洋底深部真菌利用褐煤产甲烷的生化过程及代谢机理

Integrated Ocean Drilling Program (IODP) Expedition 337 mainly focused on the exploration of subseafloor lignite, microorganism and its interaction mechanism. It was the first time for the discovery of low matured coal beds, microbial methane, and microorganisms with high number of species and richness compared to those in the other geological layers at ~2 km below the ocean floor. As a sole representative of mainland China in the Expedition 337, the applicant was responsible for the research of cultural fungi within the cored samples. Total 27 fungal species have been obtained from the samples, and Schizophyllum commune, which was isolated from brown coal-bed, was proved to be able to convert lignite to methane under anaerobic condition. This result suggests that the interaction among “lignite-fungi-methane” may exist in the deep subseafloor environments, which remains unclear. In this project, we are going to study the biochemical reaction, metabolic pathways, and key genes, which are related to methane production from lignite degradation by Schizophyllum commune under anaerobic conditions using many methods such as multi-omics, isotope tracing, gene editing techniques. In addition, we will also conduct an experiment to study the diversity and methane production activity of methanogenic fungi in the subseafloor lignite samples under a controlled culture condition based on the in situ environmental factors. The implementation of the project, not only could discover a new mechanism of methane production by microorganisms, expand our knowledge about the theory of biogenic coal bed methane formation; but also has a profound impact on the understanding about biogeochemical function in deep subseafloor environments as well as the origin and evolution of methane in the natural ecosystems.

IODP337航次（2012）是一次以探索洋底褐煤、微生物及其互作机理为主要目的的国际科学大洋钻探行动。该航次在深达2km的洋底发现了低成熟度的褐煤层、微生物成因甲烷、种类和数量均较其它地质层显著丰富的微生物。申请人作为该航次的唯一中国大陆代表，承担该航次样品中真菌的研究。已分离获得27种可培养真菌，还发现来源于褐煤层的裂褶菌能够厌氧转化褐煤形成甲烷，提示洋底深部环境可能存在“褐煤-真菌-甲烷”关联，但关联机制不清楚。本项目拟运用多维组学、同位素示踪、基因编辑等手段，研究产甲烷裂褶菌厌氧降解褐煤形成甲烷的生化反应、代谢途径和关键基因，并模拟原位环境条件，研究洋底褐煤样品中产甲烷真菌的多样性及甲烷代谢活性。该项目的实施，不仅有可能发现新的微生物产甲烷机制，丰富和发展煤层气生物成因相关理论；还将对认识洋底深部环境的生物地球化学作用以及自然生态系统中甲烷的起源、演化过程等产生深远影响。

以一株分自约2km深的洋底含煤沉积物中的裂褶菌为研究对象，运用同位素示踪、生化和组学等多种检测分析手段，研究了该菌厌氧利用褐煤产甲烷途径及分子机理，探究了其在厌氧环境中的生长发育机制，为人类了解洋底深部生物圈生命及其生态学作用提供科学依据，主要结果如下：.（1）发现并证明裂褶菌等木腐真菌普遍具有厌氧产甲烷能力, 其产甲烷量与所处沉积层深度和地质环境无关；（2）证明裂褶菌拥有不同于古菌的厌氧产甲烷代谢途径，其产甲烷能力具有底物依赖性，只能利用苯酚、苯甲酸及其衍生物以及葡萄糖产生甲烷；（3）证明卤甲烷是裂褶菌厌氧产甲烷的前体物质，裂褶菌利用不同底物产卤甲烷和甲烷的比例为17:1~77:1；（4）根据全球真菌年产氯甲烷20~40万吨以及真菌产卤甲烷与甲烷的比例为17:1~77:1，估算真菌甲烷年产量约0.26~2.35万吨，约占全球陆地甲烷释放量的0.004~0.118%；（5）初步证实卤甲烷合成基因mct1和分解基因dh3是决定裂褶菌合成甲烷的关键基因；（6）推测裂褶菌厌氧降解褐煤或木质素产甲烷过程，涉及底物分解和苯酚或苯甲酸类中间产物的产生，这些中间产物在漆酶的作用下失去一个电子形成自由基，然后在膜结合MCT酶的催化下合成卤甲烷；卤甲烷在脱卤酶的作用下分解生成甲基自由基，甲基自由基与代谢过程中产生的氢自由基结合，形成甲烷；（7）解析了裂褶菌降解褐煤或木质素机制，漆酶、细胞色素P450、酚羟化酶、环氧化物水解酶、谷胱甘肽S-转移酶等参与了褐煤或木质素的厌氧降解过程；（8）探究了裂褶菌厌氧生长与发育特性，发现其在厌氧条件下无法完成有性生殖，提示深部生物圈的真菌可能以菌丝形式存在和生长，导致进化缓慢；（9）发现真菌在厌氧条件下采用调节线粒体数量、提高乙醇和氨基酸合成代谢而获得能量；（10）证明分支氨基酸有缓解缺氧对裂褶菌有性生殖形成的抑制作用。 .总之，洋底真菌拥有适应厌氧生态系统的生长、繁殖以及产能机制，不仅在全球碳循环中发挥重要作用，而且对全球甲烷的源汇平衡有重要影响。

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