Role of Dienelactone Hydrolases (DLHs) in PET Biodegradation and Biofilm Formation by Maribacter dokdonensis and Arenibacter palladensis

Abstract

Global plastics pollution represents a major environmental challenge, and we have only a very limited knowledge of marine microorganisms involved in possible remediation. Marine Bacteroidota, particularly members of Flavobacteria, are known for their ability to hydrolyze a wide range of different algal polymers. More recently, some representatives have been identified to encode PET-active enzymes. PET degradation is primarily conducted by non-specific and secreted enzymes of esterase class (EC 3.1.1-.). They are either designated as carboxylesterases (EC 3.1.1.1), poly(ethylene terephthalate) hydrolases (EC 3.1.1.101), lipases (EC 3.1.1.3) or cutinases (EC 3.1.1.74). Notably, among the currently known and functional PETases no dienelactone hydolases DLHs (3.1.1.45) are listed. Dienelactone hydrolases are enzymes that play a central role in the degradation of cyclic esters. In the current study, we show that bacteria affiliated with the Bacteroidota (class Flavobacteria) harbor DLHs acting on PET foil and powder. We report on the isolation of two marine bacterial strains, Arenibacter palladensis UHH-Hm9b and Maribacter dokdonensis UHH-5R5, forming biofilms on PET foil and releasing µM amounts of terephthalic acid after 5-7 days. Genome sequencing and functional analyses enables the identification of two secreted DLHs, designated PET93 and PET94, involved in PET degradation. While their predicted active sites and substrates binding pockets were identical to previously published PETases, both enzymes differed largely in their structural features from known PETases and represented novel scaffolds. Moreover, they lacked the typical porC-domain that is characteristic of known PETases from Flavobacteria.

For the two recombinant enzymes PET93 and PET94, activity on PET as well as the primary degradation products Bis(2-Hydroxyethyl) terephthalate (BHET) and Mono- (Hydroxyethyl) terephthalate (MHET) was demonstrated. Both enzymes were able to hydrolyze PET foil and powder at low but significant rates. These findings represent the first described DLHs with activity on plastics. This demonstrates that Flavobacteria harbor a greater diversity of PET-active, albeit unspecific, enzymes than previously assumed. In addition, the PET94 gene is positioned downstream of Fur-like Transcritiponal regulator, providing a rationale for testing iron supplementation in this experiment. Indeed, this study demonstrated that BHET degradation was enhanced upon iron supplementation, with up to 9-fold increases in MHET concentration detected by UHPLC. However, transcriptome analysis revealed no expression of PET94 under tested conditions, suggesting iron may modulate PET degradation at the post-transcriptional level, or that the applied concentrations were insufficient to trigger transcriptional activation. In summary, this study highlights the importance of expanding the currently known biodiversity of PET-hydrolyzing enzymes within marine Bacteroidota phylum. PET93, PET94, and their homologs within this phylum are found in diverse climatic zones worldwide, indicating a potentially important role in the degradation of plastic particles in marine ecosystems.