Putative Mechanisms for Initial Reaction of Anaerobic Benzene Degradation Presumably Involving a Flavoprotein

Biodegradation of benzene is most challenging among the plethora of hydrocarbons due to its high chemical stability. While aerobic bacteria employ O₂-dependent oxygenases to overcome the high energy barrier, anaerobic benzene degradation remains enigmatic. Here we achieve an anaerobic enrichment, which is clonal with respect to the benzene-degrading, sulphate-reducing, marine bacterium BzS1, from a previously reported enrichment culture. We investigate this clonal enrichment culture by metabolite and proteogenomic analyses. BzS1 has a tailor-made catabolic network for utilising benzene, toluene, and acetate. Concerning the initial reaction of anaerobic benzene degradation, labelling experiments render previously suggested direct carboxylation (to benzoate) or methylation (to toluene) unlikely. Accordingly, a benzoate-CoA ligase is not encoded in the genome and proteins of anaerobic toluene degradation are not detectable in benzene-utilising cells. Rather, detection of labelled 3-phenylpropanoate hints at addition of benzene to a C₃-cosubstrate. Moreover, a presumptively heterotrimeric flavoprotein is present at remarkably high abundance (~⅕ of covered proteome) during anaerobic growth with benzene. This protein is related to glycolate oxidase and flavoproteins of the VOA/PCMH family. Taken together, these findings open the possibility of considering flavin cofactor involvement for the initial reaction using, for example, enolpyruvate (central metabolite) or glutaconyl-CoA (intermediate of benzoyl-CoA pathway) as cosubstrate.