MmoD和MmoG在甲烷氧化酶sMMO合成中的作用及机理

Methane, the principal component of natural gas and biogas, is a cheap, abundant, and renewable energy and carbon source. However, methane is also the second most prevalent greenhouse gas. Methane-oxidizing bacteria (MOB) are able to use methane as their sole source of carbon and energy and are responsible for the assimilation of methane in the carbon cycle. Soluble methane monooxygenase (sMMO) catalyzes the conversion of methane to methanol, the initial and key step of methane assimilation. Historically, slow growth rate and the lack of efficient genetic manipulation method have been the limiting factors in analyzing the gene functions of MOB. Thus, the regulation mechanism of sMMO synthesis is not revealed yet. The gene clusters of sMMO from different hosts involve eight conserved genes, all of which are essential to the synthesis of active sMMO. Among these genes, mmoXYBZC encode the components required for activity; the product of mmoR is involved in the transcriptional regulation of sMMO gene cluster; however, the roles of mmoD and mmoG are still unknown. Based on current reports and our preliminary data, we speculate that the function of MmoD and MmoG is facilitating the folding and assembly of sMMO. To demonstrate this, we firstly determine the roles of MmoD and MmoG in strain 5GB1C which grows fast and is genetically tractable. How MmoD and MmoG work is investigated in detail through analysis of protein-protein interactions, proteins co-synthesis in vivo and cell-free synthesis of target proteins. Then, to investigate whether the function of MmoD and MmoG is conserved among methanotrophs, the MmoD and MmoG from other two methanotrophs OB3b and Bath will be studied using the same strategy. Finally, based on the findings, we will functionally synthesize sMMO in E. coli. This project will elucidate the post-translational regulation mechanism of sMMO synthesis and pave the way for the construction of E. coli cells which can convert methane to value-added compounds.

甲烷氧化酶sMMO来自甲烷氧化菌，催化甲烷生成甲醇——碳循环中甲烷同化的起始步骤。该酶不仅具有重要的理论和生态价值，还在甲烷减排和高效利用方面被寄予厚望。sMMO的结构和催化机制已被阐明，但其合成的调控机理还不清楚。合成有活性的sMMO需要8个保守的基因，其中， mmoXYBZC编码sMMO的各个组份，mmoR涉及转录调控，而mmoD和mmoG的角色还未知。综合已有报道和项目组前期数据，推测MmoD和MmoG的主要功能都是帮助sMMO折叠或组装。本项目首先在菌株5GB1C中通过失活蛋白清除系统、解析蛋白间相互作用和体外蛋白合成等策略阐明其MmoD和MmoG的功能；为了检验结论的普遍性，鉴定另外两株甲烷氧化菌中的MmoD和MmoG的功能；最后，依据上面的结论，尝试在大肠杆菌中合成sMMO。预期将阐明sMMO合成调控的关键机制，实现在大肠杆菌中功能性合成sMMO，为该酶的改造和应用奠定基础。

可溶性甲烷单加氧酶sMMO可以催化甲烷生成甲醇(甲烷氧化菌同化甲烷的起始步骤)。该酶不仅具有重要的理论和生态价值，也在甲烷减排和高效利用方面被寄予厚望。sMMO基因簇中mmoXYBZC编码sMMO的各个组份，而mmoD和mmoG的功能则未知，项目以此为科学问题开展了系列研究。首先，在甲烷氧化菌模式菌株Methylomicrobium buryatense 5GB1，M. alcaliphilum 20Z和Methylomonas sp. LW13中建立了基于反向筛选标记pheSAG的基因无痕敲除方法，该方法的阳性率超过90%；接着在菌株5GB1C中，通过生理生化、遗传以及分子生物学等手段证明了MmoG是sMMO羟化酶组份大亚基MmoX以及sMMO基因簇转录激活蛋白MmoR的折叠伴侣，MmoD则在MmoG下游发挥作用，极有可能作为sMMO羟化酶组份MMOH的组装伴侣，帮助MMOH进行正确组装。同时以菌株LW13为材料，平行验证了MmoG和MmoD的功能；最后，运用大肠杆菌作为宿主，尝试异源表达sMMO，通过多种优化实现了大肠杆菌中sMMO的功能性表达。项目的实施阐明了sMMO合成中的调控机制，为sMMO的改造及开发应用奠定了理论基础。