Grantee Research Project Results
2003 Progress Report: Biological Effects of Epoxy Fatty Acids
EPA Grant Number: R829479C003 aka R829479C011Subproject: 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: Klieforth, Barbara I
Project Period: August 7, 2003 through September 30, 2003
Project Period Covered by this Report: August 7, 2003 through September 30, 2004
RFA: The Consortium for Plant Biotechnology Research, Inc., Environmental Research and Technology Transfer Program (2001) RFA Text | Recipients Lists
Research Category: Hazardous Waste/Remediation , Targeted Research
Objective:
The objectives of this research project are 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; and (3) test the effects of these epoxy fatty acids on model bacteria.
Progress Summary:
Initially, we prepared gram quantities of Vernonia oil, castor oil, purified vernolic acid, free fatty acids from Vernonia oil, and methyl-vernolate. Vernonia oil is approximately 70 percent of a 12,13-epoxy fatty acid, vernolic acid, and castor oil is approximately 90 percent of another oxygenated fatty acid, the 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 such effects of these compounds as yet have not been investigated. These oxygenated fatty acids were added to corn at ppm levels and tested against corn insect pests by John Sedlacek and a student at Kentucky State University (see Annual Report for STAR Grant No. R829479C011). 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 all five compounds, each in excess of 100 g for Kentucky State University's insect feeding studies. The castor oil was purchased from a local drug store ("castor oil USP"). The 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 the last two compounds was trivernolin, which was purified from the Vernonia oil by recrystallization 11 times. The vernolic acid content in the trivernolin preparation is about 98 percent based on gas chromatograph-mass spectrometer 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 from thin-layer chromatography analysis that we believe to be a dihydroxy fatty acid, possibly produced from vernolic acid as a result of the acidification process, even though the acetic acid used is a weak acid. The identity of this compound, whether dihydroxy fatty acid or something else, will be investigated further. Methyl vernolate was made by sodium methoxide transmethylation from trivernolin.
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. It is anticipated that this combination of genes, when highly expressed in developing soybean seeds, should lead to soybean seeds with at least moderate accumulation of vernolic acid in its oil. Transgenic somatic embryos were matured, some were germinated, and whole plants were grown in the greenhouse. The plants should produce mature seeds soon, and these will be analyzed for vernolic acid content. Those with appreciable accumulation will be grown for another generation to accumulate more seed for insect feeding studies. We also have made transgenic yeast expressing both of these genes, and these also will be added to insect diets and E. coli growth media. Additionally, we have produced engineered yeast with epoxygenase genes that produce isomers of vernolic acid thought to have different biological effects, such as higher insecticidal activity than vernolic acid.
Future Activities:
Higher concentrations of Vernonia oil, castor oil, purified vernolic acid, free fatty acids from Vernonia oil, and methyl-vernolate will be tested for effects on storage insects and vegetable insect pests by Sedlacek, et al., at Kentucky State University. These compounds also will be tested for effects on growth of Escherichia coli cultures at the University of Kentucky. The accumulation of alternative epoxy fatty acids in the engineered yeast from the above work will be investigated, and those with accumulation will be used in insect and E. coli growth studies. Likewise, the progeny of transgenic soybean plant seeds will be tested for epoxy fatty levels, and those with significant levels (5 percent or more) will be added to insect diets and bacterial growth media.
Journal Articles:
No journal articles submitted with this report: View all 4 publications for this subprojectSupplemental Keywords:
pest control, hydroxy fatty acids, vernolic acid, insects, bactericides., 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, bacteriacidesRelevant Websites:
http://www.uky.edu/Agriculture/Agronomy/PLBC/research.html Exit
http://www.cpbr.org Exit
Progress and Final Reports:
Original AbstractMain 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
The perspectives, information and conclusions conveyed in research project abstracts, progress reports, final reports, journal abstracts and journal publications convey the viewpoints of the principal investigator and may not represent the views and policies of ORD and EPA. Conclusions drawn by the principal investigators have not been reviewed by the Agency.
Project Research Results
Main Center: R829479
208 publications for this center
44 journal articles for this center