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

古细菌Ca. Argoarchaeum和Ca. Ethanoperedens催化乙烷的完全厌氧氧化以获得其细胞能量，并且在此过程中产生的电子被转移到伴侣细菌。因此，这些厌氧微生物控制着海底第二丰富的气体成分的流量，避免其释放到大气中。我们发现，乙烷是由一种新的发散的甲基辅酶M还原酶，称为乙基辅酶M还原酶（ECR）的变体激活。该酶捕获乙烷作为硫醇加合物乙基辅酶M，其将逐渐转化为乙酰辅酶A，代谢的转盘。以下导致乙酰辅酶A的反应是未知的，需要未知的生物化学。我们最近成功培育了快速生长的乙烷氧化剂Ca。Ethanoperedens将提供生物材料，以破译哪些酶和化学反应操作乙基辅酶M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）确定催化途径的酶和（iii）破译这些反应背后的分子机制。这个为期3年的项目将由两名PI，一名博士后，一名博士生和一名代谢物分析专家指导。我们将培养含有大部分Ca的微生物聚生体。在发酵罐中的Ethanoperedens使我们能够产生大量的生物质代谢活性。使用稳定同位素标记和代谢物分析的组合，我们将确定在乙基辅酶M氧化为乙酰辅酶A的过程中形成的中间体。基因组，转录组和蛋白质组的比较分析将使我们能够确定候选酶催化形成这样的中间体。将使用乙基-CoM、不同的电子受体和其他中间体，在细胞提取物和不同的蛋白质组分中测定和确认这些酶催化的拟定反应。酶将从微生物聚生体中天然分离，因为它们可能含有不可预见的辅因子，这是ECR表征已经实现的一项具有挑战性的任务。它们的结构将通过结晶和X射线衍射获得。乙烷降解菌的这种转变的阐明将完成这一分解代谢途径的知识，并可能扩展到其他短链和长链烷烃氧化古菌的一些共同原则。这一整体工作包是为基于二氧化碳减排的乙烷生物生产的生物技术发展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和将可再生能源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.