Grantee Research Project Results
Final Report: Interdisciplinary Biotechnology Program in Environmental Dehalogenation
EPA Grant Number: R827612E02Title: Interdisciplinary Biotechnology Program in Environmental Dehalogenation
Investigators: Sawyer, Roger , Woodin, Sarah A. , Lincoln, David E. , Lovell, Charles R. , Little, T. Scott , Morris, James T. , Yoch, Duane , Dawson, John , Marton, Laszlo , Leiboda, Lukasz , Czako, Mihaly , Chen, Yung-Pin
Institution: University of South Carolina at Columbia
EPA Project Officer: Chung, Serena
Project Period: June 1, 1999 through May 30, 2002
Project Amount: $246,999
RFA: EPSCoR (Experimental Program to Stimulate Competitive Research) (1998) RFA Text | Recipients Lists
Research Category: EPSCoR (The Experimental Program to Stimulate Competitive Research)
Objective:
The specific objectives of this research project were to:
1. Optimize the catalytic properties of the dehaloperoxidase (DHP) from the polychaete sea worm Amphitrite ornate.
2. Characterize the DHPs from the plant Spartina alterniflora and a bacterial isolate. Determine substrate range and gene and protein sequence, optimize the purification procedures, and characterize the enzymes using kinetics, spectroscopy, and x-ray crystallography.
3. Determine consensus sequences and regions important for function and substrate specificity.
4. Tailor the nucleotide sequence of the DHP gene that shows greatest stability to fit the consensus sequence required for broader substrate specificity determined by careful analysis of the three-dimensional to create a single biocatalyst of superior qualities.
5. Improve human resources and physical infrastructure for phytoremediation research.
Summary/Accomplishments (Outputs/Outcomes):
Haloaromatic compounds present a most severe environmental problem due to their relative stability and toxicity to vertebrates. The goal of this research project was to characterize the haloaromatic degrading enzymes, DHPs from the sea worm Amphitrite ornata and the salt marsh plant S. alterniflora, and use the kinetic, spectroscopic, x-ray crystallographic, amino acid, and DNA sequence data to help create a single biocatalyst of superior qualities.
We report on the successful cloning, sequencing of the full length, protein-coding sequence, and detailed three-dimensional structure for DHP obtained using a reverse-genetic approach and a cDNA library from marine worm A. ornata (Lebioda, et al., 1999). . The coding sequence has been incorporated into bacterial and plant gene expression systems. Although the enzyme was not active when expressed in Escherichia coli, transgenic plants expressing this gene exhibit enhanced degradation activity against 2,4,6-trichlorophenol, a priority pollutant. The Spartina DHP activity is attributable to multiple enzymes, some of which are comparable in molecular weight to the worm enzyme. The gene-coding region is yet to be isolated from a cDNA library prepared from Spartina root tissue. A system for introducing foreign genes into Spartina has been developed.
Additional objectives of this research project were to improve human resources and physical infrastructure. Accordingly, part of the U.S. Environmental Protection Agency/EPSCoR funds were used as a contribution towards the start-up fund for a new tenure track faculty position. After a nationwide search for a new tenure track faculty member, a specialist of plant uptake and transport processes, Dr. Erin Connolly was hired to enhance the productivity of the existing team (start date August 16, 2000). A full-time research professor, Dr. Yung Pin Chen, was hired at the start of the award to conduct the most critical initial experiments on the purification and characterization of the Spartina DHP.
The participating three new graduate students have met regularly to exchange information and discuss progress of the joined projects. The new students associated with the tenure track and research faculty will conduct rotations in the laboratory of the new participating faculty member. Five undergraduate research assistants, two undergraduate Howard-Hughes fellows, and two high-school summer interns worked in various aspects of the project.
The significance of these achievements is that such genetically modified plants can be used in haloaromatic clean-up technologies. Environmental programs, including brownfields, waste prevention, and hazardous waste cleanup may be interested in these achievements because these are the foundations of useful technologies for environmental protection. To improve human resources development and physical infrastructure, a critical mass of scientific talent including eight faculty, a research associate professor, Dr. Chen, and a staff scientist, Dr. Czako, and four graduate students widened their existing cooperation, and addressed this very important national priority. This program has had a major impact on human resource development and education by enhancing the quality of research and teaching at the University of South Carolina. It also has stimulated cooperation between scientists from different disciplines. Important partnerships with local industry already have been established, generating additional grant applications and funding.
Journal Articles on this Report : 7 Displayed | Download in RIS Format
| Other project views: | All 13 publications | 7 publications in selected types | All 7 journal articles |
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Connolly EL, Guerinot ML. Iron stress in plants. Genome Biology 2002;3(8):1024.1-1024.4. |
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Connolly EL, Fett JP, Guerinot ML. Expression of the IRT1 metal transporter is controlled by metals at the levels of transcript and protein accumulation. The Plant Cell 2002;14(6):1347-1357. |
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Czako M, Marton L. A heartwood pigment in Dalbergia cell cultures. Phytochemistry 2001;57(6):1013-1022. |
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Han KP, Woodin SA, Lincoln DE, Fielman KT, Ely B. Amphitrite ornata, a marine worm, contains two dehaloperoxidase genes. Marine Biotechnology 2001;3(3):287-292. |
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He YK, Sun JG, Feng XZ, Czako M, Marton L. Differential mercury volatilization by tobacco organs expressing a modified bacterial merA gene. Cell Research 2001;11(3):231-236. |
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LaCount MW, Zhang E, Chen YP, Han K, Whitton MM, Lincoln DE, Woodin SA, Lebioda L. The crystal structure and amino acid sequence of dehaloperoxidase from Amphitrite ornata indicate common ancestry with globins. Journal of Biological Chemistry 2000;275(25):18712-18716. |
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Lebioda L, LaCount MW, Zhang E, Chen YP, Han K, Whitton MM, Lincoln DE, Woodin SA. An enzymatic globin from a marine worm -- Brief communications. Nature 1999;401(6752):445. |
R827612E01 (Final) R827612E02 (Final) R824776 (1999) R824776 (Final) |
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Supplemental Keywords:
halo-organics, dehaloperoxidase, DHP, protein structure, genetic/protein engineering, transgenic plants, polychaete sea worm, Amphitrite ornate, Spartina alterniflora, bacterial isolate, kinetics, spectroscopy, x-ray crystallography, nucleotide sequence, phytoremediation., Scientific Discipline, Geographic Area, Waste, Water, TREATMENT/CONTROL, Contaminated Sediments, Environmental Chemistry, Remediation, Treatment Technologies, State, Chemistry, Technology, Bioremediation, Environmental Engineering, environmental technology, biodegradation, contaminated sediment, remediation technologies, chemical contaminants, catalysts, bioremediation of soils, engineering, halogenated organic contaminants, biotechnology, dehalogenation, X-ray crystallography, contaminants in soil, South Carolina (SC), assessment methodsProgress and Final Reports:
Original AbstractThe 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.