Given the current inability to degrade trinitrotoluene (TNT) aerobically with existing dioxygenases, and the accumulation of aminodinitrotoluenes (ADNT) through natural attenuation of TNT, are using an innovative protein engineering approach to create dioxygenases that initiate aerobic attack on ADNT and/or TNT. The enhanced dioxygenases will be coupled to existing degradative enzymes to yield the first aerobic pathway for TNT and ADNT mineralization (complete conversion to innocuous compounds). The inherent broad substrate range of the wild-type nitroarene dioxygenases should allow DNA shuffling to generate enzymes with enhanced activity against TNT/ADNT. The dramatic modifications afforded by shuffling mutagenesis should overcome the steric barriers that remain for TNT derivatives as substrates. The approach will be to expand the substrate range of nitroarene dioxygenases to allow oxidative transformation of ADNT and/or TNT by concentrating on shuffling the gene encoding the large subunit of the terminal oxygenase; this protein dictates the catalytic specificity of the dioxygenase. DNA shuffling will be used to create variants based on the family of related three-component dioxygenases to create chimeric dioxygenase enzymes (molecular breeding). By swapping domains among various related enzymes, even greater advances in enzymatic activity can be obtained.

Building on a recent $715,000 grant from NSF, a team from UConn and Colorado State University will metabolically engineer a bacterium for the degradation of chlorinated ethenes such as tetrachloroethylene (PCE) and trichloroethylene (TCE). The specific objectives are to (1) use DNA shuffling to create an optimized monooxygenase that degrades PCE, TCE, and mixtures of chlorinated solvents, (2) clone this monooxygenase into the chromosome (3) clone an epoxide hydrolase into the chromosome to reduce toxicity of the chlorinated aliphatic degradation products which include unstable epoxides (4) evaluate the effects of this genetic engineering on host cell physiology using 2-dimensional protein electrophoresis, and (5) evaluate the fate of the modified cells and introduced genes in soil and bioreactor settings. One of the host strains, designed for field application, will be a biosurfactant producer modified to decrease adhesive tendencies; the other bacterium will be unaltered and is intended for use in bioreactors. Adding new metabolic capabilities to an organism is only part of metabolic engineering. To fully achieve the goal of creating a more useful organism, the effects of the genetic modification(s) and the functioning of the new strain must be evaluated. In this project, bioinformatics (2-D protein electrophoresis) will be used to determine the impacts of the new pathway (monooxygenase and epoxide hydrolase) on cellular physiology, and the ability of the field application organism to function in soil environments will be evaluated. Finally, the genes of the optimized monooxygenases will be sequenced and compared to the wild-type enzymes to probe enzyme structure/function relationships. The kinetics of the biodegradation reactions will also be discerned so that bioreactors may be designed.

Man has polluted his environment, and bacteria have a vast potential for cleansing it. We recently have discovered the only known aerobic enzyme which can degrade tetrachloroethylene (PCE), toluene-o-xylene monooxygenase (ToMO) of the soil bacterium Pseudomonas stutzeri OX1 (Nature Biotechnology, July, 2000); hence, for the first time, all the chlorinated ethenes may be degraded by a single enzyme (e.g., trichloroethylene, cis-1,2-dichloroethylene, trans-1,2-dichloroethylene, 1,1-dichloroethylene, and vinyl chloride). In an effort to create a stable, competitive recombinant bacterium to remediate soil and groundwater of these chlorinated wastes, it is proposed integrate the genes for ToMO into the chromosome of a tree-colonizing bacterium so that we utilize the niche environment created by plant roots (rhizosphere) which favors those microorganisms that have adapted to use the enzymes and nutrients exuded by plant roots. This bacteria-plant interaction will be utilized to facilitate PCE degradation since the plant transports the bacteria to the PCE in soil and contaminated water, breaks the soil, and provides both nutrients and oxygen for PCE degradation. Current investigations have had great success with using a related monooxygenase to create rhizoremediation systems which can degrade TCE. We also plan to create even better enzymes for use in these plant-bacterium systems by using DNA evolution, a cutting-edge, random mutagenesis process which can enhance enzymes for bioremediation. Our goals are to create enzymes which degrade PCE better as well as degrade mixtures of chlorinated ethenes more effectively. Also, we wish to study the plant/bacterium interactions necessary to create robust and competitive engineered bacteria. Along these lines, we have tagged the poplar hosts with the green fluorescent protein and wish to study these chlorinated-ethene-degrading strains in the rhizosphere using confocal microscopy. We would like also to investigate how PCE and TCE induce ToMO activity indirectly (which molecules interact with the DNA). In addition, we are studying chemotaxis of P. stutzeri OX1 toward PCE and TCE. Hence, this strain may be used to remediate sites without the addition of chemical inducers as it moves toward the contaminate.

This project will result in the creation of optimized monooxygenase enzymes for two novel synthetic routes which will create less wastes than the current synthesis methods. Using directed evolution and molecular breeding, monooxygenases will be evolved both for converting naphthalene to 1-naphthol and for creating chiral styrene epoxide. In addition, DNA microarrays will used to understand the burden of expressing these large enzymes and to optimize the Escherichia coli host for large-scale production of these chemicals. The wastes which will be reduced (or eliminated) include solvents (toxic pyridine), acids (naphthalene-1-suflonic acid and toxic hydrogen fluoride), bases (NaOH), and metal catalysts for 1-naphthol synthesis and the solvent methylene chloride for styrene epoxide synthesis (required for a Mn-catalyzed reaction and methylene chloride is toxic and a suspected carcinogen).

The specific objectives of this project are:

The Wood group has recently used directed evolution (one round of shuffling) to enhance the ability of toluene o-monooxygenase (TOM) of the soil bacterium Burkholderia cepacia G4 to degrade trichloroethylene (TCE) and to oxidize naphthalene to 1-naphthol (BES-9807146). This represents the first directed evolution of this class of enzymes as well as the first directed evolution of an enzyme containing 6 subunits; it also shows that an enzyme may be evolved simultaneously for both green chemistry and environmental remediation. These results are also the first report of DNA shuffling for remediating chlorinated ethenes. The industrially-significant conversion of naphthalene to 1-naphthol was enhanced (6 to infinite-fold) with the same beneficial mutation. No change was made in the regiospecificity of the enzyme since greater than 97% 1-naphthol was produced. In addition, the beneficial mutation of TOM-Green was identified and related to protein function: the substitution of valine to alanine at position 106 of the a -subunit of the hydroxylase was shown to alter an important "gate" amino acid.

The anticipated results of this research are the creation of two new, broadly-applicable, biological synthetic routes for two industrially-relevant sythons, 1-naphthol and styrene epoxide, and a better, basic understanding of bacterial fermentations.

To identify environmentally-innocuous, effective anti-biofouling agents, we are investigating the use of naturally-occurring marine furanones to inhibit the formation of biofilms and their resulting biofouling on ship hulls. This project will result in the formulation of a hull coating which will resist biofouling based on natural and improved furanones as well as based on new compounds identified by discovering the genes required for biofilm formation.

The specific objectives of this project are:

This work is based on the recent discoveries that bacteria in biofilms must communicate with one another using chemical signals and that furanones from the red alga inhibit the formation of biofilms by interrupting this signaling process. It is anticipated that the composition of the metal surface should not matter and that this anti-biofouling strategy will be broadly applicable to both engineering and medicine.

It has been estimated recently that the yearly corrosion damage costs are equivalent to 4.2% of the U.S. Gross National Product. Traditional organic coatings are not cost-effective; however, bacteria can coat metals with a regenerative biofilm, and this biofilm may be used to prevent corrosion. Microbiologically-induced corrosion (MIC) is a common, recurring corrosion problem in industrial systems like cooling towers, pipes and oil rigs and is caused by sulfate-reducing bacteria (SRB) which grow in anaerobic pockets of naturally-occurring, aerobic biofilms and results in pitting of the steel. The objectives of this project are to evaluate the feasibility of using aerobic biofilms to inhibit corrosion of various metals including steel. To this end, various biofilm-forming bacteria (belonging to seven different genera) have been evaluated for their corrosion inhibition (some reduce corrosion by 45-fold), and the protective architecture of the biofilms quantified in terms of the proportion of live cells, dead cells, and polysaccharide using confocal microscopy. The mechanism of corrosion inhibition appears to involve aerobic bacteria removing oxygen (as they respire) and preventing it from oxidizing the metal surface. These protective bacteria have been genetically engineered to produce antimicrobials to prevent corrosive attack by sulfate-reducing bacteria.