一株含双降解质粒的红球菌（Rhodococcus sp.）二噁英降解机理研究

Dioxin pollution is one of the severe problems in China. Biodegradation by aerobic bacteria is an important way to remove dioxins. The crucial step in degradation of dioxins is the initial dihydroxylation catalyzed by dioxygenases which are encoded by conservative dfdA or dbfA in distinct Gram positive degraders. However, a few strains harbor both dfdA and dbfA, which may expand the substrate range and improve removal activity. It is unclear that how the dioxygenase DfdA and DbfA cooperate in such strain during degradation of dioxin. Genes involved in initial ring-cleavage of dioxin are usually harbored by catabolic plasmid. It can be expected that cooperation of the catabolic plasmids in the strain favors its catabolic ability and environmental adaptation. Cooperation of the catabolic plasmids, especially the dioxin catabolic plasmids, remains to be explored. The proposed research involves the cooperation of the dioxygenase DfdA and DbfA in a dioxin-degrader Rhodococcus sp., as well as that of two dioxin catabolic plasmids which harbor dfdA and dbfA, respectively. To learn the cooperation of DfdA and DbfA, the substrate ranges of the enzymes, the synergy effects during degradation, and the transcriptional activities of dfdA and dbfA will be investigated. Based on the understanding of collaborative dioxygenases, cooperation of dioxin catabolic plasmids (pDF0 and pDF02) during degradation of dioxins as well as conjugative transfer and stable maintaining of plasmids will be elucidated. The proposed study will provide insight into dioxin biodegradation with Gram positive bacteria, and therefore contribute to the bioremediation of dioxin.

二噁英在我国有广泛的污染源与污染分布区域。有氧条件下细菌的降解作用是去除二噁英污染的重要途径，由双加氧酶催化的起始双羟化是二噁英降解的关键步骤。DfdA与DbfA分别为在不同革兰氏阳性降解菌中发现的催化起始双羟化的双加氧酶，在极少数革兰氏阳性降解菌中同时含有两种酶。本项目拟以一株含有DfdA与DbfA，且酶的编码基因分别位于不同降解质粒（pDF01与pDF02）的红球菌Rhodococcus sp. strain p52为研究对象，研究其二噁英降解机理。通过分析DfdA与DbfA底物范围的差异、降解协同性以及酶基因转录活性的差异，揭示两种双加氧酶在二噁英降解中的协作；在此基础上，进一步分析降解质粒pDF01与pDF02在二噁英降解、质粒接合迁移与稳定遗传中的协作。通过本项目的研究可为开发、利用、改造微生物更有效去除二噁英等污染物提供理论依据。

二噁英污染的生物修复广为关注，在有氧条件下通过细菌矿化极具潜在应用价值。有氧条件下细菌主要是通过有角度的双加氧酶催化起始双羟化（少数微生物通过细胞色素P450催化单羟化），随后通过外二醇双加氧酶催化间位开环裂解，再通过水解酶催化生成小分子有机物（如水杨酸、龙胆酸），后者经转化后可进入三羧酸循环。本项目以一株二噁英降解菌Rhodococcus sp. strain p52为研究对象，该菌含有两套催化起始双羟化的双加氧酶系统DfdA 与 DbfA，二者的编码基因分别位于降解质粒pDF01及pDF02。项目开展了以下研究：（1）对DfdA 与 DbfA在二噁英降解中的作用开展研究。为此分别克隆表达了编码末端加氧酶的dfdA1A2及dbfA1A2，编码外二醇双加氧酶的dfdB及flnD，及编码水解酶的dfdC及flnE。底物转化实验证明，DfdB及DfdC参与二苯并呋喃双羟化产物的间位裂解及水解。在二苯并呋喃或氯代二苯并呋喃存在时，通过转录组分析表明在二噁英降解过程中，位于pDF01上的dfdA1A2、dfdB及dfdC转录活性上调，而位于pDF02上的dbfA1A2、flnD及 flnE转录活性下调。项目也对参与催化烃类羟化的单加氧酶（如P450家族的CYP183）编码基因进行了克隆表达分析。（2）对两个二噁英降解大质粒的接合转移及稳定保持进行研究，基因组完成图测序分析揭示了其分子生物学基础。接合实验证明pDF01及pDF02可从供体菌株p52向多个属的受体菌共同转移。通过Southern杂交及荧光原位杂交，明确了在铜绿假单胞菌接合子细胞中pDF01与pDF02共存，且降解质粒可在该接合子中行使降解功能，完成对氯代二苯并呋喃的转化。进一步的实验结果表明，将菌株p52与活性污泥混合菌群混合后pDF01及pDF02可向多属细菌转移；同样在菌株p52强化的SBR中降解质粒的转移伴随着反应器中污染物降解效果的提升。同时考察了在无选择压力条件下，降解质粒pDF01及pDF02在供体菌株p52、铜绿假单胞菌接合子及活性污泥中分离的接合子中的稳定性，证明pDF01及pDF02在多种宿主菌中具相对稳定性。上述研究表明利用菌株p52进行“基因”生物强化去除二噁英污染具有应用潜力，该结果具有创新性，引起国际同行关注。