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广泛的生物学功能，但只有甲基营养细菌专门利用单碳化合物作为能量和碳的来源。甲烷营养(即，吞食甲烷)甲基营养菌作为地球上生物甲烷的汇是必不可少的，随着温室气体在这段快速的全球变化期间继续积累，了解这一点变得至关重要。对30多个甲烷菌基因组序列的比较表明，氮代谢与碳代谢密切相关，具有丰富的多样性。拟议研究的总体假设是，氮源和可获得性可预测地控制甲烷营养细菌中的碳代谢，而不考虑基因组的变异性，使我实验室的学生能够：1)确定氮代谢如何调节碳转化为工业利用的附加值终端产品；2)表征我们最近发现的依赖甲烷的反硝化途径的酶、功能和调节；以及3)开发离散的分子标记，以确定依赖甲烷的反硝化菌在调节不同环境中的温室气体通量中的存在、强度和相关性。我们基于实验室的方法利用我们管理和维护的大量基因组测序菌株集合，以及一套分析工具来测量批量和连续培养中的细胞生长，实时跟踪气体测量，基因表达水平，以及代谢物和蛋白质的生产。我们在目标1中的目标是确定氮源和有效性如何将碳转化为与工业相关的分子，如脂肪酸、类异戊二烯、氮储存聚合物、碳储存聚合物和有机酸。对不同氮源和碳源组合培养物中的核酸、蛋白质和代谢物进行生长动力学和分析，将有助于发现可用于工业开发的产品。目标2将确定依赖甲烷的硝酸盐反硝化作用的分子调控，这是我的实验室发现的一种新的生理学，它连接了两种强有力的温室气体(甲烷和一氧化二氮)和主要环境污染物(硝酸盐)在单个有机体内的代谢。利用生理学、转录学和蛋白质组学工具，我们的目标是充分表征这种活性的酶多样性和生理益处，并根据基因含量和调控特征预测特定菌株是否应该具有这种生理特性。目标3将揭示甲烷依赖的反硝化作用及其微生物在从奶牛瘤胃到融化永久冻土的不同环境中的生态相关性。反硝化甲烷氧化菌具有不同的系统发育基因，我们的目标是在以消耗甲烷为代价产生一氧化二氮的环境中找到这些基因。总之，拟议的研究充分利用了我们的甲烷营养细菌基因组测序收集和分析能力，以a)通过将工业废物转化为附加值产品与加拿大的能源部门对接，b)表征甲烷营养细菌一种新生理学的酶多样性和功能，以及c)提供工具来评估依赖甲烷的反硝化作用作为碳和氮生物地球化学循环之间的一种新联系的生态意义。除了为加拿大和更广泛的科学界开发一个潜在的有利可图和具有生态重要性的研究领域外，这项工作还为微生物学和生物技术学生提供了独特的培训体验。