Searching for new plastic-degrading enzymes from the plastisphere of alpine soils using a metagenomic mining approach.

Plastic materials, including microplastics, accumulate in all types of ecosystems, even in remote and cold environments such as the European Alps. This pollution poses a risk for the environment and humans and needs to be addressed. Using shotgun DNA metagenomics of soils collected in the eastern Swiss Alps at about 3,000 m a.s.l., we identified genes and their proteins that potentially can degrade plastics. We screened the metagenomes of the plastisphere and the bulk soil with a differential abundance analysis, conducted similarity-based screening with specific databases dedicated to putative plastic-degrading genes, and selected those genes with a high probability of signal peptides for extracellular export and a high confidence for functional domains. This procedure resulted in a final list of nine candidate genes. The lengths of the predicted proteins were between 425 and 845 amino acids, and the predicted genera producing these proteins belonged mainly to Caballeronia and Bradyrhizobium. We applied functional validation, using heterologous expression followed by enzymatic assays of the supernatant. Five of the nine proteins tested showed significantly increased activities when we used an esterase assay, and one of these five proteins from candidate genes, a hydrolase-type esterase, clearly had the highest activity, by more than double. We performed the fluorescence assays for plastic degradation of the plastic types BI-OPL and ecovio® only with proteins from the five candidate genes that were positively active in the esterase assay, but like the negative controls, these did not show any significantly increased activity. In contrast, the activity of the positive control, which contained a PLA-degrading gene insert known from the literature, was more than 20 times higher than that of the negative controls. These findings suggest that in silico screening followed by functional validation is suitable for finding new plastic-degrading enzymes. Although we only found one new esterase enzyme, our approach has the potential to be applied to any type of soil and to plastics in various ecosystems to search rapidly and efficiently for new plastic-degrading enzymes.