Evidences of aromatic degradation dominantly via the phenylacetic acid pathway in marine benthic Thermoprofundales

Thermoprofundales (Marine Benthic Group D archaea, MBG-D) is a newly proposed archaeal order and widely distributed in global marine sediment, and the members in the order may play a vital role in carbon cycling. However, the lack of pure cultures of these oeganisms has hampered the recognition of their catabolic roles. Here, by constructing high-quality metagenome-assembled genomes (MAGs) of two new subgroups of Thermoprofundales from hydrothermal sediment and predicting their catabolic pathways, we here provide genomic evidences that Thermoprofundales are capable of degrading aromatics via the phenylacetic acid (PAA) pathway. Then, the gene sequences of phenylacetyl-CoA ligase (PCL), a key enzyme for the PAA pathway, were searched in reference genomes. The widespread distribution of PCL genes among 14.9% of archaea and 75.9% of Thermoprofundales further supports the importance of the PAA pathway in archaea, particularly in Thermoprofundales where no ring-cleavage dioxygenases were found. Two PCLs from Thermoprofundales MAGs, PCLM8-3 and PCLM10-15, were able to convert PAA to phenylacetyl-CoA (PA-CoA) in vitro, demonstrating the involvement of Thermoprofundales in aromatics degradation through PAA via CoA activation. Their acid tolerance (pH 5-7), high-optimum temperatures (60 degrees C and 80 degrees C), thermostability (stable at 60 degrees C and 50 degrees C for 48 h) and broad substrate spectra imply that Thermoprofundales are capable of transforming aromatics under extreme conditions. Together with the evidence of in situ transcriptional activities for most genes related to the aromatics pathway in Thermoprofundales, these genomic, and biochemical evidences highlight the essential role of this ubiquitous and abundant archaeal order in the carbon cycle of marine sediments.