废烟叶水提液中葡萄糖对假单胞菌JY-Q尼古丁降解抑制机制及解除研究

尼古丁的污染源于烟草废弃物, 而废烟叶水提液（TWE）是其进入环境后的水体污染形式。课题组在TWE中筛选到的高尼古丁降解活性假单胞菌JY-Q具有很好的TWE耐受性，但TWE中葡萄糖却对其尼古丁降解有抑制作用。因此，阐明葡萄糖对JY-Q尼古丁代谢的抑制机制并解除葡萄糖直接和竞争抑制，对于其在TWE中能高效降解尼古丁具有重要意义。本项目拟通过关键代谢物分析，结合基因组生物信息学分析，确证JY-Q尼古丁代谢途径；通过FLRT-PCR和Hiseq转录组与蛋白组学分析，重点考察尼古丁和葡萄糖并存时，葡萄糖代谢关键酶基因和尼古丁代谢调控因子的差异表达，阐明葡萄糖存在及其代谢对尼古丁代谢的抑制机制；据此确定糖代谢途径关键酶基因和葡萄糖响应的尼古丁代谢抑制基因，并对这些基因进行敲除，以获得解除抑制的重组菌株，实现TWE尼古丁的定向降解。本研究可为微生物在复杂营养环境条件下实现定向降解提供参考。

Environmental nicotine pollution is mainly from tobacco waste. Tobacco waste aqueous extract (TWE) is the main pollution form of tobacco waste in aquatic environment. In our previous research, a strain Pseudomonas sp. JY-Q, showing high activity of degrading nicotine and excellent tolerance to TWE environment, was isolated from TWE. However, the coexistence of glucose in TWE samples showed negative effect on the nicotine degradation. Therefore, it is necessary to verify the mechanism and relieve the inhibition of glucose on the degradation of nicotine by JY-Q, so that it can efficiently directed degradation of nicotine in TWE. In the present study, the key metabolites of nicotine metabolic pathway and key enzyme gene would be evaluated, coupling with its genomic bioinformatic analysis, to verify the whole nicotine metabolic pathway of Pseudomonas sp. JY-Q; Meanwhile, the expression difference of key enzyme genes in glucose metabolism pathway and regulation factor of nicotine metabolism was examined by RTFQ PCR and Hiseq transcriptome sequencing, when nicotine and glucose was coexisted. Based on the revealed inhibition mechanism, the key genes in glucose metabolism pathway and the key regulator genes could be selected for subsequent knock out. The resultant metabolic engineering strain which might relieve the inhibition of glucose would be potential to realize the directed degradation of nicotine specifically in sugar-coexisting environment. This study would be as a model to realize the directed degradation of target pollutant be microbes in complicate nutrient environment.