An archaeal lid-containing feruloyl esterase degrades polyethylene terephthalate

The accumulation of polyethylene terephthalate (PET) plastic waste poses a significant environmental threat. While bacterial and fungal PET-degrading enzymes (PETases) have been identified, no archaeal PETase had been previously reported. The study's purpose is to characterize a novel PETase identified through metagenomic analysis, expanding the known diversity of enzymes capable of degrading this recalcitrant polymer. Understanding the diversity of PET-degrading enzymes is crucial for developing efficient bioremediation strategies to address the global plastic pollution crisis. Most studies on plastic degradation have focused on bacterial lineages, with limited attention given to archaea, despite their presence in diverse environments, including those with high plastic concentrations. The authors hypothesize that archaeal organisms may harbor novel enzymes capable of PET degradation, broadening the potential for bioremediation approaches.

The literature review highlights the growing body of research on microbial enzymes capable of degrading various synthetic polymers, including PET. Approximately 200 such enzymes have been described, encompassing esterases, amidases, and oxygenases. However, many of these enzymes exhibit low conversion rates or are only active on PET oligomers. The best-characterized bacterial PETases include IsPETase from *Ideonella sakaiensis* and the Leaf-branch Compost Cutinase (LCC). A few PET-degrading enzymes have also been identified in fungi. These enzymes share common features such as the catalytic triad (Ser-Asp-His) and the exposure of the active site to the solvent. The existing literature provides a foundation for comparing the newly discovered archaeal enzyme PET46 with its bacterial and fungal counterparts.

The study employed a combination of bioinformatics, biochemical, and structural biology techniques. A Profile Hidden Markov Model (HMM) search was used to identify putative archaeal PETases within the NCBI non-redundant database. PET46, a candidate enzyme from a *Candidatus Bathyarchaeota* archaeon, was selected for further investigation. The gene encoding PET46 was cloned, expressed, and purified for biochemical characterization. Enzyme activity assays were conducted using various substrates, including BHET, MHET, and semi-crystalline PET powder. Crystallographic analysis was performed to determine the three-dimensional structure of PET46. Docking experiments were performed to assess the interaction between PET46 and its substrates. The authors also characterized the enzyme's biochemical properties, including its temperature and pH optima, metal ion dependence, and stability in the presence of various solvents and detergents. Detailed information about the primers used, the constructs generated, and the bacterial strains used for protein production is also included.

The key findings demonstrate that PET46, an archaeal feruloyl esterase, exhibits PET-degrading activity. Its activity on semi-crystalline PET powder is comparable to that of IsPETase and LCC, while its activity on BHET and MHET is significantly higher. The crystal structure of PET46 reveals a unique lid domain, absent in most bacterial PETases, which is likely involved in substrate binding. Biochemical characterization reveals that PET46 is a promiscuous, heat-adapted hydrolase with optimal activity at 70°C and pH 7-8. The enzyme shows remarkable stability and tolerance to various solvents and detergents. The analysis shows that PET46 shares structural similarity with bacterial PETases but contains unique features, such as the presence of a lid domain common in feruloyl esterases. Docking studies provide insights into the enzyme-substrate interactions, suggesting a plausible mechanism for PET hydrolysis. Quantitative data on the amount of aromatic products (TPA, MHET, and BHET) released during PET degradation at different temperatures are presented, showing comparable PET degradation activity of PET46 with IsPETase and LCC. The study found that PET46’s activity is strongly impacted by temperature, exhibiting significantly reduced activity at lower temperatures. Mutational analysis helped reveal the roles of specific amino acids in substrate binding and catalysis.

The discovery of PET46 expands the known diversity of PET-degrading enzymes, highlighting the potential for novel biocatalytic activities in understudied microbial lineages, specifically archaea. The enzyme’s structural similarity to bacterial PETases, combined with its unique lid domain, suggests a potential evolutionary pathway from feruloyl esterases to PETases. The remarkable heat stability and broad substrate specificity of PET46 make it a promising candidate for biotechnological applications. The enzyme's tolerance to various solvents further enhances its potential for industrial use. The presence of PET46 in a marine Bathyarchaeota archaeon, a group known to thrive in anoxic environments and utilize lignin as an energy source, supports the hypothesis that these organisms may play a significant role in the degradation of plastic waste in marine ecosystems. The findings underscore the importance of exploring the vast biodiversity of microbial communities for discovering novel enzymes with potential industrial relevance.

This study successfully identified and characterized PET46, a novel archaeal PETase with promising properties for bioremediation applications. The enzyme's unique structure and biochemical characteristics, particularly its heat stability and promiscuity, offer exciting avenues for further research and development. Future studies could focus on engineering PET46 for enhanced activity and stability, paving the way for its practical application in the biodegradation of PET waste. Further research is needed to explore the ecological role of PET46 and other archaeal enzymes in degrading plastics in various environments.

The study mainly focuses on the characterization of PET46 produced heterologously in *E. coli*. While the results demonstrate its PET-degrading activity, the in vivo activity within its native archaeal host remains to be determined. Further studies are required to investigate the effects of the environmental conditions of the Guaymas Basin on the enzyme's activity. The conversion rate observed in the experiments is relatively low, highlighting the need for further enzyme engineering to enhance its efficiency.