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: 2017
• 资助国家: 加拿大
• 起止时间: 2017-01-01 至 2018-12-31
• 项目状态: 已结题
• 来源: https://www.nserc-crsng.gc.ca/ase-oro/Details-Detailles_eng.asp?id=629704
• 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宽的生物学功能，但只有甲基营养的细菌仅利用单碳化合物作为能量和碳的来源。甲共藻（即甲基营养的（即甲基嗜肉基化）甲基营养性是必不可少的，因为地球的生物甲烷下沉是必不可少的，因为在这一快速的全球变化中，温室气体继续积累，这对于理解而变得至关重要代谢。拟议研究的总体假设是，氮的来源和可用性可以预测地控制甲状腺营养细菌中的碳代谢，无论基因组变异性如何发现了甲烷依赖性的硝酸化的途径，以及3）开发离散的分子标记，以确定甲烷依赖性的丹尼替子在控制大型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，我们将策划和维持一套分析工具，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，这些培养物的培养物具有可变的氮和碳源组合，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硝酸盐的分子调节，硝酸盐是在我的实验室中发现的一种新型生理学，它连接了两种有效温室气体（甲烷和氧化甲烷和氧化甲烷）和一种主要环境污染物（硝酸盐）的代谢。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物理益处，并基于基因含量和调节特征，预测特定菌株是否应该具有这种生理学。目标3将揭示甲烷依赖性的硝酸化和在从牛umen到融化多年冻土的潜水环境中进行的微生物的生态相关性。我们旨在在已经注意到以甲烷消耗为代价的环境中找到的甲硝化甲烷营养物可能具有不同的基因。拟议的研究共同采用了我们的基因组序列收集的甲肉芽菌细菌和分析能力，a）通过将工业废物转化为增值产品，与加拿大能源部门的接触，b）表征甲氮学物质的新生理学的酶促多样性和功能，以评估甲基植物的新型工具，并在甲基植物中提供了新型工具，并具有甲基甲烷的显着性，并具有甲基甲基化的显着性。碳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。