, possibly produced from vernolic acid caused by the acidification process even though the acetic acid used is a weak acid. Methyl vernolate was made by sodium methoxide trans-methylation from trivernolin. The above compounds were given to Dr. John Sedlacek for insect feeding studies at KSU.

For Objective 2, we obtained epoxidized soybean oil and epoxidized linseed oil for testing in microorganisms. The epoxidized oils contained mixtures of various highly epoxidized triacylglyceride species. We synthesized a mixture of epoxy fatty acids from linoleic acid using a procedure that adapted a chemoenzymatic epoxidation procedure described by Rusch et al. (1996) using H2O2 and an immobilized lipase from Candida antarctica, Novozym-435R. The above mixture was thought to include 9S,10R-epoxy-12-octadecenoate, 9R,10S-epoxy-12-octadecenoate, 12R,13S-epoxy-9-octadecenoate, and vernolic acid. In animal systems, 9,10-epoxy-12-octadecenoate is termed leukotoxin and 12,13-epoxy-9-octadecenoate, is termed isoleukotoxin. They are believed to be produced from linoleic acid by cytochrome P450 gene as well as via reactive oxygen species generated by neutrophil respiratory burst oxidase. Leukotoxin and isoleukotoxin can be hydrolyzed further to corresponding diols by soluble epoxide hydrolase. It has been shown that the toxicity of leukotoxin and isoleukotoxin is not caused by epoxides, but by their respective 9,10- and 12,13-diol metabolites. Using a baculovirus expression system, it was demonstrated that leukotoxin is cytotoxic only in the presence of soluble epoxide hydrolase. At the cellular level, leukotoxin diol causes mitochondrial dysfunction through activation of the mitochondrial permeability transition, causing a release of cytochrome C and, subsequently, cell death. We also synthesized diol of vernolic acid using aqueous 2 percent hydrochloric acid in water/tetrahydrofuran (vol/vol, 50/50) at room temperature for 6 hours.

For Objective 2, we also cloned two P450 epoxygenase genes, CYP2C9 from human and CYP2C2 from rabbit, into a yeast vector pESC. Because P450 epoxygenases requires cytochrome P450 reductase to function, a soybean cytochrome P450 reductase was cloned and inserted into the same yeast expression vector with the above two epoxygenase genes. To test if adding redox components will help the expression of P450 epoxygenase in our rapid yeast testing system, five plasmids, containing either P450 CYP2C2 alone, P450 CYP2C9 alone, each together with soybean cytochrome P450 reductase, or no insert as a control, were introduced into the host yeast strain. So far, we have not detected epoxy fatty acids above the vector control levels from the above yeast expression studies. We were, therefore, unable to extract epoxy fatty acids from the engineered yeast for our testing.

Additionally for Objective 2, soybeans were transformed with a Stokesia epoxygenase responsible for biosynthesis of vernolic acid from developing Stokesia seeds and a Vernonia diacylglycerol acyltransferase involved in the final step of synthesis of Vernonia oil in developing Vernonia seeds. Transgenic soybean somatic embryos of one line had 0.9 percent vernolic acid produced, but the level was not high enough for our testing. Many transgenic soybean somatic embryos from 17 independent lines were matured, some were germinated, and whole plants were grown in the greenhouse. The plants from six independent transgenic lines produced mature seeds that were analyzed for oil content and fatty acid profiles. The total oil contents of the transgenic seeds with the two genes were the same or slightly lower than those of the vector control seeds. Unfortunately, no vernolic acid has been detected from the transgenic seeds yet. Transgenic seeds carrying the two genes did have altered fatty acid profiles; in many cases, oleic acid dramatically increased and linoleic acid decreased accordingly. In some of the transgenic seeds, oleic acid was higher than linoleic acid. The vector control seeds did not show altered fatty acid profiles. The changes in fatty acid profiles might have been caused by the incorporated Stokesia epoxygenase. These results indicate that simplyincorporating these two genes for epoxy fatty acid synthesis and accumulation is not sufficient for accumulation of vernolic acid in soybean seed oil. More fundamental studies on the natural accumulator Vernonia and the transformed soybean are needed for developing high epoxy accumulating soybean oil. We were, therefore, unable to extract epoxy fatty acids from the transgenic soybean seeds for testing in insects or microorganisms.

For Objective 3, we tested the effects of Vernonia oil, castor oil, soybean oil, vernolic acid, methyl vernolate, hydrolyzed castor oil, and hydrolyzed soybean oil on Escherichia coli growth in LB media at 37ºC with shaking at about 170 rpm. No compound added, soybean oil, and hydrolyzed soybean oil served as controls. Both high and low concentrations (10% and 0.2% of the media, respectively) of the compounds were tested. Ethanol (95%) was used at 15 percent and 2 percent (final concentration) for the high and low oil treatments, respectively, for initially dissolving the compounds and helping disperse the oil compounds in the media. Tween 20 at the concentration of 0.1 percent (for high oil treatments) and 0.05 percent (for low oil treatments) was used to help the test compounds mix with the media. The inoculum used was 50 :L of overnight culture of E. coli at 37ºC for 1 mL test media. The 10 percent vernolic acid, 10 percent hydrolyzed castor oil, 10 percent soybean oil, and 10 percent methyl vernolate stopped the growth of E. coli whereas all other treatments had no effect on the growth of the bacteria, including 10 percent Vernonia oil, 10 percent castor oil, and all 0.2 percent oil compound treatments compared to the control with no compound added. At 0.2 percent oil, there was no problem getting a good mixture, but 10 percent oil was too much for the oil to completely mix with the media. Ten percent of hydrolyzed castor oil, 10 percent hydrolyzed soybean oil, and 10 percent vernolic acid killed E. coli in LB, evident from subsequent plating results on LB plates. Because the high concentrations of Vernonia oil did not affect the growth of E. coli, while high concentrations of vernolic acid, hydrolyzed castor oil, and hydrolyzed soybean oil did, the effect was probably not caused by the epoxide functional groups tested, but by the free fatty acids that can act as detergents. The acidity of the fatty acids at high concentrations was probably not favorable for E. coli. Ten percent of methyl vernolate did not kill the E. coli, but it did stop the growth of E. coli, based on the cell pellet size after centrifugation and the results from plating as described above. It is not clear why methyl vernolate showed some effect on the growth of the E. coli. Epoxidized soybean oil, epoxidized linseed oil, vernolic acid diol, and partially epoxidized linoleic acid subsequently were tested at 5 percent concentration for their effects on the growth of E. coli in LB media at similar conditions as the high concentration testing described above. Optical density values at 600 nm were taken for 20-fold diluted cultures at 0, 12, 19, 36, and 43 hours after inoculation. The epoxidized linseed oil, epoxidized soybean oil, and partially epoxidized linoleic acid did not affect the growth of the E. coli. A diol derivative of vernolic acid, however, stopped the growth of E. coli. The toxicity of vernolic acid diol was probably caused by a similar mechanism to that found in the animal system.

For Objective 3, we also have tested the effects of Vernonia oil, castor oil, soybean oil, vernolic acid, methyl vernolate, hydrolyzed castor oil, hydrolyzed soybean oil, epoxidized soybean oil, epoxidized linseed oil, partially epoxidized linoleic acid, and vernolic acid diol on the growth of baker’s yeast (Saccharomyces cerevisiae) at 30ºC with shaking at about 170 rpm. The concentration of the compounds was 5 percent of the yeast growth media YPAD. The compounds were dissolved in ethanol and dispersed in the media. Tween 20 also was used to bring the test compounds into the solution. Ninety-five percent ethanol at 10 percent concentration was used for the study. Samples were tested at 5 percent dilutions. Ethanol and Tween 20 were used for the high concentration of the compound testing in E. coli as described above. The results showed that vernolic acid diol stopped the growth of the yeast, which was probably through a similar mechanism as for animal cells and E. coli as described previously. Epoxidized linseed oil slightly slowed the growth of the yeast. Because of the high content of epoxidized linolenic acid, epoxidized linseed oil probably has a lot of reactive oxygenated species that could affect the yeast, as a higher microorganism, more than E. coli. All other compounds tested did not affect the growth of the yeast significantly.

Dr. Sedlacek’s group at KSU tested the effects of epoxy and hydroxy fatty acids and triacylglyceride (oil) containing epoxy and hydroxy fatty acids as glycerol esters on four important pests of stored corn—the flour mill beetle, maize weevil, sawtoothed grain beetle, and red flour beetle. The epoxy fatty acid, vernolic acid, and oil containing vernolic acid increased mortality of the flour mill beetle and the maize weevil, but the hydroxy fatty acid, ricinoleic acid, or oil containing ricinoleic acid had little impact. Both the hydroxy and epoxy fatty acids and oils containing these oxygenated fatty acids reduced the emergence of all four storage grain pests. The LD50s are being determined and the impact of diols will be tested in coming months.


  • Epoxy and hydroxyl groups in fatty acids and epoxidized oil are not toxic to microorganisms such as E. coli and yeast.
  • Hydrolyzed epoxy group-diol is highly toxic to microorganisms, including E. coli and yeast.
  • High levels of fatty acids can stop the growth of and kill E. coli because of their physical effects on membranes and detergency and also possibly acidity, but no effect was found on yeast.
  • High levels of epoxy and hydroxy fatty acids appear to increase mortality and reduce emergence in important storage insect pests.

Journal Articles:

No journal articles submitted with this report: View all 4 publications for this subproject

Supplemental Keywords:

pest control, hydroxy fatty acids, vernolic acid, insects, bactericides, sustainable industry/business, treatment/control, agricultural engineering, environmental engineering, geochemistry, new/innovative technologies, technology, Agrobacterium, bacteriacides, bioenergy, bioengineering, biotechnology, engineered plant tissues, epoxy fatty acids, genetics, in vitro enzymes, oilseed improvement, plant genes,, Scientific Discipline, TREATMENT/CONTROL, Sustainable Industry/Business, Geochemistry, Technology, New/Innovative technologies, Environmental Engineering, Agricultural Engineering, agrobacterium, bioengineering, genetics, epoxy fatty acids, in vitro enzymes, engineered plant tissues, oilseed improvement, plant genes, engineered plant tisues, biotechnology, remediation, bacteriacides

Relevant Websites: Exit Exit

Main Center Abstract and Reports:

R829479    The Consortium for Plant Biotechnology Research, Inc., Environmental Research and Technology Transfer Program

Subprojects under this Center: (EPA does not fund or establish subprojects; EPA awards and manages the overall grant for this center).
R829479C001 Plant Genes and Agrobacterium T-DNA Integration
R829479C002 Designing Promoters for Precision Targeting of Gene Expression
R829479C003 aka R829479C011 Biological Effects of Epoxy Fatty Acids
R829479C004 Negative Sense Viral Vectors for Improved Expression of Foreign Genes in Insects and Plants
R829479C005 Development of Novel Plastics From Agricultural Oils
R829479C006 Conversion of Paper Sludge to Ethanol
R829479C007 Enhanced Production of Biodegradable Plastics in Plants
R829479C008 Engineering Design of Stable Immobilized Enzymes for the Hydrolysis and Transesterification of Triglycerides
R829479C009 Discovery and Evaluation of SNP Variation in Resistance-Gene Analogs and Other Candidate Genes in Cotton
R829479C010 Woody Biomass Crops for Bioremediating Hydrocarbons and Metals
R829479C011 Biological Effects of Epoxy Fatty Acids
R829479C012 High Strength Degradable Plastics From Starch and Poly(lactic acid)
R829479C013 Development of Herbicide-Tolerant Energy and Biomass Crops
R829479C014 Identification of Receptors of Bacillus Thuringiensis Toxins in Midguts of the European Corn Borer
R829479C015 Coordinated Expression of Multiple Anti-Pest Proteins
R829479C016 A Novel Fermentation Process for Butyric Acid and Butanol Production from Plant Biomass
R829479C017 Molecular Improvement of an Environmentally Friendly Turfgrass
R829479C018 Woody Biomass Crops for Bioremediating Hydrocarbons and Metals. II.
R829479C019 Transgenic Plants for Bioremediation of Atrazine and Related Herbicides
R829479C020 Root Exudate Biostimulation for Polyaromatic Hydrocarbon Phytoremediation
R829479C021 Phytoremediation of Heavy Metal Contamination by Metallohistins, a New Class of Plant Metal-Binding Proteins
R829479C022 Development of Herbicide-Tolerant Energy and Biomass Crops
R829479C023 A Novel Fermentation Process for Butyric Acid and Butanol Production from Plant Biomass
R829479C024 Development of Vectors for the Stoichiometric Accumulation of Multiple Proteins in Transgenic Crops
R829479C025 Chemical Induction of Disease Resistance in Trees
R829479C026 Development of Herbicide-Tolerant Hardwoods
R829479C027 Environmentally Superior Soybean Genome Development
R829479C028 Development of Efficient Methods for the Genetic Transformation of Willow and Cottonwood for Increased Remediation of Pollutants
R829479C029 Development of Tightly Regulated Ecdysone Receptor-Based Gene Switches for Use in Agriculture
R829479C030 Engineered Plant Virus Proteins for Biotechnology