2005 Progress Report: Enhanced Production of Biodegradable Plastics in Plants

EPA Grant Number: R829479C007
Subproject: this is subproject number 007 , 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: Enhanced Production of Biodegradable Plastics in Plants
Investigators: Mooney, Brian Patrick
Institution: University of Missouri - Columbia
EPA Project Officer: Lasat, Mitch
Project Period: June 1, 2003 through December 31, 2006 (Extended to December 31, 2007)
Project Period Covered by this Report: June 1, 2005 through December 31, 2006
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) confirm the plastid-targeting of the chimeric Arabidopsis thaliana branched-chain dehydrogenase alpha and beta subunits (AtBCE1 α and AtBCE1 β); (2) confirm the protein-protein association of the chimeric AtBCE1 enzyme (composed of 2α and 2β subunits) with the endogenous plastid dihydrolipoyl acteyltransferase (E2) enzyme; and (3) generate transgenic plants expressing plastid-targeted AtBCE1 that produce elevated amounts of propionyl-CoA.

Progress Summary:

Objective 3

We have generated plant transformation constructs that allow chloroplast targeting of the alpha and beta subunits of the branched-chain alpha-ketoacid dehydrogenase (AtBCE1 α and AtBCE1 β). These constructs were used to transform Arabidopsis thaliana plants. As a result of restriction enzyme site constraints, the two subunits had to be cloned into separate transformation vectors. Plants then were transformed with the individual constructs and co-transformed with both constructs. Transgenic plants containing the alpha subunit only were generated and shown to contain the transformation construct by polymerase chain reaction (PCR). Additionally, some plants were demonstrated to survive on both antibiotics encoded by the transformation vectors and, presumably, have been co-transformed with the alpha and beta subunits. We subsequently confirmed that both constructs were present in the transgenic plants using PCR followed by DNA sequencing of the amplified products. We are now in the process of confirming that the two proteins encoded by the transformation vectors under control of the 35S promoter are being expressed. Sixteen independent transformation events (transgenic plants) are being evaluated for AtBCE1α/β expression by Western blotting. Protein has been isolated from whole leaves as well as from chloroplasts prepared from developing leaves. We have antibodies to both subunits that are being employed in the Western blotting. We have some preliminary data that suggest at least the alpha subunit is expressed, but the experiments are ongoing.

Future Activities:

We will continue to attempt to confirm expression of the AtBCE1 α and AtBCE1 β subunits in the transgenic plants we have generated. Additionally, we plan to take a proteomics approach to confirm expression. The difference gel electrophoresis (DIGE) technique will be employed to label two samples prior to analysis by two-dimensional (2D) electrophoresis. These two samples (wild-type and transgenic plants) then are separated in the same 2D gel. Similar to a microarray, proteins that are expressed differentially show up as red or green spots on the gel, whereas those unchanged in their expression are yellow. This technique will be coupled to mass spectrometry to identify the differentially expressed proteins. This will be useful to confirm that the AtBCE1α and β are expressed and to determine if there are other unforeseen changes in protein expression occurring in the transgenic plants that could potentially affect their utility for production of biodegradable plastics.

As the funding for these experiments ends on December 31, 2005, we hope to confirm the expression of the subunits quickly. Following this, we need to determine if the biodegradable plastics seed chemicals (propionyl-CoA and acetyl-CoA) are being produced at a level sufficient to support biodegradable plastic production. If we get to this step, it is anticipated that we will attempt to license our technology (through the University of Missouri) to Metabolix (Cambridge, MA). Metabolix holds all the patents for biodegradable plastic production in plants that were previously held by Monsanto. An alternative to licensing our technology to Metabolix is to secure additional funding from this company or a federal agency to continue the work by crossing our transgenic plants with those already expressing the bacterial enzymes required for biodegradable plastic production.

Journal Articles:

No journal articles submitted with this report: View all 6 publications for this subproject

Supplemental Keywords:

sustainable industry, waste, agricultural engineering, bioremediation, environmental engineering, new technology, innovative technology, bioaccumulation, biodegradation, bioenergy, bioengineering, biotechnology, phytoremediation, plant biotechnology,, Scientific Discipline, TREATMENT/CONTROL, Sustainable Industry/Business, Geochemistry, Genetics, Technology, New/Innovative technologies, Environmental Engineering, Agricultural Engineering, bioengineering, biodegradable plastics, plant genes, biotechnology, plant biotechnology, novel plastics

Relevant Websites:

http://www.cpbr.org Exit

Progress and Final Reports:

Original Abstract
  • 2003 Progress Report
  • 2004 Progress Report
  • 2006
  • Final Report

  • 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