Topic Area: 2f. Challenges in Application of Bioremediation Tools

Formatted Title:

Identification of Benzene Activation Enzymes in a Methanogenic Enrichment Culture

Background/Objectives:

Anaerobic benzene degradation was first discovered in 1980, reversing a long-held tenet that aromatic hydrocarbons were recalcitrant under anaerobic conditions. Notorious for its carcinogenicity, stringent environmental regulations, and persistence in anoxic environments, benzene has been the subject of numerous studies seeking to elucidate how benzene degradation is catalyzed in the absence of oxygen. Carboxylation has been proposed as a possible mechanism, but associated functional genes (abcA, benzoyl-CoA ligase) have only been identified in select benzene-degrading species. Isotopic fractionation studies and molecular analyses suggest that at least one other biochemical mechanism exists in nature. Here, we used a metagenomic and proteomic approach to identify novel benzene activation enzymes in a methanogenic enrichment culture. By presenting this work, we hope to engage the research community to help confirm the roles of these enzymes and to search through their own cultures for homologous features.

Approach/Activities:

DGG-B is a strictly anoxic enrichment culture that degrades benzene into methane and CO₂. It contains two closely related strains of benzene-degrading Desulfobacterota (ORM2a and ORM2b) and >100 other microorganisms with various predicted functions. Benzene is the only known substrate that supports the growth of ORM2a and ORM2b. In this study, the metagenome of DGG-B was sequenced and used to reconstruct the genomes of 74 DGG-B species, including the complete (closed) genome of ORM2a. Next, three rounds of proteomics experiments were used to identify putative benzene degradation enzymes and the genomes that encode for them. Automated annotation pipelines were used to assign protein predicted functions. Additional sequence-based and structural prediction tools (e.g., CATH v. 4.4, AlphaFold 2, and DeepFRI) were used glean more information into the molecular functions of select high abundance proteins.

Nancy Bawa

Affiliation

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Identification of Benzene Activation Enzymes in a Methanogenic Enrichment Culture