Biochemistry and Genetics of Anaerobic Alkane Metabolism: Interrogation of Sulfate-Reducing Isolates and Enrichments Using Genome-Enabled and Proteomic Approaches

厌氧烷烃代谢的生物化学和遗传学：使用基因组和蛋白质组方法探究硫酸盐还原分离物和富集

• 批准号: 0921265
• 负责人: Amy Callaghan
• 金额: $ 72.52万
• 依托单位: University of Oklahoma Norman Campus
• 依托单位国家: 美国
• 项目类别: Standard Grant
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-07-15 至 2013-09-30
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=0921265&HistoricalAwards=false
• 关键词: Biochemistry; Genetics; Anaerobic; Alkane; Metabolism

This award is funded under the American Recovery and Reinvestment Act of 2009 (Public Law 111-5).While the mechanisms and genetics of aerobic hydrocarbon biodegradation have been explored for decades, very little is known about the comparable activities of these environmentally important processes in anaerobic bacteria, which are difficult to isolate and characterize. Alkanes are the least reactive class of hydrocarbons due to their apolar sigma bonds. In the absence of high temperatures, high pressures, metal catalysts or UV light, biotransformation plays the dominant role in environmental alkane transformation. These reactions are responsible for a significant component of the carbon cycle with practical implications for energy production and environmental biotechnology. To date, there are two known pathways of anaerobic alkane biotransformation: addition to fumarate and a putative carboxylation pathway. Sulfate-reducing strains Desulfatibacillum alkenivorans AK-01 and Desulfococcus oleovorans Hxd3 serve as model organisms for these two mechanisms, respectively, and the genome sequences of both strains were recently completed. Three major research areas are under investigation: 1) AK-01 activates alkanes via addition of the sub-terminal carbon of the alkane across the double bond of fumarate. This finding is analogous to a mechanism for the anaerobic activation of toluene by the glycyl radical enzyme, benzylsuccinate synthase (Bss). Recent work by the PIs demonstrated that strain AK-01 contains a gene (assA1) that encodes the catalytic subunit of a glycyl radical enzyme (alkylsuccinate synthase) and that expression of this gene correlates with growth on hexadecane. The genetics and regulation of the putative assA1 operon in strain AK-01 will be investigated; 2) Unlike AK-01, Hxd3 activates alkanes at the C3 carbon via addition of inorganic carbon. To date, this pathway has not been fully elucidated, and the requisite genes and enzymes have not been identified. Genome-enabled proteomic analysis will be used to identify proteins for which expression correlates with growth on alkanes in order to further elucidate this pathway; and 3) The biochemistry and genetics of anaerobic paraffin degradation are unknown. It is hypothesized that these compounds are activated by similar mechanisms that are involved in the degradation of short- and medium-chain alkanes. Therefore, the biochemistry and genetics of anaerobic paraffin degradation will be investigated via metabolite profiling and metagenomic characterization of an anaerobic, paraffin-degrading enrichment culture. Broader Impacts. The availability of the complete genome sequences for the model organisms Hxd3 and AK-01, coupled with the unique expertise of the involved research team and recent advances in technology, have created an opportunity to provide significant insight to the physiology, ecology, and functional genomics of anaerobic alkane degraders. Overall, the project will significantly improve our understanding of the biology of anaerobic environments. It will also provide a solid foundation for understanding the novel biochemical processes involved in anaerobic alkane metabolism and will be the first investigation of anaerobic alkane degradation that examines genome-level data. As part of this project, a broader effort to integrate the research into the education of high-school, undergraduate, and graduate students will be made by providing research opportunities and mentoring. Students will be trained in molecular biology, functional genomics, proteomics and metabolite profiling. Educational outreach to the community will be achieved through the K-20 Center for Education and Community Renewal at the University of Oklahoma by: 1) providing training opportunities for one high school student each summer to participate in a research program through the center's GEAR UP program, 2) participating in the Teacher Learning program, which aims to disseminate science teaching tools to K-12 teachers in Oklahoma, and 3) participation in the annual Student Research and Performance Day which aims to provide community K-12 students opportunities to be exposed to university research, interact with scientists, and learn about career options.

该奖项是根据2009年美国复苏和再投资法案（公法111-5）资助的。虽然好氧碳氢化合物生物降解的机制和遗传学已经探索了几十年，但对厌氧细菌中这些环境重要过程的可比活动知之甚少，这些过程很难分离和表征。 烷烃由于其非极性σ键而成为最不活泼的烃类。 在没有高温、高压、金属催化剂或紫外光的情况下，生物转化在环境烷烃转化中起主导作用。这些反应是碳循环的重要组成部分，对能源生产和环境生物技术具有实际意义。迄今为止，有两种已知的厌氧烷烃生物转化途径：添加到富马酸和推定的羧化途径。 硫酸盐还原菌株Desulfatibacillum alkenivorans AK-01和Desulfococcus oleovorans Hxd 3分别作为这两种机制的模式生物，最近完成了这两种菌株的基因组序列。 目前正在研究三个主要的研究领域：1）AK-01通过将烷烃的亚末端碳加成到富马酸酯的双键上来活化烷烃。 这一发现类似于由甘氨酰自由基酶苄基琥珀酸合酶（Bss）对甲苯进行厌氧活化的机制。 PI最近的工作表明，菌株AK-01含有编码甘氨酰自由基酶（烷基琥珀酸合酶）催化亚基的基因（assA 1），该基因的表达与十六烷上的生长相关。 将研究菌株AK-01中推定的assA 1操纵子的遗传学和调控; 2）与AK-01不同，Hxd 3通过添加无机碳在C3碳处活化烷烃。 到目前为止，这一途径尚未完全阐明，所需的基因和酶尚未确定。 基因组使能的蛋白质组学分析将用于鉴定表达与烷烃上的生长相关的蛋白质，以进一步阐明该途径;以及3）厌氧石蜡降解的生物化学和遗传学是未知的。 据推测，这些化合物是由类似的机制，参与短链和中链烷烃的降解激活。因此，厌氧石蜡降解的生物化学和遗传学将通过厌氧石蜡降解富集培养物的代谢物谱分析和宏基因组学表征来研究。 更广泛的影响。模式生物Hxd 3和AK-01的完整基因组序列的可用性，加上相关研究团队的独特专业知识和最新技术进展，为厌氧烷烃降解菌的生理学，生态学和功能基因组学提供了重要的见解。 总的来说，该项目将大大提高我们对厌氧环境生物学的理解。 它还将为了解厌氧烷烃代谢中涉及的新生化过程提供坚实的基础，并将是第一次研究基因组水平数据的厌氧烷烃降解。 作为该项目的一部分，将通过提供研究机会和指导，更广泛地努力将研究纳入高中，本科和研究生的教育。 学生将接受分子生物学、功能基因组学、蛋白质组学和代谢物分析方面的培训。 通过俄克拉荷马州大学的K-20教育和社区更新中心，将通过以下方式实现对社区的教育推广：1）每年夏天为一名高中生提供培训机会，通过该中心的GEAR UP计划参加一个研究项目，2）参加教师学习计划，该计划旨在向俄克拉荷马州的K-12教师传播科学教学工具，以及3）参加一年一度的学生研究和表演日，旨在为社区K-12学生提供接触大学研究，与科学家互动以及了解职业选择的机会。