细菌分解代谢2-氯-4-硝基酚污染物的机理研究

Chlorinated nitroaromatic compounds are persistent environmental pollutants that have been introduced into the environment due to the industrial development and the anthropogenic activities. Micro-organisms play the most important roles in the degradation of aromatics and the carbon recycling in the earth, through a variety of adaptive evolution mechanism and the diversity of metabolic capacity. The research focusing on the metabolic mechanism will be greatly helpful not only for our fully understanding of the diversity of microbial metabolic pathways, but also for the taking full advantages of the diversities and for the biodegradation, biocatalysis, biotransformation and bioremediation. .In this study, two 2-chloro-4-nitrophenol utilizers, Rhodococcus sp. DSM45091 and Burkholderia sp. DSM 23195 were selected to illustrate the molecular mechanism of bacterial aromatic degradation. The two strains are gram positive and gram negative respectively, with diverse aromatic degradation abilities, and can fully carbonized 2-chloro-4-nitropheol via different pathways. The aims of this study are to clone and identify the gene clusters involved in 2-chloro-4-nitropheol degradation in these two strains, via gene knockout, in vitro expression and enzymatic characterization. The results will contribute greatly to illustrate the mechanisms for the aromatic biodegradation from molecular, biochemical, and genetic levels. Furthermore, using 2-chloro-4-nitrophenol as a model, we will explore the evolution for the diversity of the microbial metabolism of aromatics and their adaptive evolution strategy.

面对种类繁多的芳烃污染物，微生物通过多种适应性进化机制而获得多样性的代谢能力。对其代谢机理研究，不仅丰富了对微生物代谢途径多样性的认识，也是从事生物降解、生物催化、生物转化以及生物修复等相关研究的前提。具有多种芳烃污染物代谢能力的革兰氏阳性的Rhodococcus sp. DSM45091和革兰氏阴性的Burkholderia sp. DSM 23195菌株能够通过两个完全不同的途径矿化污染物2-氯-4-硝基酚，本研究拟克隆这两株菌株中参与2-氯-4-硝基酚及其它芳烃污染物代谢的基因簇，结合基因敲除、体外表达、酶活测定等方法鉴定其生理学功能，首次从分子、生化、遗传学水平上阐明这两株菌株的代谢机理。并以这两株菌作为革兰氏阳性和阴性菌的代表，以2-氯-4-硝基酚为模式污染物，探索微生物代谢芳烃污染物的代谢机理的多样性及其适应性进化策略。

2-氯-4-硝基酚（2CNP）是一种重要的化工原料，常用于医药、染料和农药等化工产品的合成，并且被大量释放到环境中并造成严重的环境污染。关于2CNP的微生物降解目前仅仅停留在菌株的筛选以及代谢途径的鉴定，没有任何分子生物学和生化水平的相关报道。本研究选取革兰氏阴性菌Burkholderia sp. strain SJ98 (DSM23195) 和革兰氏阳性菌Rhodococcus imtechensis RKJ 300 (DSM45091)，首次从分子生物学和生化水平上对2CNP的代谢途径进行研究。产物鉴定结果表明SJ98菌株降解2CNP生成中间代谢产物2-氯-对苯二醌和2-氯-对苯二酚。从SJ98菌株中克隆了参与2-氯-4-硝基酚代谢的核苷酸序列（pnpABA1CDEF基因簇）。操纵子结构分析显示pnp基因簇的全部基因位于同一个操纵子，转录水平分析显示2-氯-4-硝基酚和4-硝基酚都能诱导pnp基因簇的转录。酶学分析和产物鉴定证明PnpA可以催化2-氯-4-硝基酚和4-硝基酚的单加氧反应，并分别生成2-氯-对苯二醌和对苯二醌。PnpB可以催化2-氯-对苯二醌和对苯二醌的还原，并分别生成2-氯-对苯二酚和对苯二酚。而且PnpB在体外可以明显增强PnpA的活力。基因敲除和互补证实pnpA基因是菌株SJ98降解2CNP和4-硝基酚所必需的基因。通过顺序催化还发现PnpCD、PnpE和PnpF同时参与了2CNP和4-硝基酚的下游代谢途径。Rhodococcus imtechensis RKJ 300 (DSM45091) 降解2CNP以偏苯三酚为开环底物。从RKJ 300菌株中克隆并鉴定了参与2CNP代谢的核苷酸序列（pnpA1A2BC基因簇）。酶学分析和产物鉴定证实PnpA1A2除了可以催化4-硝基酚氧化生成对苯二酚和偏苯三酚外，还能催化2-氯-4-硝基酚的氧化，并检测到2-氯-对苯二酚和偏苯三酚的生成。此外，PnpA1A2还能分别催化2-氯-对苯二酚和对苯二酚的单加氧反应，并且都生成偏苯三酚。基因敲除互补证实pnpA1编码基因是RKJ300菌株利用2CNP和4-硝基酚所必需的基因。本研究首次在分子生物学和生物化学水平上阐明了2CNP的微生物降解机制。并且证实了无论是在革兰氏阴性菌还是革兰氏阳性菌株中，2CNP和4-硝基酚在代谢时都共用一套完整的基因簇。