Directed Evolution of Aromatic Dioxygenases for Trinitrotoluene and Aminodinitrotoluene Degradation

[01:14126] Smets本研究项目的目的是利用一种创新的蛋白质工程方法来制造双加氧酶，对TNT自然衰减的产物三硝基甲苯（TNT）和氨基二硝基甲苯（ADNT）发起有氧攻击。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洗牌将分别用于创建两个2,4-二硝基甲苯（DNT）双加氧酶的变体。Burkholdria sp.菌株DNT将作为初始宿主，为TNT/ADNT降解创造一个完整的途径。将开始建立一个完整的、成功的ADNT和/或TNT矿化途径。因此，预期转化产品的下游加工的酶的步骤也将被解决。利用一种新的定向进化方法和代谢工程，该项目旨在创建TNT/ADNT生物矿化的第一个氧化酶和途径。构建的菌株对于制定可持续的爆炸物污染环境修复战略具有不可估量的价值。＊＊＊

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. ***