Multistep Enzymatic Low-density Polyethylene Degradation via Phenylalanine Monooxygenase and Isocitrate Lyase in Pseudomonas aeruginosa

Plastics have become indispensable in modern industries; however, their resistance to natural degradation poses environmental challenges. Biological degradation technologies employing microorganisms offer promising solutions. Here, we analyzed the transcriptome and proteome of Pseudomonas aeruginosa, a plastic-degrading microorganism found in the gut of superworms, to identify the genes and enzymes upregulated during polyethylene degradation. Functional analyses of these upregulated genes and enzymes using the Kyoto Encyclopedia of Genes and Genomes and Gene Ontology databases revealed an increase in lipid and hydrophobic amino acid metabolism, suggesting their involvement in polyethylene degradation. Based on these analyses, we identified phenylalanine monooxygenase, which is capable of oxidizing plastics, and isocitrate lyase, which is involved in C-C bond cleavage. To investigate the involvement of these enzymes in polyethylene degradation, phhA and aceA were transformed into Escherichia coli, and the enzymes were produced and purified. The purified enzymes were then reacted with polyethylene and analyzed. The results revealed the formation of hydroxyl (-OH) and C-O groups on the polyethylene surface after treatment with phenylalanine monooxygenase, confirming its ability to oxidize polyethylene. Isocitrate lyase alone did not affect polyethylene production; however, when combined with phenylalanine monooxygenase, it contributed to a reduction in molecular weight. This suggests a two-stage process of polyethylene degradation involving oxidation and depolymerization that requires sequential action of multiple enzymes. Thus, we identified the enzymes involved in each stage and demonstrated the degradation ability of polyethylene using purified enzymes.