Directed Evolution of Aromatic Dioxygenases for Trinitrotoluene and Aminodinitrotoluene Degradation

0114126 Smets该研究项目的目的是使用创新的蛋白质工程方法来创建双加氧酶，启动对三硝基甲苯（TNT）和氨基二硝基甲苯（ADNT）的有氧攻击，TNT的天然衰减产物。 该方法将扩大硝基芳烃双加氧酶的底物范围，以允许ADNT和/或TNT的氧化转化。 编码末端加氧酶大亚基的基因将是这项工作的重点。 DNA改组将被用来创建两个2，4-二硝基甲苯（DNT）双加氧酶单独的变体。 伯克霍尔德氏菌属菌株DNT将用作初始宿主，以创建TNT/ADNT降解的完整途径。 将开始创建完整、成功的ADNT和/或TNT矿化途径。 因此，还将讨论预期转化产物下游加工的酶促步骤。 利用一种新的定向进化方法和代谢工程，该项目旨在创建第一个用于TNT/ADNT生物矿化的氧化酶和途径。 构建的菌株将是非常宝贵的，在制定可持续的战略，修复爆炸物污染的环境。***

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. ***