ETHOX – Reveal the missing steps in archaeal ethane oxidation – the ETHyl-CoM OXidation to acetyl-CoA

古生菌Ca.Argoararum和CA.乙烷菌催化乙烷的完全厌氧氧化以获得它们的细胞能量，在这个过程中产生的电子被转移到伙伴细菌。因此，这些厌氧微生物控制着海底第二丰富的气体成分的通量，避免其在大气中释放。我们发现乙烷被甲基辅酶M还原酶的一种新的发散变异体激活，称为乙基辅酶M还原酶(ECR)。这种酶捕获乙烷作为硫醇加合物乙基-COM，将逐步转化为乙酰辅酶A，新陈代谢的转盘。下列导致乙酰辅酶A的反应是未知的，需要未知的生物化学。我公司近期培育成功的快速生长的乙烷氧化剂钙。乙酸乙酯将提供生物材料来破译哪些酶和化学反应将乙基-COM氧化成乙酰-辅酶A。在这里，我们建议(I)揭示未知的中间体，(Ii)确定催化该途径的酶，(Iii)破译它们在这些反应中的分子机制。这个为期3年的项目将由两名学生指导，一名博士后，一名博士后和一名代谢物分析专家。我们将培养以钙为主的微生物联合体。发酵罐中的乙酸乙酯使我们能够生产大量具有代谢活性的生物质。利用稳定同位素标记和代谢物分析相结合的方法，我们将鉴定乙基乙酸乙酯氧化成乙酰辅酶A过程中形成的中间产物。对基因组、转录组和蛋白质组的比较分析将使我们能够确定催化这些中间体形成的候选酶。这些酶催化的反应将在细胞抽提物和不同的蛋白质组分中使用乙基-COM、不同的电子受体和额外的中间体进行测试和确认。这些酶将从微生物联合体中自然分离出来，因为它们可能含有不可预见的辅因子，这对于ECR表征来说已经是一项具有挑战性的任务。它们的结构将通过结晶和X射线衍射法获得。对乙烷降解物中这种转化的阐明将完善对这一分解代谢途径的认识，并可能扩展到其他短链和长链烷烃氧化古生菌的一些共同原理。这一总体工作方案是为以二氧化碳减排为基础的乙烷生物生产的生物技术开发铺平道路的先决条件，这一过程将满足我们现代社会捕获温室气体并将来自可再生来源的能量储存到稳定分子中的迫切需求。

The archaea Ca. Argoarchaeum and Ca. Ethanoperedens catalyze the complete anaerobic oxidation of ethane to derive their cellular energy, and the electrons generated during this process are transferred to a partner bacterium. Therefore, these anaerobic microorganisms control the fluxes of the second most abundant gas component in the seafloor, avoiding its release in the atmosphere. We showed that ethane is activated by a novel divergent variant of methyl-coenzyme M reductases, described as ethyl-coenzyme M reductase (ECR). This enzyme captures ethane as the thiol adduct ethyl-CoM that will be progressively transformed into acetyl-CoA, the turntable of the metabolism. The following reactions leading to acetyl-CoA are unknown and will require uncharted biochemistry. Our recent cultivation success of the fast-growing ethane oxidizer Ca. Ethanoperedens will provide biological material to decipher which enzymes and chemical reactions operate the ethyl-CoM oxidation into acetyl-CoA. Here we propose to (i) reveal the unknown intermediates, (ii) identify the enzymes catalyzing the pathway and (iii) decipher their molecular mechanisms underlying these reactions. This 3-year project will be guided by the two Pl, a postdoc, a PhD student and an expert in metabolite analysis. We will cultivate the microbial consortium containing a majority of Ca. Ethanoperedens in a fermenter allowing us to produce large amounts of biomass metabolically active. Using a combination of stable isotope labelling and metabolite analysis we will identify the intermediates formed during the oxidation of ethyl-CoM to acetyl-CoA. Comparative analyses of genomes, transcriptomes and proteomes will allow us to identify candidate enzymes catalyzing the formation of such intermediates. The proposed reaction catalyzed by these enzymes will be assayed and confirmed in cell extracts and in different protein fractions using ethyl-CoM, different electron acceptors and additional intermediates. The enzymes will be natively isolated from the microbial consortia because they might contain unforeseen cofactors, a challenging task already achieved for the ECR characterization. Their structures will be obtained by crystallization and x-ray diffraction. The elucidation of this transformation in ethane degraders will complete the knowledge of this catabolic pathway and might be extended to some common principles for other short- and long-chain alkane oxidizing archaea. This overall work package is a prerequisite to pave avenues towards the biotechnological development of ethane biological production based on CO2 reduction, a process that would answer the urgent needs of our modern society to capture the greenhouse gas and store energy from renewable sources into a stable molecule.