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

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.

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。

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。本项目完成了Pseudomonas sp. JY-Q全基因组的测序，继而进行比较基因组学分析，确定了JY-Q尼古丁代谢途径是属于吡咯烷途径。而且发现JY-Q除了存在一条尼古丁代谢吡咯烷途径上游基因簇nic1（有2个重复基因簇）以外，还包含特殊的尼古丁代谢遗传架构：（1）存在两条相隔较远的高度同源重复基因簇nic2-1和nic2-2 (AA098_21325-AA098_21340和AA098_03885- AA098_03900)，两者介导尼古丁吡咯烷下游代谢途径；（2）还有一条烟酸降解簇na (AA098_17770-AA098_ 17790)，其与nic2存在部分功能重叠，即将2，5-二羟基吡啶降解至富马酸。探究了JY-Q中同源基因簇nic1-1和nic1-2对尼古丁代谢转化的贡献差异。分别选取nic1-1和nic1-2簇中的关键基因nicA2和nox（编码尼古丁胺加氧酶）进行敲除及回补试验。发现nic1-2簇对尼古丁的转化代谢贡献要高于nic1-1簇。E. coli BL21异源表达得到了NicA2和NOX的两个重组酶，发现Nox的比酶活较NicA2低，但Nox的pH和温度稳定性，尤其是对底物亲和力，均比NicA2强。通过FLRT-PCR和Hiseq转录组与蛋白组学分析，发现葡萄糖存在及其代谢对尼古丁代谢的影响。 利用KEGG进行基因组分析，确定糖代谢途径关键酶基因和尼古丁代谢抑制基因，并对这些基因（gck、gdh-12490、gdh-22860、gdh-11910和gdh-05800）进行敲除，获得累积缺失的突变菌Q/5∆，提高了突变株在葡萄糖和尼古丁共存环境的降解尼古丁能力，为实现其在复杂环境如TWE中尼古丁的定向降解打下基础。