Program Element 3: Biomolecular Science and Engineering

Fundamental research in molecular and structural biology to enhance our understanding of bioremediation and improve the efficacy of bioremedial organisms and identify novel remedial genes.

PROGRAM OBJECTIVE

To use molecular biology to enhance our understanding of bioremediation and to genetically modify molecules and organisms to improve bioremedial activities.

BACKGROUND

The potential of molecular manipulation to enhance bioremediation in the field remains untapped.

It is generally accepted that the great diversity of organisms living in soil and ground water have evolved the biochemical tools to detoxify many anthropogenic contaminants of concern to DOE. The enzymatic pathway which degrades trichloroethylene (TCE) is one example. At some contaminated sites, resident organisms lack critical enzymes for degradative processes or are inhibited by some component of the mixed contamination. Selection techniques in genetics that modify organisms have been used to improve remedial affects on the environment, for example, bacterial-enhanced oil recovery. No molecular manipulations have yet been applied to bioremediation in the field, and the real potential of this methodology remains untapped.

The use of genetically engineered organisms in the environment has been limited by public perception and the concerns of regulatory agencies about the potential risks of releasing genetically altered organisms into the environment. In addition, engineered organisms' inability to compete with natural soil populations is an issue that must be addressed. Accordingly, the technology associated with the regulation and containment of such organisms and the direct use of biomolecules extracted or removed from cell-free systems are critical areas for research.

APPROACH

Molecular biologists can isolate and characterize genes and gene products that will enhance the bioremediation of mixed contaminants.

Using the information and data gained from the program elements on Community Dynamics and Biotransformation and Biodegradation, scientists will gain a better understanding of the active, critical resident soil and subsurface populations. They will identify the molecules, enzymes, and pathways most effective for bioremediation of mixed contaminants and determine how techniques in molecular and structural biology can improve the effectiveness of these organisms. Molecular biologists can isolate and characterize genes and gene products that will enhance the bioremediation of mixed contaminants. They will conduct structural studies of bioremedial enzymes and other molecules to build a basis for understanding and manipulating molecular activities for bioremediation purposes. They will also develop expression systems for remedially active molecules for pathway engineering, structure and function studies, and large-scale, cell-free systems. In addition, molecular biologists will use rational design and random mutagenesis techniques to modify activity and substrate binding specificity once the structural aspects of molecules with remedial potential are identified and characterized. All of these efforts combined should lead to the application, testing, and evaluation of improved bioremedial activity with respect to the stability, efficacy, and safety of recombinant organisms and new or engineered molecules in the field research center.

Accordingly, the biomolecular engineering program will concentrate on five areas:

1. Analysis of genes and regulatory elements for critical molecules in bioremediation.

2. Structure and function of bioremedial molecules.

3. Genetic selection and engineering of improved bioremedial molecules and organisms.

4. Pathway engineering.

5. Cell-free systems for bioremediation.

Subelement 3.1: Analysis of Genes, Proteins, and Regulatory Elements for Critical Molecules in Bioremediation

Fundamental research to identify genes and promoters that affect bioremedial activity.

Objective

Identify, clone, and sequence novel genes and promoters important to the bioremediation of mixed contaminants.

Goals

Three-Year

Identify and characterize the important bioremedial genes and proteins by cloning and sequencing.

Identify critical promoter elements that induce or regulate bioremedial activity of mixed contaminants.

Five-Year

Develop expression systems, including transformation systems of soil organisms, for bioremedial genes and proteins.

Identify gene products, including substances that enhance the ability of organisms to live under contaminated conditions, that affect the survivability of bioremedially active organisms in mixed contaminants.

Ten-Year

Evaluate and compare recombinant organisms against natural populations for bioremedial efficacy in the field research center.

Subelement 3.2: Structure and Function of Bioremedial Molecules

Fundamental research to determine the three-dimensional structure and critical components of molecules of bioremedial value for sites with mixed contaminants.

Objective

Obtain structure and function information pertinent to bioremediation that will, for example, facilitate the design and expression of improved molecules and our understanding of enzymatic mechanisms for the detoxification of contaminants.

Goals

Three-Year

Develop strategies for overproduction of bioremedial molecules by prokaryotes and eukaryotes for structural analyses of these molecules.

Five-Year

Study the structural information about bioremedial molecules provided by x-ray diffraction patterns, and nuclear magnetic resonance. Use structural information to predict and test rational strategies for improvements in bioremedial molecules.

Ten-Year

Test designed bioremedial molecules for their efficacy in situ and ex situ.

Subelement 3.3: Genetic Selection and Engineering of Improved Bioremedial Molecules and Organisms

Fundamental research in microbiology to genetically engineer and enhance molecules and organisms for bioremediation.

Objective

Generate molecules and organisms through mutagenesis and selection with improved bioremedial activity.

Goals

Three-Year

Generate and choose mutants for improved bioremedial activity through mutagenesis, directed evolution, and other means.

Examine robust members of the contaminated soil community for their potential as hosts and carriers for horizontal gene transfer and for developing alternative transformation systems.

Five-Year

Design and develop, in genetically modified organisms, survival selection pressure systems for expression of heterologous remedial genes in the field.

Develop genetically modified organisms that are more predictable in terms of performance and ecological behavior in sites containing mixed contaminants by using alternative, nonantibiotic selection and suicide determinants.

Ten-Year

Test useful, predictive, and competitive genetically modified organisms for applications in the field and for large scale bioprocessing engineering.

Subelement 3.4: Pathway Engineering

Fundamental research to identify, isolate, and mix genes involved in bioremedial pathways.

Objective

To construct ("quilt") or enhance a bioremedial pathway by identifying actives genes from different prokaryote and eukaryote organisms and then inserting those genes into one of more organisms.

Goals

Three-Year

Identify genes from different organisms that can work together to transform the intermediate compounds in a bioremediation pathway from toxic contaminants to nontoxic endpoints.

Five-Year

Create improved enzymatic pathways for bioremediation by combining optimized genes from different sources into a single organism.

Combine activities from different (engineered) organisms to create extracellular enzymatic pathways.

Ten-Year

Evaluate genetically engineered organisms for their effectiveness in the field and their ability to outperform natural organisms.

Subelement 3.5: Cell-Free Systems for Bioremediation

Fundamental research to evaluate the use of cell-free systems (enzymes and biochelators) for bioremedial activity.

Objective

Examine cell-free systems that may have an application or delivery advantage over whole cells.

Goals

Three-Year

Identify molecules that have potential for bioremediating mixed contaminants in cell-free systems.

Using information from other program subelements, identify, express, and evaluate the applicability for in situ bioremediation of mixed contaminants.

Five-Year

Design experiments to test how well cell-free molecules perform in the soil matrix and in bioreactors.

Develop methods to produce high yields of these molecules to test their efficacy, durability, reproducibility, and economy as bioremedial agents.

Investigate the feasibility for large-scale production and recovery of these molecules.

Ten-Year

Test cell-free bioremedial molecules for their efficacy in situ and ex situ.

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