Bioprospecting for polyesterase activity relevant for PET degradation in marine Enterobacterales isolates

Abstract

Plastics have quickly become an integral part of modern life. Due to excessive production and improper waste disposal, they are recognized as contaminants present in practically all habitat types. Although there are several polymers, polyethylene terephthalate (PET) is of particular concern due to its abundance in the environment. There is a need for a solution that is both cost-effective and ecologically friendly to address this pollutant. The use of microbial depolymerizing enzymes could offer a biological avenue for plastic degradation, though the full potential of these enzymes is yet to be uncovered. The purpose of this study was to use (1) plate-based screening methods to investigate the plastic degradation potential of marine bacteria from the order Enterobacterales collected from various organismal and environmental sources, and (2) perform genome-based analysis to identify polyesterases potentially related to PET degradation. 126 bacterial isolates were obtained from the strain collection of RD3, Research Unit Marine Symbioses-GEOMAR-and sequentially tested for esterase and polyesterase activity, in combination here referred to as PETase-like activity. The results show that members of the microbial families Alteromonadaceae, Shewanellaceae, and Vibrionaceae, derived from marine sponges and bryozoans, are the most promising candidates within the order Enterobacterales. Furthermore, 389 putative hydrolases from the α/β superfamily were identified in 23 analyzed genomes, of which 22 were sequenced for this study. Several candidates showed similarities with known PETases, indicating underlying enzymatic potential within the order Enterobacterales for PET degradation.

Keywords: PETase-like activity; bryozoans; marine bacteria; order Enterobacterales; sponges.

Figure 1.. Graphical reference for activity classification on agar-plate substrate media. Clearance area category example after 10 days on tributyrin agar plates. (A) no activity, (B) low–activity, (C) medium–activity, and (D) high–activity. This reference area was used as an approximate to classify activity for all media tested.

Figure 2.. Phylogenetic tree and enzymatic activity screening results for all isolates screened (n = 126). Phylogenetic relatedness of the 5 tested families of the order Enterobacterales is displayed. The screening results are displayed from the inside out for the respective activities on TRI, PCD, PCL and BHET. As outgroup we used DSM 8802^T Bacillus halotolerans.

Figure 3.. Distribution of isolates obtained from different sources and their respective taxonomic affiliation within the order Enterobacterales. The stacked bar plot shows the number of isolates tested by their origin sources, where isolates obtained from bryozoans and sponges build the majority. The plot also shows the counting results in relation to bacterial family representatives per source.

Figure 4.. Maximum–likelihood (ML) phylogenetic tree for esterase sequences from Hal099-E and Hal280-CE using the reference dataset of PETase enzymes. The light–yellow background highlights all reference sequences listed in Table 2, and with light–blue the two protein sequences of interest are shown. The matched relatedness between the sequences is indicated by colour coded purple circles and yellow triangles, as determined by the LED database analyses.

Figure 5.. Amino acid sequence alignment of Hal280-CE polyesterase and Mors1. Hal280-CE was suggested as a suitable structural homolog for Mors1 by the LED server. Amino acid residues coloured in red correspond to the ones strictly conserved between Mors1 and Hal280-CE, while residues highlighted in yellow show areas with an average level of homology. The pentapeptide motif G-x1-S-x2-G is framed and highlighted by a purple rectangle.

Figure 6.. Amino acid sequence alignment of Hal099-E and PmC. Hal099 was suggested as a suitable structural homolog for PmC by the LED server. Amino acid residues coloured in red correspond to the ones strictly conserved between PmC and Hal099, while residues highlighted in yellow show areas with an average level of homology. The pentapeptide motif in G-x1-S-x2-G is fully preserved and shown and framed with the purple rectangle.

Figure 7.. Substrate activity per tested source. The stacked bar plot shows the activity count accordingly to the tested media for lipase (TRI), polyesterase (PCD and PCL), carboxylesterase–like (BHET), and absence of activity where the count is depicted by origin source.