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

EAGER：致力于开发用于甲烷生产和转化为液体燃料的工程微生物和酶系统

• 批准号: 1646895
• 负责人: Steven Mansoorabadi
• 金额: $ 10.62万
• 依托单位: Auburn University
• 依托单位国家: 美国
• 项目类别: Standard Grant
• 财政年份: 2017
• 资助国家: 美国
• 起止时间: 2017-01-01 至 2017-12-31
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=1646895&HistoricalAwards=false
• 关键词: EAGER; Towards; Development; Engineered; Microorganisms

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.

1646895(Mansoorabadi)这项研究旨在确定重新设计甲基辅酶M还原酶所需的修改，该酶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。该技术将促进现场处理页岩气甲烷，以替代天然气燃烧或昂贵的储存或运输选择。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)转化为杂二硫化物、CoB-S-COM和甲烷。近年来，厌氧甲烷营养古菌(ANME)被发现能够催化甲烷的厌氧氧化反应。AOM被认为至少在一定程度上与甲烷生成相反，与Mcr的同系物催化了该途径的第一步，即用CoB-S-S-COM活化甲烷。因此，ANME在天然气到液体燃料转换策略中的应用潜力很大。不幸的是，迄今为止还没有获得ANME的纯培养，这在很大程度上是因为它们的生长速度异常缓慢，以及它们与硫酸盐还原细菌的同养关系，这限制了它们在大规模转化过程中的使用。一种有吸引力的替代方案是将AOM途径及其关键酶MCR改造成更适合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(也不能激活)的形式产生。该项目将通过识别和表征生产成熟全息MCR所需的基因和相应的酶来解决这一不足。MCR的活性在很大程度上依赖于唯一的含镍四吡咯辅酶F430。除了含有辅酶F430外，MCR的活性位点还包含几个前所未有的翻译后修饰(PTM)。这些PTM在MCR催化中扮演的确切角色尚不清楚，负责它们形成的基因的身份也不清楚。利用比较基因组学研究确定了在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，该项目将通过开发一种表达系统来描述这些基因在生产具有功能活性的MCR中所起的作用：1)体内合成辅酶F430；2)研究MCR PTM；以及3)异源生产全息MCR。除了促进使用MCR生产可再生生物甲烷和将页岩气转化为液体燃料和其他高价值化学品外，该项目还将为本科生和研究生提供跨学科培训机会，并为K-12学生提供教育丰富机会。该奖项由新兴前沿和多学科活动ENG办公室共同资助。