Regulatory Interplay between Nitrogen and Methane Metabolism: New Frontiers in Product Discovery, Physiology and Ecology of Methanotrophic Bacteria

氮和甲烷代谢之间的调节相互作用：甲烷氧化细菌产品发现、生理学和生态学的新领域

• 批准号: RGPIN-2014-03745
• 负责人: Stein, Lisa
• 金额: $ 3.42万
• 依托单位: University of Alberta
• 依托单位国家: 加拿大
• 项目类别: Discovery Grants Program - Individual
• 财政年份: 2018
• 资助国家: 加拿大
• 起止时间: 2018-01-01 至 2019-12-31
• 项目状态: 已结题
• 来源: https://www.nserc-crsng.gc.ca/ase-oro/Details-Detailles_eng.asp?id=665900
• 关键词: Regulatory; Interplay; between; Nitrogen; Methane

Metabolism of single-carbon compounds is an ancient and widespread biological function, yet only methylotrophic bacteria exclusively utilize single-carbon compounds as sources of energy and carbon. Methanotrophic (i.e. methane-eating) methylotrophs are essential as the Earth's biological methane sink, which is becoming critical to understand as greenhouse gases continue to accumulate during this period of rapid global change. Comparison of over 30 methanotroph genome sequences revealed a rich diversity of inventory for nitrogen metabolism that is intimately interwoven with carbon metabolism. The overarching hypothesis of the proposed research is that nitrogen source and availability predictably control carbon metabolism in methanotrophic bacteria, regardless of genomic variability, enabling students in my laboratory to: 1) determine how nitrogen metabolism regulates carbon transformations into value-added end products for industrial exploitation, 2) characterize enzymes, functionality, and regulation for our recently discovered pathway of methane-dependent denitrification, and 3) develop discrete molecular markers to determine the presence, strength, and relevance of methane-dependent denitrifiers in regulating greenhouse gas fluxes in diverse environments. Our laboratory-based approaches utilize a large genome-sequenced strain collection that we curate and maintain along with a suite of analytical tools to measure cell growth in batch and continuous culture, real-time trace gas measurements, gene expression levels, and metabolite and protein production. Our goal in Objective 1 is to determine how N-source and availability gate carbon into molecules of industrial relevance like fatty acids, isoprenoids, N-storage polymers, C-storage polymers, and organic acids. Growth kinetics and analysis of nucleic acids, proteins, and metabolites from cultures grown with variable combinations of nitrogen and carbon sources will enable discovery of products for industrial exploitation. Objective 2 will determine the molecular regulation of methane-dependent denitrification from nitrate, a novel physiology discovered in my laboratory, that connects the metabolism of two potent greenhouse gases (methane and nitrous oxide) and a major environmental pollutant (nitrate) within a single organism. Using physiological, transcriptomic and proteomic tools, our aim is to fully characterize the enzymatic diversity and physiological benefit of this activity and to predict, based on gene content and regulatory features, whether a particular strain should have this physiology. Objective 3 will reveal the ecological relevance of methane-dependent denitrification and the microorganisms that perform it in diverse environments from cow rumen to thawing permafrost. Denitrifying methanotrophs possess phylogenetically distinct genes that we aim to find in environments where nitrous oxide production at the expense of methane consumption has been noted. Together, the proposed research fully employs our genome-sequenced collection of methanotrophic bacteria and analytical capacity to a) interface with the energy sector in Canada by converting industrial waste products into value-added products, b) characterize the enzymatic diversity and function of a novel physiology in methanotrophic bacteria, and c) provide tools to assess the ecological significance of methane-dependent denitrification as a novel linkage between the carbon and nitrogen biogeochemical cycles. In addition to developing a potentially lucrative and ecologically important area of research for Canada and the broader scientific community, this work offers a unique training experience for students of microbiology and biotechnology.

单碳化合物的代谢是一种古老而广泛的生物学功能，但只有甲基营养细菌仅利用单碳化合物作为能量和碳的来源。由于地球的生物甲烷下沉，甲烷营养性（即甲烷饮食）是必不可少的，因为在这一快速的全球变化时期，随着温室气体继续积累，这对于理解而变得至关重要。比较30多种甲烷营养基因组序列表明，氮代谢的库存多样性多样性与碳代谢紧密相互交织。拟议的研究的总体假设是，氮的来源和可用性可以预见地控制甲烷型细菌中的碳代谢，无论基因组的可变性如何甲烷依赖性的反硝化和3）发展离散的分子标记，以确定甲烷依赖性反状体在调节各种环境中温室气通量中的存在，强度和相关性。 我们的基于实验室的方法采用了大型基因组取消的菌株收集，我们将策划并保持一套分析工具，以测量批处理和连续培养，实时痕量气体测量，基因表达水平以及代谢物和蛋白质产生的细胞生长。 我们在目标1中的目标是确定N源和可用性栅​​极碳如何进入工业相关性分子（例如脂肪酸，类异on-，N储存聚合物，C储存聚合物和有机酸）。生长动力学和核酸，蛋白质和代谢产物的分析，这些培养物生长具有氮和碳源的可变组合，将使能够发现用于工业开发的产品。目标2将确定硝酸甲烷依赖性反硝化的分子调节，硝酸盐是在我的实验室中发现的一种新型生理学，它连接了两种有效的温室气体（甲烷和氧化物）的代谢和一种主要环境污染物（硝酸盐）。使用生理，转录组和蛋白质组学工具，我们的目的是充分表征该活动的酶促多样性和生理益处，并根据基因含量和调节特征进行预测，是否应该具有这种生理学。目标3将揭示甲烷依赖性反硝化的生态相关性和在从牛umen到融化的多种环境中执行的微生物。非硝化甲烷营养具有系统发育不同的基因，我们旨在在以甲烷消耗为代价的情况下发现的环境中找到的环境。拟议的研究共同采用了我们的基因组序列收集的甲烷营养细菌和分析能力，a）通过将工业废物转化为增值产品，与加拿大能源部门的互动，b）酶多样性和表征新型生理学的酶促多样性和功能，以评估甲基植物的新型脱甲素化剂量，属于甲基甲基甲基甲基甲基甲基甲烷的依赖性，属于甲基甲基甲基甲基甲基甲基的含量，甲基含量依赖于甲基甲烷的含量，属于甲基甲基甲基甲基甲基甲烷的差异，并具有甲基化的显着性。和氮生物地球化学周期。除了为加拿大和更广泛的科学界开发潜在的有利可图且在生态上重要的研究领域外，这项工作还为微生物学和生物技术的学生提供了独特的培训经验。