甲烷单加氧酶pMMO铜响应的转录调控机制

Methane-methane oxidizing bacteria-pMMO, is a hot keywords combination. As an important energy and greenhouse gas, methane is a star molecular. In nature, the assimilation of methane is an important part of the carbon cycle of the earth, and it is responsible by the methane oxidizing bacteria (MOB). Methane monooxygenase pMMO catalyzes methane to methanol, the initial and key step of methane assimilation. pMMO exists in almost all known MOB, and is the main enzyme system for the oxidation of methane in nature. Because of its ecological significance and potential application value, the catalytic mechanism of pMMO and the mechanism of expression and regulation have attracted much attention. Reported studies and our previous data indicate that, as the active central ion of pMMO, copper (Cu2+) could significantly enhance the transcriptional level of pMMO gene cluster pmoCAB. The transcriptional regulation mechanism is of great interest but remains unknown. To reveal this mechanism, the transcriptional units of pmoCAB will be analyzed in strain 5GB1C firstly; then gene(s) encodes the transcription regulator of pmoCAB will be cloned; finally, characteristics of the transcriptional regulator will be identified. This project expected to reveal the transcriptional regulation mechanism of pMMO enhancing expression by Cu2+, and provide a theoretical basis for studying the ecological behavior of MOB and constructing efficient methanogenic transformation strains.

甲烷—甲烷氧化菌—pMMO一直是个热点关键词组合。作为重要的能源与温室气体，甲烷是个明星分子。在自然界中，甲烷的同化是地球碳循环的重要环节，由甲烷氧化菌负责。甲烷单加氧酶pMMO催化甲烷生成甲醇—甲烷同化的起始也是最重要的步骤。pMMO存在于几乎所有已知的甲烷氧化菌中，是自然界中氧化甲烷的主要酶系。由于其重要的生态学意义及潜在的应用价值，pMMO催化机制及表达调控机理备受关注。现有报道和项目组前期数据表明，作为pMMO的活性中心离子，Cu2+可以显著提高pMMO基因簇pmoCAB的转录水平，但其中的转录调控机制仍然未知。本项目拟以菌株5GB1C作为研究材料，首先解析pmo的转录结构；然后克隆pmo的转录调控蛋白基因；最后对该调控蛋白的转录调控特性进行鉴定。预期将揭示Cu2+提高pMMO表达量的转录调控机制，为研究甲烷氧化菌的生态学行为以及构建高效的甲烷转化菌株提供理论基础。

甲烷单加氧酶pMMO催化甲烷生成甲醇，是自然界中氧化甲烷的主要酶系。铜离子可以显著提高pMMO基因簇的转录水平，但其中的转录调控机制仍然未知。本项目以此为科学问题，研究表明：（1）菌株Methylotuvimicrobium buryatense 5GB1C中pmoCAB基因簇存在两个启动子，分别位于pmoC基因前和pmoA基因前；PpmoC的转录起始位点为起始密码子前96位的“A”碱基；PpmoA的转录起始位点为其起始密码子前40位的“A”碱基。pmoCAB基因簇的转录存在两种转录单元结构，即pmoCAB和pmoAB；（2）意外发现菌株Methylotuvimicrobium buryatense 5GB1C中存在一个Flavin-based类型的荧光蛋白基因（EQU24_15305），其编码的蛋白在绿色荧光蛋白GFP激发条件下也可以发出绿色荧光。该蛋白有潜力开发成适用于甲烷氧化菌的一个较好的报告基因系统；（3）通过DNA Affinity途径鉴定了8个与pmoC和pmoA启动子结合且置信度较高的转录调控蛋白，从中进一步验证确定其中EQU24_14530基因编码Crp/Fnr类型的转录调控蛋白与PpmoC启动子相互作用参与pmoC基因的转录调控；（4）体外实验表明Crp蛋白不直接响应铜离子信号，因此菌株中可能还存在其他的master regulator响应铜信号，这值得进一步研究。项目的实施为pMMO的进一步开发应用奠定了基础。