G. metallireducens与M. barkeri DIET方式耦合还原CO2产甲烷机理解析

Methanogens can use the electrons obtained directly from the partner of its co-cultures for the reduction of carbon dioxide to methane via direct interspecies electron transfer (DIET), which greatly improves the efficiency of methane production. However, neither has the mechanism through which the electron carried by reduced ferridoxin (Fdred) within electron donor's microorganisms is transferred to extracellular electron acceptors been revealed, nor has the mechanism through which methanogens directly use the electrons obtained from extracellular electron donors for the reduction of carbon dioxide to methane been analyzed to date. . Therefore, the aim of this project is to reveal the mechanism through which the electron carried by Fdred within Geobacter metallireducens is transferred to extracellular electron donors via analyzing Geobacter sulfurreducens Δehr mutant and Geobacter metallireducens Δact mutant metabolizing acetate, and to demonstrate the mechanism through which methanogens directly use the electrons derived from G. metallireducens to reduce carbon dioxide to methane by analyzing the mix-cultures of G. metallireducens and Methanosarcina barkeri mutants (Δech, ΔfpoF, Δfpo, Δfrh, Δvho and Δcytc mutant) stoichiometrically converting propanol to methane with the co-culture of G. metallireducens and M. barkeri as a model co-culture. After that, the mechanism for co-culture of G. metallireducens and M. barkeri stoichiometrically reducing carbon dioxide to methane via DIET will be elucidated by two aspects mentioned above.

产甲烷菌可通过种间直接电子传递（DIET）方式从与其共生的微生物中获取电子还原CO2产甲烷，从而极大地提高了产甲烷的效率。但是，其共生体内Fdred携带的电子传递到胞外机制及产甲烷菌直接利用胞外电子还原CO2产甲烷机制尚不清楚。因此，本项目拟以G.metallireducens和M.barkeri的co-culture为模型，通过G.sulfurreducens ehr及G.metallireducens act基因突变株代谢乙酸研究以解析G.metallireducens胞内Fdred携带的电子传递到胞外机制，然后通过M.barkeri ech、fpoF、fpo、frh、vho及cytc基因突变株与G.metallireducens耦合代谢丙醇研究以揭示M.barkeri直接利用胞外电子还原CO2产甲烷机制，进而从以上两方面阐明该co-culture通过DIET方式耦合产甲烷机理。

厌氧产甲烷体系中微生物间的电子传递，一直以来被认为是通过种间H2/甲酸转移来实现。然而，近年来研究发现某些电活性微生物与产甲烷菌之间存在可替代种间H2/甲酸转移以实现电子传递的种间直接电子传递（Direct Interspecies Electron Transfer，DIET）。通过DIET方式，电活性产甲烷菌可从与其共生的微生物中直接获得的电子还原CO2产甲烷，该方式极大地提高产甲烷的效率和产甲烷的量。虽然近年来微生物种间直接电子传递耦合还原CO2研究取得了很大进展，但是目前对电活性产甲烷菌通过DIET方式耦合产甲烷的机制还缺乏深入研究。本项目以Geobacter metallireducens GS-15与Methanosarcina barkeri 800 共培养物为研究对象，以解析M. barkeri 800 通过DIET方式耦合还原CO2产甲烷的机制，研究内容主要包括M. barkeri 800直接接受胞外电子的机制、M. barkeri胞内Fdred、F420H2、CoM-SH及CoB-SH产生机制。结果表明：1）镶嵌在M. barkeri外膜表面的细胞色素b在直接接受胞外电子中起重要作用；2）胞外电子通过M. barkeri外膜表面的细胞色素b将电子传递给氧化态的甲基吩嗪（MP）将其还原还原态的甲基吩嗪（MPH2），然后异二硫键还原酶（HdrED）利用MPH2将CoM-S-S-CoB还原为CoM-SH与CoB-SH；3）在质子跨膜势能的作用下产甲烷菌膜上的辅酶F420脱氢酶（Fpo）利用MPH2将氧化态的辅酶F420还原为还原态的辅酶F420（F420H2）；4）F420还原酶（Frh）将产生的部分F420H2氧化并产生H2，而Ech-hydrogenase利用产生的H2将氧化态的铁氧还原蛋白（Fdox）还原为还原态铁氧还原蛋白（Fdred）。该研究初步揭示了M. barkeri 800通过DIET方式耦合产甲烷的机制，为电辅助电活性产甲烷菌还原二氧化碳产甲烷的研究奠定了理论基础。