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

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。虽然好氧烃生物降解的机制和遗传学已经被探索了几十年，但人们对厌氧细菌中这些对环境重要的过程的可比活动知之甚少，这些过程很难分离和表征。烷烃是最不活跃的一类碳氢化合物，因为它们的非极性西格马键。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影响。到目前为止，已知的厌氧烷烃生物转化有两条途径：富马酸加成途径和推测的羧化途径。硫酸盐还原菌株嗜酸脱硫杆菌AK-01和油酸脱硫球菌Hxd3分别作为这两种机制的模式菌，并于最近完成了两株菌的基因组测序。主要研究了三个方面：1)AK-01通过在富马酸双键上加成烷烃的亚端碳来活化烷烃。这一发现类似于甘氨酰自由基酶--丁二酸苄酯合成酶(BSS)对甲苯的厌氧活化机制。PIS最近的工作表明，AK-01菌株含有一个基因(AssA1)，它编码甘氨酰自由基酶(烷基琥珀酸合成酶)的催化亚单位，该基因的表达与在十六烷上的生长有关。将研究AK-01菌株中可能的assA1操纵子的遗传学和调控；2)与AK-01不同，Hxd3通过添加无机碳激活C3碳上的烷烃。到目前为止，这一途径还没有完全阐明，也没有鉴定出必要的基因和酶。基因组蛋白质组分析将被用来确定其表达与烷烃生长相关的蛋白质，以进一步阐明这一途径；以及3)厌氧石蜡降解的生化和遗传学尚不清楚。据推测，这些化合物被参与降解短链和中链烷烃的类似机制激活。因此，厌氧降蜡的生物化学和遗传学将通过对厌氧降蜡增菌培养的代谢物图谱和元基因组学特征进行研究。更广泛的影响。模式生物Hxd3和AK-01的完整基因组序列的获得，加上相关研究团队的独特专业知识和最新技术进步，创造了一个机会，为厌氧烷烃降解菌的生理学、生态学和功能基因组学提供重要的见解。总体而言，该项目将显著提高我们对厌氧环境生物学的理解。它还将为理解厌氧烷烃代谢中涉及的新的生化过程提供坚实的基础，并将是首次研究基因组水平的厌氧烷烃降解。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整合到高中、本科生和研究生的教育中。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图谱方面的培训。通过俄克拉荷马大学K-20教育和社区更新中心，将实现对社区的教育推广：1)每年夏天通过该中心的Gear Up计划为一名高中生提供参加研究计划的培训机会；2)参与旨在向俄克拉荷马州K-12教师传播科学教学工具的教师学习计划；以及3)参与一年一度的学生研究和表现日，旨在为社区K-12学生提供接触大学研究、与科学家互动和了解职业选择的机会。

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.