Acidovorax sp. JS3050分解代谢3,4-二氯硝基苯的机理研究

Continuous evolution of metabolic pathways of microorganisms in the polluted environment are gradually adapting themself to the capability of degrading the synthetic chemicals. Compound 3,4-dichloronitrobenzene (3,4DCNB) is a toxic and persistent synthetic pollutant, which is a key starting material for the production of diuron and other herbicides. Up to now, no dichloronitrobenzene degrading bacteria or metabolic pathways have been reported. In a previous work, Acidovorax sp. JS3050 was isolated from the polluted soil and can grow with 3,4-dichloronitrobenzene as sole carbon, nitrogen and energy source. This strain was confirmed to metabolize 3,4DCNB by oxidation pathway and releases nitrite, and its metabolic activity of 3,4DCNB is inducible. This research aims to demonstrate the microbial 3,4DCNB catabolic pathway and key genes involved in this pathway. Genome sequencing, transcriptome sequencing, transposon insertion mutant libraries and other approaches will be used to search for the genes involved. Through gene expression, protein purification and products identification, the function of candidate genes will be characterized. Gene knockout and complementation will also help to clarify the 3,4DCNB metabolic pathways in this strain. This research is helpful for understanding the unknown metabolic pathways of dichloronitroaromatics, enhancing our understanding of the diversity and evolution of nitroarene metabolic pathways, as well as has potential application for bioremediation of 3,4DCNB contaminated environment.

污染环境中的微生物不断的适应性进化而逐渐具有了降解人工合成化合物的能力。具有毒性且难以降解的3,4-二氯硝基苯是生产敌草隆和其它除草剂的关键原料，为一种重要污染物。但迄今为止尚无任何二氯硝基苯降解菌更无其代谢途径的报道。前期工作中从污染土样中分离到一株能以3,4-二氯硝基苯为唯一碳源、氮源和能源生长的细菌Acidovorax sp. JS3050。该菌株在3,4-二氯硝基苯诱导下通过氧化途径代谢该物质，并释放亚硝酸根。本研究旨在通过基因组测序、差异转录组、插入突变文库等方法寻找代谢相关基因；通过基因表达与纯化，酶学研究，反应产物鉴定来确认基因功能；同时结合基因敲除与互补阐明该菌株降解3,4-二氯硝基苯的代谢途径。该研究有助于对尚未知晓的二氯硝基芳烃的代谢途径的认识，增强人们对硝基芳烃代谢途径多样性及进化的理解，并且对3,4-二氯硝基苯污染环境的生物修复具有潜在的应用价值。

硝基芳烃化合物被广泛应用于化学中间体、炸药、农药和染料的工业生产中，并由此引发了严重的水体和土壤污染。由于苯环的稳定性以及硝基的吸电子作用，该类化合物化学性质较为稳定且易在环境中长期积累，具有生殖毒性、致突变、致畸、致癌等特性，严重威胁人类的健康和生存环境。目前已经分离到多株微生物能够分别完全矿化多种硝基芳烃污染物并作为唯一的碳源、氮源和能源生长。这为硝基芳烃污染的生物治理和生物修复提供了可能。污染环境中的微生物不断的适应性进化而逐渐具有了降解人工合成化合物的能力。具有毒性且难以降解的3,4-二氯硝基苯（3,4DCNB）是生产敌草隆和其它除草剂的关键原料，是一种重要的污染物。但迄今为止尚无任何二氯硝基苯降解菌更无其代谢途径的报道。前期工作中从污染土样中分离到一株能以3,4-二氯硝基苯为唯一碳源、氮源和能源生长的细菌Diaphorobacter sp. strain JS3050。该菌株在该物质诱导下利用氧化途径代谢3,4DCNB，并释放亚硝酸根。本研究通过基因组测序等方法初步找到了相关基因；通过基因表达，酶学研究，反应产物鉴定确认了基因的功能。由染色体编码的三组分硝基芳烃双加氧酶DcnAaAbAcAd将3,4DCNB等化学计量数的转化为4,5-二氯邻苯二酚，随后由氯邻苯二酚1,2-双加氧酶DcnC催化4,5-二氯邻苯二酚的开环反应而逐渐进入三羧酸循环。该研究揭示了尚未知晓的二氯硝基芳烃的微生物代谢途径，增强了人们对硝基芳烃代谢途径多样性及进化的认识，并且对3,4DCNB污染环境的生物修复具有潜在的应用价值。