Directed Evolution of Aromatic Dioxygenases for Trinitrotoluene and Aminodinitrotoluene Degradation

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0114126 Smets The objective of this research project is to use an innovative protein engineering approach to create dioxygenases that initiate aerobic attack on trinitrotoluene (TNT) and aminodinitrotoluenes (ADNT), products of natural attenuation of TNT. The approach will be to expand the substrate range of nitroarene dioxygenases to allow oxidative transformation of ADNT and/or TNT. The gene encoding the large subunit of the terminal oxygenase will be the focus of this work. DNA shuffling will be used to create variants of two 2,4-dinitrotoluene (DNT) dioxygenases individually. Burkholdria sp. strain DNT will be used as the initial host to create a complete pathway for TNT/ADNT degradation. Creation of a complete, successful ADNT and/or TNT mineralization pathway will be initiated. Hence, the enzymatic steps for the down-stream processing of expected transformation products will also be addressed. Using a novel, directed-evolution approach and metabolic engineering, this project aims to create the first oxidative enzyme and pathway for the biomineralization of TNT/ADNT. The constructed strain will be invaluable in developing sustainable strategies for remediation of explosives contaminated environments. ***