Anaerobic microbial consortia and enzymes for biotransformation of aromatic compounds

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This Discovery Grant proposal is focused elucidating the mechanisms for the anaerobic microbial transformation of the widely used industrial chemical benzene. Benzene is the simplest unsubstituted aromatic hydrocarbon, is toxic and carcinogenic, and is the structure at the heart of more complex polyaromatic hydrocarbons (PAHs) also of environmental concern. Intriguingly, unlike very closely related compounds like methylated benzene (toluene) or carboxylated benzene (benzoate), benzene is remarkably more difficult to biodegrade in the absence of oxygen. The pathways and enzymes for anaerobic toluene and benzoate biodegradation are now well characterized. In contrast, the mechanism for anaerobic benzene oxidation is still unknown, although significant progress has been made in the past few years, particularly in sustaining anaerobic transformation reactions in laboratory cultures. For many years, my laboratory has been studying the complex cellular interactions and reaction pathways in anaerobic enrichment cultures that degraded benzene in the absence of oxygen, under denitrifying or methanogenic conditions. We have identified two distinct organisms responsible for anaerobic benzene oxidation. We have collected metagenomic sequence data from these organisms and have a list of candidate genes for enzymes that may catalyze the reactions in the initial steps of benzene activation. Transcriptional and proteomic data have further narrowed down the options for relevant enzymes. Based on preliminary data, the two organisms appear to use different reaction mechanisms - one of which is a carboxylation - though neither has been biochemically proven yet. The specific objective of this discovery program is to biochemically characterize the enzymes involved in anaerobic benzene oxidation. This will be achieved using native polyacrylamide gel electrophoresis, anaerobic enzyme assays and heterologous expression of candidate proteins. These enzymes constitute a novel metabolic pathway and novel biochemistry for the transformation or detoxification of benzene and other important chemicals, including pesticides, herbicides, polyaromatic hydrocarbons, pharmaceuticals and lignin, that are all derivatives of benzene.