极小单胞菌T2代谢尼古丁的分子机制研究

Nicotine is very harmful to human. It has been treated as Toxic Release Inventory. Therefore, it is important to remove nicotine from tobacco waste, or even convert it to valuable compounds. Microbial treatment is one of the recommended technologies for the rapid decomposition of tobacco waste. Pusillimonas sp. T2 could use nicotine as sole nitrogen and carbon source, which was a newly isolated bacterium by our laboratory previously. The nicotine-degrading pathway contained two shunt pathways by Pusillimonas sp. T2, which was different from any other strains repotred. In this study, we want to obtain the complete gene cluster involved in nicotine degradation by genomic DNA sequencing, bioinformatics analysis, and transposon-mutagenesis; confirm the biological functions of every single gene by gene knockout and complementation or overexpression in vitro; purify the key enzymes in the shunt pathways and determine the kinetics; analyze the differences and relationships of the gene cluster among the different nicotine degrading strains. And in this study, for the first time, it will be elucidated that the machanism for the degradation of nicotine by Pusillimonas sp. T2 at molecular, biochemical, and genetic levels. And the diversities of nicotine catabolism will also be expored. Moreover, the research on mechanisms of nicotine degradation will be greatly helpful to expore the diverse capability for the microbial bioremediation, green biotransformation, and illustrate the adaptive evolution strategies of microbes using nicotine as a model.

尼古丁作为一种有毒的危险物，给人体健康及生存环境带来极大威胁，利用微生物方法从烟草废弃物中去除尼古丁或者将其转化为有价值的化合物具有重要意义。申请人筛选到一株新型可彻底矿化尼古丁的极小单胞菌Pusillimonas sp. T2。该菌降解尼古丁的下游途径含有两条支路途径，与以往报道均不同。本项目拟以该菌株为对象，通过基因组测序、生物信息学分析预测、转座子随机插入突变等方法获得完整的尼古丁代谢基因簇；结合基因敲除互补、体外表达等方法鉴定各基因的生理学功能；纯化支路代谢途径中的关键酶并研究酶学特性；与已报道的降解基因（簇）比较并分析其差异和进化关系。本研究将首次从分子、生化和遗传学水平上阐明极小单胞菌Pusillimonas sp. T2完整的代谢基因簇，并揭示尼古丁微生物代谢途径多样性、代谢基因（簇）多样性和进化策略，并为尼古丁引起的环境污染的微生物降解修复及绿色转化提供理论和技术依据。

环境污染是21世纪全人类所面临的共同问题。尼古丁和其他吡啶类衍生物在自然环境中广泛存在，对人类均具有一定程度的毒害作用。微生物在消除该类物质的污染或者将其变废为宝的过程中发挥着不可替代的作用。本项目围绕极小单胞菌T2及其他降解菌株，通过结合中间代谢产物鉴定、基因组测序与生物信息学分析、转座子随机插入突变与染色体步移、基因敲除与回补等技术，揭示细菌降解尼古丁等吡啶类环境有毒物质的微生物代谢机制。测定了极小单胞菌T2全基因组，发现了2,5-二羟基吡啶的两个同工酶基因和一个钼辅因子合成必需基因；分离了首株既能降解尼古丁又能降解烟酸的高效降解菌假单胞菌JQ581；测定了申氏杆菌HZN7的全基因组，鉴定了降解尼古丁的完整基因簇，同时发现尼古丁降解菌HZN7能够利用同一组基因（nctA和nctB）降解去甲基尼古丁转化，并首次鉴定了3个转化产物的化学结构；以2,5-二羟基吡啶为中心点，分离了代谢性能优良的粪产碱菌JQ135，克隆了5-羟基吡啶-2-羧酸关键降解酶基因hpaM；同时筛选到世界上首株能够降解可替宁的纯培养细菌JQ2195，初步测定了其中间代谢产物和第一步的粗酶酶活力。本项目较为深入地探索了微生物降解吡啶类污染物的分子机制，为该类物质的污染修复和潜在的绿色转化提供了新的种质资源和理论依据。