An Ultra-Sensitive Comamonas thiooxidans Biosensor for the Rapid Detection of Enzymatic Polyethylene Terephthalate (PET) Degradation.

Polyethylene terephthalate (PET) is a prevalent synthetic polymer that is known to contaminate marine and terrestrial environments. Currently, only a limited number of PET-active microorganisms and enzymes (PETases) are known. This is in part linked to the lack of highly sensitive function-based screening assays for PET-active enzymes. Here, we report on the construction of a fluorescent biosensor based on Comamonas thiooxidans strain S23. C. thiooxidans S23 transports and metabolizes TPA, one of the main breakdown products of PET, using a specific tripartite tricarboxylate transporter (TTT) and various mono- and dioxygenases encoded in its genome in a conserved operon ranging from tphC-tphA1. TphR, an IclR-type transcriptional regulator is found upstream of the tphC-tphA1 cluster where TPA induces transcription of tphC-tphA1 up to 88-fold in exponentially growing cells. In the present study, we show that the C. thiooxidans S23 wild-type strain, carrying the sfGFP gene fused to the tphC promoter, senses TPA at concentrations as low as 10 µM. Moreover, a deletion mutant lacking the catabolic genes involved in TPA degradation thphA2-A1 (ΔtphA2A3BA1) is up to 10,000-fold more sensitive and detects TPA concentrations in the nanomolar range. This is, to our knowledge, the most sensitive reporter strain for TPA and we demonstrate that it can be used for the detection of enzymatic PET breakdown products. IMPORTANCE Plastics and microplastics accumulate in all ecological niches. The construction of more sensitive biosensors allows to monitor and screen potential PET degradation in natural environments and industrial samples. These strains will also be a valuable tool for functional screenings of novel PETase candidates and variants or monitoring of PET recycling processes using biocatalysts. Thereby they help us to enrich the known biodiversity and efficiency of PET degrading organisms and enzymes and understand their contribution to environmental plastic degradation.

The metagenome-derived esterase PET40 is highly promiscuous and hydrolyses polyethylene terephthalate (PET).

Polyethylene terephthalate (PET) is a widely used synthetic polymer and known to contaminate marine and terrestrial ecosystems. Only few PET-active microorganisms and enzymes (PETases) are currently known, and it is debated whether degradation activity for PET originates from promiscuous enzymes with broad substrate spectra that primarily act on natural polymers or other bulky substrates, or whether microorganisms evolved their genetic makeup to accepting PET as a carbon source. Here, we present a predicted diene lactone hydrolase designated PET40, which acts on a broad spectrum of substrates, including PET. It is the first esterase with activity on PET from a GC-rich Gram-positive Amycolatopsis species belonging to the Pseudonocardiaceae (Actinobacteria). It is highly conserved within the genera Amycolatopsis and Streptomyces. PET40 was identified by sequence-based metagenome search using a PETase-specific hidden Markov model. Besides acting on PET, PET40 has a versatile substrate spectrum, hydrolyzing δ-lactones, ß-lactam antibiotics, the polyester-polyurethane Impranil® DLN, and various para-nitrophenyl ester substrates. Molecular docking suggests that the PET degradative activity is likely a result of the promiscuity of PET40, as potential binding modes were found for substrates encompassing mono(2-hydroxyethyl) terephthalate, bis(2-hydroxyethyl) terephthalate, and a PET trimer. We also solved the crystal structure of the inactive PET40 variant S178A to 1.60 Å resolution. PET40 is active throughout a wide pH (pH 4-10) and temperature range (4-65 °C) and remarkably stable in the presence of 5% SDS, making it a promising enzyme as a starting point for further investigations and optimization approaches.

Metagenomic Screening of a Novel PET Esterase via In Vitro Expression System.

Metagenomic screening is a widely applied biotechnological approach for screening of novel industrial enzymes. The traditional method of metagenomic screening is based on the functional analyses of heterologously expressed environmental genes in a suitable host, which is the bottleneck of this method. To avoid limitation from the clone-dependent system, an in vitro expression technology has been developed in combination with next-generation sequencing and bioinformatics. First, the sequence profile of a target enzyme, e.g., poly(ethylene terephthalate) esterase in this protocol, is constructed according to the sequences of well-characterized enzymes. Then, the sequence screening is performed with this computationally generated profile among all available metagenomic databases. Afterwards, the candidate genes are synthesized and expressed in vitro with RNA polymerase and translation machinery from special cell extract. Finally, such in vitro produced enzymes are directly applied for the functional analyses. Comparing to the traditional screening methods, this in vitro screening technology can not only save time and materials, but also be easily developed for high-throughput screening with an automatic pipetting robot.

The PET-Degrading Potential of Global Metagenomes: From In Silico Mining to Active Enzymes.

Against the background of the steadily increasing amount of plastic waste in the sea and on land, it is more important than ever to find ways out of this situation. In recent years, microorganisms have been discovered that are capable of degrading artificial polymers such as polyethylene terephthalate (PET). Even if the turnover rates of the enzymes responsible for this reaction may be too low to solve the global plastic pollution problem, it is still of great societal interest to find microorganisms that are able to degrade the polymer. The corresponding enzymes, PET esterases (PETases) can be used in biotechnological processes and could contribute to a resource-saving circular economy. In this chapter, we present a sequence-based in silico screening method to find new PETases in metagenomic datasets. This method can easily be adapted to find other enzyme classes. We also list a number of assays that can be used to test the enzymes for activity on PET as well as other substrates.

An archaeal lid-containing feruloyl esterase degrades polyethylene terephthalate.

Polyethylene terephthalate (PET) is a commodity polymer known to globally contaminate marine and terrestrial environments. Today, around 80 bacterial and fungal PET-active enzymes (PETases) are known, originating from four bacterial and two fungal phyla. In contrast, no archaeal enzyme had been identified to degrade PET. Here we report on the structural and biochemical characterization of PET46 (RLI42440.1), an archaeal promiscuous feruloyl esterase exhibiting degradation activity on semi-crystalline PET powder comparable to IsPETase and LCC (wildtypes), and higher activity on bis-, and mono-(2-hydroxyethyl) terephthalate (BHET and MHET). The enzyme, found by a sequence-based metagenome search, is derived from a non-cultivated, deep-sea Candidatus Bathyarchaeota archaeon. Biochemical characterization demonstrated that PET46 is a promiscuous, heat-adapted hydrolase. Its crystal structure was solved at a resolution of 1.71 Å. It shares the core alpha/beta-hydrolase fold with bacterial PETases, but contains a unique lid common in feruloyl esterases, which is involved in substrate binding. Thus, our study widens the currently known diversity of PET-hydrolyzing enzymes, by demonstrating PET depolymerization by a plant cell wall-degrading esterase.

The Bacteroidetes Aequorivita sp. and Kaistella jeonii Produce Promiscuous Esterases With PET-Hydrolyzing Activity.

Certain members of the Actinobacteria and Proteobacteria are known to degrade polyethylene terephthalate (PET). Here, we describe the first functional PET-active enzymes from the Bacteroidetes phylum. Using a PETase-specific Hidden-Markov-Model- (HMM-) based search algorithm, we identified several PETase candidates from Flavobacteriaceae and Porphyromonadaceae. Among them, two promiscuous and cold-active esterases derived from Aequorivita sp. (PET27) and Kaistella jeonii (PET30) showed depolymerizing activity on polycaprolactone (PCL), amorphous PET foil and on the polyester polyurethane Impranil® DLN. PET27 is a 37.8 kDa enzyme that released an average of 174.4 nmol terephthalic acid (TPA) after 120 h at 30°C from a 7 mg PET foil platelet in a 200 µl reaction volume, 38-times more than PET30 (37.4 kDa) released under the same conditions. The crystal structure of PET30 without its C-terminal Por-domain (PET30ΔPorC) was solved at 2.1 Å and displays high structural similarity to the IsPETase. PET30 shows a Phe-Met-Tyr substrate binding motif, which seems to be a unique feature, as IsPETase, LCC and PET2 all contain Tyr-Met-Trp binding residues, while PET27 possesses a Phe-Met-Trp motif that is identical to Cut190. Microscopic analyses showed that K. jeonii cells are indeed able to bind on and colonize PET surfaces after a few days of incubation. Homologs of PET27 and PET30 were detected in metagenomes, predominantly aquatic habitats, encompassing a wide range of different global climate zones and suggesting a hitherto unknown influence of this bacterial phylum on man-made polymer degradation.