New Insights into the Function and Global Distribution of Polyethylene Terephthalate (PET)-Degrading Bacteria and Enzymes in Marine and Terrestrial Metagenomes

Abstract

Polyethylene terephthalate (PET) is one of the most important synthetic polymers used today. Unfortunately, the polymers accumulate in nature and to date no highly active enzymes are known that can degrade it at high velocity. Enzymes involved in PET degradation are mainly α- and β-hydrolases, like cutinases and related enzymes (EC 3.1.1). Currently, only a small number of such enzymes are well characterized. In this work, a search algorithm was developed that identified 504 possible PET hydrolase candidate genes from various databases. A further global search that comprised more than 16 Gb of sequence information within 108 marine and 25 terrestrial metagenomes obtained from the Integrated Microbial Genome (IMG) database detected 349 putative PET hydrolases. Heterologous expression of four such candidate enzymes verified the function of these enzymes and confirmed the usefulness of the developed search algorithm. In this way, two novel and thermostable enzymes with high potential for downstream application were partially characterized. Clustering of 504 novel enzyme candidates based on amino acid similarities indicated that PET hydrolases mainly occur in the phyla of Actinobacteria, Proteobacteria, and Bacteroidetes. Within the Proteobacteria, the Betaproteobacteria, Deltaproteobacteria, and Gammaproteobacteria were the main hosts. Remarkably enough, in the marine environment, bacteria affiliated with the phylum Bacteroidetes appear to be the main hosts of PET hydrolase genes, rather than Actinobacteria or Proteobacteria, as observed for the terrestrial metagenomes. Our data further imply that PET hydrolases are truly rare enzymes. The highest occurrence of 1.5 hits/Mb was observed in sequences from a sample site containing crude oil.IMPORTANCE Polyethylene terephthalate (PET) accumulates in our environment without significant microbial conversion. Although a few PET hydrolases are already known, it is still unknown how frequently they appear and with which main bacterial phyla they are affiliated. In this study, deep sequence mining of protein databases and metagenomes demonstrated that PET hydrolases indeed occur at very low frequencies in the environment. Furthermore, it was possible to link them to phyla that were previously not known to harbor such enzymes. This work contributes novel knowledge on the phylogenetic relationships, the recent evolution, and the global distribution of PET hydrolases. Finally, we describe the biochemical traits of four novel PET hydrolases.

Bio-upcycling of polyethylene terephthalate

“…For PET, the abundance of polyester hydrolases in the environment was investigated in detail. The results suggest that this enzyme activity is rather rare, but more frequent in crude oil rich environments 26,59,60 . Currently further metagenome and mechanistic studies of this important enzyme class are carried out by the scientific community, most likely discovering protein family members with superb activities or at least interesting amino acid variations.…”

A Novel Polyester Hydrolase From the Marine Bacterium Pseudomonas aestusnigri – Structural and Functional Insights

“…For PET degradation, comprehensive activitybased screening studies are missing. However, a bioinformatics study using a hidden Markov model succeeded to identify PET hydrolase genes using the UniProtKB database and more than 100 metagenome datasets, many of which originated from marine sources (Danso et al, 2018). The reported frequency of PET hydrolases was, dependent on the origin of the metagenomic sample, between 0.0001 and 1.5 hits per megabases of sequence, with the highest hit rate in a metagenome obtained from an oil polluted environment (Danso et al, 2018).…”

“…However, a bioinformatics study using a hidden Markov model succeeded to identify PET hydrolase genes using the UniProtKB database and more than 100 metagenome datasets, many of which originated from marine sources (Danso et al, 2018). The reported frequency of PET hydrolases was, dependent on the origin of the metagenomic sample, between 0.0001 and 1.5 hits per megabases of sequence, with the highest hit rate in a metagenome obtained from an oil polluted environment (Danso et al, 2018). In contrast to the marine origin of many predicted PET hydrolases, most of the PET degrading enzymes studied so far originate from terrestrial sources, with Cut190 from Saccharomonospora viridis (Kawai et al, 2014), Tha_Cut1 from Thermobifida alba (Ribitsch et al, 2012), Thc_Cut1 and Thc_Cut2 from T. cellulosilytica (Ribitsch et al, 2012), Tfu_0883, Tfu_0882 and TfCut2 from T. fusca (Chen et al, 2010;Roth et al, 2014) and LCC identified from a leafbranch compost metagenome (Sulaiman et al, 2012) for example.…”

Genes for a Circular and Sustainable Bio-PET Economy

“…a more accessible active site) display the highest activity against PET [34]. A recent bioinformatic analysis has investigated the distribution of genes encoding for homologs of these esterases in terrestrial and marine metagenomes and has allowed to identify 504 new hydrolases [35]. The two main conclusions of this study are: i) genes potentially encoding polyester hydrolases are rare, and ii) their taxonomic distribution seems to be related to the niche studied, with Actinobacteria or Proteobacteria being more prominent hosts in terrestrial environments whereas Bacteroidetes are the most frequent hosts in marine metagenomes [35].…”