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

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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.