EAGER: Towards the Development of Engineered Microorganisms and Enzyme Systems for Methane Production and Conversion to Liquid Fuel

1646895（Mansoorabadi）该研究旨在确定重新设计甲基辅酶M还原酶（MCR）所需的修饰，MCR是甲烷生物形成和厌氧氧化的关键酶，用于页岩气活化和随后转化为液体燃料和高价值化学品。该技术将促进页岩气甲烷的现场处理，作为天然气燃烧或昂贵的储存或运输选项的替代方案。甲基辅酶M还原酶（MCR）是甲烷生物形成和厌氧氧化的关键酶，甲烷是一种强有力的温室气体和生物燃料。MCR催化辅酶B（CoB-SH）和甲基辅酶M（CH 3-S-CoM）转化为混合杂二硫化物CoB-S-S-CoM和甲烷。近年来，厌氧嗜甲烷古菌（ANME）被证明可以催化甲烷的厌氧氧化（AOM）。AOM被认为至少部分地作为甲烷生成的逆作用，其中MCR的同系物催化该途径中的第一步，即用CoB-S-S-CoM活化甲烷。因此，ANME在天然气至液体燃料转化策略中的使用具有巨大潜力。不幸的是，迄今为止还没有获得ANME的纯培养物，这在很大程度上是由于它们异常缓慢的生长速率以及它们与硫酸盐还原菌的互养关联，这限制了它们在大规模转化过程中的使用。一个有吸引力的替代方案是将AOM途径及其关键酶MCR工程化为更适合用于天然气生物转化的微生物。然而，MCR目前不能在异源宿主中以活性（或可活化）形式产生。该项目将通过识别和表征生产成熟holo MCR所需的基因和相应的酶来解决这一缺陷。MCR的活性主要依赖于独特的含镍四吡咯辅酶F430。除了容纳辅酶F430外，MCR的活性位点还包含几种前所未有的翻译后修饰（PTM）。这些PTM在MCR催化中发挥的确切作用是未知的，负责其形成的基因的身份也是未知的。利用比较基因组学研究来鉴定所有产甲烷菌中保守的几个基因，这些基因是参与MCR成熟的优秀候选基因。因此，该项目将通过开发用于以下目的的表达系统来描述这些基因在功能活性MCR生产中的作用：1）辅酶F430的体内合成; 2）MCR PTM的研究;和3）holo MCR的异源生产。除了促进MCR在可再生生物甲烷生产和页岩气转化为液体燃料和其他高价值化学品中的使用外，该项目还将为本科生和研究生提供跨学科培训机会，以及为K-12学生提供教育丰富机会。该奖项由ENG新兴前沿和多学科活动办公室共同资助。

1646895 (Mansoorabadi)The study is aimed at identifying the modifications needed to re-engineer methyl-coenzyme M reductase (MCR), a key enzyme in the biological formation and anaerobic oxidation of methane, for shale gas activation and subsequent conversion to liquid fuel and high-value chemicals. The technology would facilitate on-site processing of shale gas methane as an alternative to either gas flaring or costly storage or transporting options.Methyl-coenzyme M reductase (MCR) is the key enzyme in the biological formation and anaerobic oxidation of methane, a potent greenhouse gas and biofuel. MCR catalyzes the conversion of coenzyme B (CoB-SH) and methyl-coenzyme M (CH3-S-CoM) to the mixed heterodisulfide, CoB-S-S-CoM, and methane. Recently, anaerobic methanotrophic archaea (ANME) have been shown to catalyze the anaerobic oxidation of methane (AOM). AOM is thought to operate, at least in part, as the reverse of methanogenesis, with a homolog of MCR catalyzing the first step in the pathway, the activation of methane with CoB-S-S-CoM. Thus, there is great potential for the use of ANME in natural gas-to-liquid fuel conversion strategies. Unfortunately, no pure culture of an ANME has been obtained to date, due in large part to their exceptionally slow growth rates and their syntrophic association with sulfate-reducing bacteria, which limits their use in large-scale conversion processes. An attractive alternative is to engineer the AOM pathway and its key enzyme, MCR, into a more suitable microorganism for use in the bioconversion of natural gas. However, MCR cannot currently be produced in an active (nor activatable) form in a heterologous host. The project will address this deficiency by identifying and characterizing the genes and corresponding enzymes required for the production of mature holo MCR. The activity of MCR is critically dependent on the unique nickel-containing tetrapyrrole, coenzyme F430. In addition to housing coenzyme F430, the active site of MCR contains several unprecedented post-translational modifications (PTMs). The exact roles these PTMs play in MCR catalysis are unknown, as are the identities of the genes responsible for their formation. A comparative genomics investigation was utilized to identify several genes conserved in all methanogens that are excellent candidates to be involved in MCR maturation. The project will thus delineate the roles these genes play in the production of functionally active MCR by developing an expression system for the: 1) In vivo synthesis of coenzyme F430; 2) Investigation of MCR PTM; and 3) Heterologous production of holo MCR. In addition to facilitating the use of MCR in the production of renewable biomethane and the conversion of shale gas to liquid fuel and other high-value chemicals, the project will offer interdisciplinary training opportunities for undergraduate and graduate students, as well as educational enrichment opportunities for K-12 students.The award is co-funded by the ENG Office of Emerging Frontiers and Multidisciplinary Activities.