环境有害物尼古丁的假单胞菌分解代谢机制

The N-heterocyclic compounds found in industrial wastes are carcinogenic, teratogenic, and mutagenic. Nicotine, an important N-heterocyclic compound, is abundant in the discharge from tobacco manufacturing plants and is a toxic and hazardous environmental toxicant. Nicotine degradation has drawn much attention during the last decades. Many bacteria that can degrade nicotine have been isolated and characterized. Two pathways underlying nicotine degradation have been identified in bacteria, namely, the pyridine pathway and pyrrolidine pathway, with the latter being more significant because of its partial similarity to the pyrrolidine pathway in human beings. Pseudomonas can efficiently degrade nicotine via the pyrrolidine pathway. However, this pathway has not yet been completely elucidated..The proposed project will be performed on the basis of our previous genomic and proteomic research on Pseudomonas putida S16 and will be dedicated to systematic investigation of the complete pyrrolidine pathway and examination of the key genes in this pathway. Gene knockout, gene cloning, and activity analysis will be used in the functional verification of the key genes. To investigate the route by which certain genes are acquired, mobile genetic elements such as genomic islands will be predicted by bioinformatic analysis, followed by functional analysis. The mobility, integration, excision, and circularization of genomic islands will also be studied. Analysis of S16 genomic islands and the patterns of horizontal gene transfer in Pseudomonas will aid in elucidating the genetic code responsible for the degradative ability of S16. The molecular regulatory mechanism of nicotine-degradative genes will be investigated by identifying specific/global regulators by using DNA affinity pull-down, followed by functional analysis involving insertional mutations. These regulators will be purified and analyzed using DNA footprinting, gel-shift assay, and crystal structure resolution to determine DNA-binding sites. The key enzymes in nicotine metabolism will be expressed and purified for in-depth study of their biochemical properties and catalytic mechanism, that is, factors such as catalytic type, reaction mechanism, substrate specificity, enzyme stability, Km and kcat values, maximum reaction rate, cofactors, and the transient response process will be examined. Structural biology-based methods such as crystallization, X-ray diffraction, and site-directed mutagenesis will also be used to analyze enzyme active sites. .The research results of this proposed project would clarify the genetic and biochemical mechanisms underlying nicotine degradation by Pseudomonas, which have puzzled scientists for decades. The proposal also has important scientific and practical significance with respect to exploiting microbial resources for biocatalysis of heterocycles and microbial remediation of the environmental pollution caused by the tobacco industry.

N-杂环化合物存在于工农业废弃物中，具有致癌、致畸、致突变等性质。尼古丁作为重要N-杂环化合物的一种，广泛存在烟草加工废弃物中，是十分有毒的环境有害物。假单胞属菌能以一条部分类似于人体代谢的吡咯途径代谢尼古丁，该途径至今还没有被揭示清楚。本项目拟在前期完成的恶臭假单胞菌S16全基因组测序和注释基础上，结合分析已获得的蛋白组学数据，系统探索尼古丁代谢途径中的关键基因，揭示其完整的代谢途径；研究其尼古丁降解基因的分子调控机制；对S16基因组岛进行分析，研究尼古丁代谢能力获得的遗传机制；针对代谢关键酶，进行催化机制的研究，进一步利用结构生物学手段对酶的晶体结构进行解析，深入研究其生物化学催化机制。本项目研究成果对详细揭示困惑科学家几十年的假单胞菌代谢尼古丁的遗传和生化机制科学问题、深度挖掘杂环分解代谢微生物资源用于生物催化和利用该类微生物修复被烟草工业所污染的环境等方面具有重要科学与实践意义。

环境污染是人类面临的严峻问题，环境保护与污染防治是实现国家经济和环境可持续发展战略的重要前提。中国作为世界烟草生产大国，每年会产生大量烟草废弃物-尼古丁，对水体和土壤造成了严重污染。微生物降解被认为是最有潜力的修复方法，高效降解有害污染物的微生物制品研究，能够为环境保护与污染防治提供技术支撑。本项目利用分子生物学、结构生物学、生物信息学等多学科手段，解决了尼古丁微生物降解的关键科学问题。首次从分子水平系统阐明了困惑科学家近60年的假单胞菌代谢尼古丁的吡咯代谢主途径的机制，发现该途径与人体代谢尼古丁的2' 羟基化途径类似。克隆调控蛋白NicR2，提出新的HTH型调控蛋白结合DNA模型，填补了假单胞菌代谢尼古丁的调控机制领域空白。阐明了黄素单加氧酶Pnao、2,5-DHP双加氧酶和HSP羟化酶等尼古丁代谢途径关键酶的催化机理，并解析了马来酸异构酶、N-甲酰马来酰胺酸脱甲酰基酶、马来酰胺酸脱氨基酶和调控蛋白NicR2的结构。在完整解析S16代谢尼古丁的分子生物学途径，定位所有降解关键基因的基础上，本项目提出了尼古丁代谢高值中间物的制备，实现变废为宝的策略，实现了一种以废次烟叶为底物生物转化制备3-琥珀酰吡啶的方法，为吡啶类生物活性物质合成提供了更多的出发产物选择。共发表SCI论文14篇，包括微生物学领域顶级期刊Mol Microbiol 4篇，遗传学领域主流期刊PLoS Genetics 1篇，生化领域顶级期刊JBC1篇等。由于在相关领域作出的创新性科学贡献，团队负责人分别2014年和2015年被遴选为美国医学生物工程院（AIMBE）fellow和美国国家发明家科学院（NAI）fellow。团队还培养出国家优秀青年基金获得者1名，上海市曙光学者1名，科学中国人2016年度人物杰出青年科学家奖”。前期结果《难降解环境有害物的微生物代谢》获得2016年获得教育部自然科学一等奖。