Polyethylene Terephthalate Hydrolase Research Articles

Harnessing xylitol utilization in Escherichia coli for antibiotic-free plasmid and recombinant protein production

Modern genetic medicine such as RNA vaccines are of increasing interest and many of these products require plasmid. There is an increasing interest in developing an antibiotic-free plasmid maintenance system which can potentially speed up clinical trial evaluation and increase product safety. We have previously reported that wild-type E. coli strain cannot utilize xylitol unless a single gene (xylitol dehydrogenase: gutB) was over-expressed. Chemically defined medium formulation was made possible with xylitol as the main carbon source. The present study focused on replacing the antibiotic resistance gene on plasmid with gutB (1056 bp) for maintenance in regular E. coli strains, DH5α and BL21DE3. Four plasmids with different replication origins (pMB1 and pAC) were constructed and selected using the xylitol-based, chemically defined medium. The workflow needs 24 h for colony formation and another 24 h for cell propagation in liquid medium. The plasmid titer and productivity were similar to that obtained using a conventional plasmid and the Luria-Bertani (LB) medium containing an antibiotic. The new plasmid system was also used to produce two recombinant proteins without antibiotics. One of the proteins (a polyethylene terephthalate hydrolase) had ∼5 times higher specific activity when it is produced using the new system compared with the conventional one. The new plasmid selection/maintenance method reported here could be useful to academic research and industrial bio-manufacturing processes.

Expression, purification and characterization of a novel bis (hydroxyethyl) terephthalate hydrolase from Hydrogenobacter thermophilus

PET (polyethylene terephthalate) is one of the most important petrochemicals that is widely used in mineral water bottles, food and beverage packaging and textile industry. Because of its stability under environmental conditions, the massive amount of PET wastes caused serious environmental pollution. The use of enzymes to depolymerize PET wastes and upcycling is one of the important directions for plastics pollution control, among which the key is the depolymerization efficiency of PET by PET hydrolase. BHET (bis(hydroxyethyl) terephthalate) is the main intermediate of PET hydrolysis, its accumulation can hinder the degradation efficiency of PET hydrolase significantly, and the synergistic use of PET hydrolase and BHET hydrolase can improve the PET hydrolysis efficiency. In this study, a dienolactone hydrolase from Hydrogenobacter thermophilus which can degrade BHET (HtBHETase) was identified. After heterologous expression in Escherichia coli and purification, the enzymatic properties of HtBHETase were studied. HtBHETase shows higher catalytic activity towards esters with short carbon chains such as p-nitrophenol acetate. The optimal pH and temperature of the reaction with BHET were 5.0 and 55 ℃, respectively. HtBHETase exhibited excellent thermostability, and retained over 80% residual activity after treatment at 80 ℃ for 1 hour. These results indicate that HtBHETase has potential in biological PET depolymerization, which may facilitate the enzymatic degradation of PET.

Rational Design of Disulfide Bridges in BbPETaseCD for Enhancing the Enzymatic Performance in PET Degradation.

Polyethylene terephthalate (PET) is one of the most prevalent transparent thermoplastics. It is commonly utilized due to its low cost and high durability. With the massive accumulation of waste PET, however, serious environmental pollution has become a global problem. Compared to traditional chemical degradation, biodegradation of PET catalyzed by PET hydrolase (PETase) is more environmentally friendly and energy-efficient. BbPETaseCD from the Burkholderiales bacterium is a PETase that shows favorable properties for application in the biodegradation of PET. To enhance the enzymatic performance of this enzyme, this work focuses on the rational design of disulfide bridges in BbPETaseCD. We utilized two computational algorithms to predict the probable disulfide-bridge mutations in BbPETaseCD, and five variants were acquired from the computations. Among these, the N364C/D418C variant with one additional disulfide bond showed higher expression than the wild-type enzyme (WT) and the best enzymatic performance. The melting temperature (Tm) of the N364C/D418C variant presented an increase of 14.8 °C over that of WT (56.5 °C), indicating that the additional disulfide bond significantly raised the thermodynamic stability of the enzyme. Kinetic experiments at different temperatures also demonstrated the thermal stability increase of the variant. The variant also showed significantly increased activity over WT when using bis(hydroxyethyl) terephthalate (BHET) as the substrate. More remarkably, the N364C/D418C variant exhibited approximately an 11-fold increase over the WT enzyme in the long-term (14 days) degradation of PET films. The results prove that the rationally designed disulfide bond significantly improved the enzymatic performance of the enzyme for PET degradation.

Rational redesign of thermophilic PET hydrolase LCCICCG to enhance hydrolysis of high crystallinity polyethylene terephthalates

Polyethylene terephthalate (PET)-degrading enzymes represent a promising solution to the plastic pollution. However, PET-degrading enzymes, even thermophilic PETase, can effectively degrade low-crystallinity (∼8%) PETs, but exhibit weak depolymerization of more common, high-crystallinity (30–50%) PETs. Here, based on the thermophilic PETase, LCCICCG, we proposed two strategies for rational redesign of LCCICCG using the machine learning tool, Preoptem, combined with evolutionary analysis. Six single-point mutants (S32L, D18T, S98R, T157P, E173Q, N213P) were obtained that exhibit higher catalytic efficiency towards PET powder than wild-type LCCICCG at 75 °C. Additionally, the optimal temperature for degrading 39.07% crystalline PET increased from 65 °C in the wild-type LCCICCG to between 75 and 80 °C in the LCCICCG_I6M mutant that carries all six single-point mutations. Especially, the LCCICCG_I6M mutant has a significantly higher degradation effect on some commonly used bottle-grade plastic powders at 75–80 °C than that of wild type. The enzymatic digestion of ground 31.30% crystalline PET water bottles by LCCICCG_I6M yielded 31.91 ± 0.99 mM soluble products in 24 h, which was 3.64 times that of LCCICCG (8.77 ± 1.52 mM). Overall, this study provides a feasible route for engineering thermostable enzymes that can degrade high-crystallinity PET plastic.

Enantioselectivity and origin of enhanced efficiency in polyethylene terephthalate hydrolases catalyzed depolymerization

Biotechnology is one of the most promising strategies to resolve the global crisis of plastic pollution. A clear understanding of the core enzyme mechanisms in the biotransformation process is critical for rational enzyme engineering and for practical, industrial-scale applications. Herein, we systematically examined and evidenced a largely unexplored piece in the depolymerization mechanism catalyzed by polyethylene terephthalate (PET) hydrolases: their enantioselectivity. We found that all the short-lived tetrahedron intermediates (IM3 and IM8) possess S-type chirality in six representative PET hydrolases. For instance, the binding percentage ratio of pro-S:pro-R is 57:21 in FAST-PETase, while pro-S binding leads to a much lower average energy barrier (5.2 kcal/mol) than pro-R binding (33.1 kcal/mol). Key structural features (e.g. the angle for Ser@H1-His@N1-PET@O2 and distance for His@N1-PET@O2) that significantly modulate the enantioselectivity were identified. The origin of the energy landscape variation between wild-type IsPETase and mutant FAST-PETase was also unveiled via analysis of key features, the distortion/interaction energy, and non-covalent bond interactions. This study supplies the missing piece in the mechanism for depolymerization catalyzed by PET hydrolases, and will aid in the rational design of enzymes for industrial recycling of PET plastic waste.

A New PETase from the Human Saliva Metagenome and Its Functional Modification via Genetic Code Expansion in Bacteria.

The discovery and engineering of new plastic degrading enzymes is an important challenge in chemical biotechnology to enable transition to a more sustainable and circular plastics economy. This field has so far yielded a range of enzymes and microbial pathways for the recycling and valorization of plastic waste. New research from Uttamapinant et al. reports the discovery of a novel polyethylene terephthalate (PET) hydrolase from the human saliva metagenome that displays improved properties and catalytic performance over previously characterized PET hydrolases (PETases). The authors also demonstrate the site-specific incorporation of a photocaged unnatural amino acid, 2,3-diaminopropionic acid (DAP), which upon photodecaging enables covalent binding of DAP to the PET surface. Thus, this work highlights metagenomic datasets as an untapped source of new PET degrading enzymes and the chemical modification of PETases via genetic code expansion, enabling new biotechnologies for the circular plastics economy.

A New PETase from the Human Saliva Metagenome and Its Functional Modification via Genetic Code Expansion in Bacteria.

The discovery and engineering of new plastic degrading enzymes is an important challenge in chemical biotechnology to enable transition to a more sustainable and circular plastics economy. This field has so far yielded a range of enzymes and microbial pathways for the recycling and valorization of plastic waste. New research from Uttamapinant et al. reports the discovery of a novel polyethylene terephthalate (PET) hydrolase from the human saliva metagenome that displays improved properties and catalytic performance over previously characterized PET hydrolases (PETases). The authors also demonstrate the site-specific incorporation of a photocaged unnatural amino acid, 2,3-diaminopropionic acid (DAP), which upon photodecaging enables covalent binding of DAP to the PET surface. Thus, this work highlights metagenomic datasets as an untapped source of new PET degrading enzymes and the chemical modification of PETases via genetic code expansion, enabling new biotechnologies for the circular plastics economy.

You get what you screen for: a benchmark analysis of leaf branch compost cutinase variants for polyethylene terephthalate (PET) degradation

Reference enzymes allow PET hydrolases and protein engineering approaches to be compared across different substrates, their properties and reaction conditions. Using this concept, semi-rational protein design led to an improved PET hydrolase variant.

Impacts of QM region sizes and conformation numbers on modelling enzyme reactions: a case study of polyethylene terephthalate hydrolase.

A quantum mechanics/molecular mechanics (QM/MM) approach is a broadly used tool in computational enzymology. Treating the QM region with a high-level DFT method is one of the important branches. Here, taking leaf-branch compost cutinase-catalyzed polyethylene terephthalate depolymerization as an example, the convergence behavior of energy barriers as well as key structural and charge features with respect to the size of the QM region (up to 1000 atoms) is systematically investigated. BP86/6-31G(d)//CHARMM and M06-2X/6-311G(d,p)//CHARMM level of theories were applied for geometry optimizations and single-point energy calculations, respectively. Six independent enzyme conformations for all the four catalytic steps (steps (i)-(iv)) were considered. Most of the twenty-four cases show that at least 500 QM atoms are needed while only two rare cases show that ∼100 QM atoms are sufficient for convergence when only a single conformation was considered. This explains why most previous studies showed that 500 or more QM atoms are required while a few others showed that ∼100 QM atoms are sufficient for DFT/MM calculations. More importantly, average energy barriers and key structural/charge features from six conformations show an accelerated convergence than that in a single conformation. For instance, to reach energy barrier convergence (within 2.0 kcal mol-1) for step (ii), only ∼100 QM atoms are required if six conformations are considered while 500 or more QM atoms are needed with a single conformation. The convergence is accelerated to be more rapid if hundreds and thousands of conformations were considered, which aligns with previous findings that only several dozens of QM atoms are required for convergence with semi-empirical QM/MM MD simulations.

Efficient depolymerization of polyethylene terephthalate (PET) and polyethylene furanoate by engineered PET hydrolase Cut190

The enzymatic recycling of polyethylene terephthalate (PET) can be a promising approach to tackle the problem of plastic waste. The thermostability and activity of PET-hydrolyzing enzymes are still insufficient for practical application. Pretreatment of PET waste is needed for bio-recycling. Here, we analyzed the degradation of PET films, packages, and bottles using the newly engineered cutinase Cut190. Using gel permeation chromatography and high-performance liquid chromatography, the degradation of PET films by the Cut190 variant was shown to proceed via a repeating two-step hydrolysis process; initial endo-type scission of a surface polymer chain, followed by exo-type hydrolysis to produce mono/bis(2-hydroxyethyl) terephthalate and terephthalate from the ends of fragmented polymer molecules. Amorphous PET powders were degraded more than twofold higher than amorphous PET film with the same weight. Moreover, homogenization of post-consumer PET products, such as packages and bottles, increased their degradability, indicating the importance of surface area for the enzymatic hydrolysis of PET. In addition, it was required to maintain an alkaline pH to enable continuous enzymatic hydrolysis, by increasing the buffer concentration (HEPES, pH 9.0) depending on the level of the acidic products formed. The cationic surfactant dodecyltrimethylammonium chloride promoted PET degradation via adsorption on the PET surface and binding to the anionic surface of the Cut190 variant. The Cut190 variant also hydrolyzed polyethylene furanoate. Using the best performing Cut190 variant (L136F/Q138A/S226P/R228S/D250C-E296C/Q123H/N202H/K305del/L306del/N307del) and amorphous PET powders, more than 90 mM degradation products were obtained in 3 days and approximately 80 mM in 1 day.Graphical

An Overview into Polyethylene Terephthalate (PET) Hydrolases and Efforts in Tailoring Enzymes for Improved Plastic Degradation.

Plastic or microplastic pollution is a global threat affecting ecosystems, with the current generation reaching as much as 400 metric tons per/year. Soil ecosystems comprising agricultural lands act as microplastics sinks, though the impact could be unexpectedly more far-reaching. This is troubling as most plastic forms, such as polyethylene terephthalate (PET), formed from polymerized terephthalic acid (TPA) and ethylene glycol (EG) monomers, are non-biodegradable environmental pollutants. The current approach to use mechanical, thermal, and chemical-based treatments to reduce PET waste remains cost-prohibitive and could potentially produce toxic secondary pollutants. Thus, better remediation methods must be developed to deal with plastic pollutants in marine and terrestrial environments. Enzymatic treatments could be a plausible avenue to overcome plastic pollutants, given the near-ambient conditions under which enzymes function without the need for chemicals. The discovery of several PET hydrolases, along with further modification of the enzymes, has considerably aided efforts to improve their ability to degrade the ester bond of PET. Hence, this review emphasizes PET-degrading microbial hydrolases and their contribution to alleviating environmental microplastics. Information on the molecular and degradation mechanisms of PET is also highlighted in this review, which might be useful in the future rational engineering of PET-hydrolyzing enzymes.

Biodegrading plastics with a synthetic non-biodegradable enzyme

<h2>Summary</h2> Biodegradation of plastics through the use of enzymes provides an attractive, environmentally friendly solution to dispose of and recycle plastic wastes. Although many natural and engineered plastic-degrading enzymes have been developed, their practical applications are hindered by their biodegradation in open environments. We reasoned that most popularly used plastics, such as polyethylene terephthalate (PET), are achiral and hence can be hydrolyzed by both the l- and d-amino acid versions of plastic-degrading enzymes. Here, we chemically synthesized a mirror-image version of the 231-aa PET hydrolase, which is capable of efficiently degrading a variety of plastics such as PET, polybutylene terephthalate (PBT), and polybutylene succinate (PBS) but meanwhile itself is resistant to biodegradation by soil extract and seawater from the environment. The synthetic mirror-image PET hydrolase demonstrates the potential of a new class of plastic-degrading, non-biodegradable mirror-image enzymes to help address the plastic pollution problem in our homochiral biosphere.

Performance of PET hydrolases with tethered binding modules in large-scale applications

In this issue of Chem Catalysis , Graham et al. report on how carbohydrate-binding modules tethered to polyethylene terephthalate (PET) hydrolases affect enzymatic plastic degradation. With their work, they stand out from previous studies performed in the low solid-loading range by considering real-world applications and concisely delineating the enzyme performance—yielding a valuable outcome for the field of enzymatic plastic degradation. In this issue of Chem Catalysis , Graham et al. report on how carbohydrate-binding modules tethered to polyethylene terephthalate (PET) hydrolases affect enzymatic plastic degradation. With their work, they stand out from previous studies performed in the low solid-loading range by considering real-world applications and concisely delineating the enzyme performance—yielding a valuable outcome for the field of enzymatic plastic degradation.

Valorization of Polyethylene Terephthalate to Muconic Acid by Engineering Pseudomonas Putida.

Plastic waste is rapidly accumulating in the environment and becoming a huge global challenge. Many studies have highlighted the role of microbial metabolic engineering for the valorization of polyethylene terephthalate (PET) waste. In this study, we proposed a new conceptual scheme for upcycling of PET. We constructed a multifunctional Pseudomonas putida KT2440 to simultaneously secrete PET hydrolase LCC, a leaf-branch compost cutinase, and synthesize muconic acid (MA) using the PET hydrolysate. The final product MA and extracellular LCC can be separated from the supernatant of the culture by ultrafiltration, and the latter was used for the next round of PET hydrolysis. A total of 0.50 g MA was produced from 1 g PET in each cycle of the whole biological processes, reaching 68% of the theoretical conversion. This new conceptual scheme for the valorization of PET waste should have advantages over existing PET upcycling schemes and provides new ideas for the utilization of other macromolecular resources that are difficult to decompose, such as lignin.

Structural insight and engineering of a plastic degrading hydrolase Ple629

Polyethylene terephthalate (PET) is one of the most abundantly produced synthetic polyesters. The vast number of waste plastics including PET has challenged the waste management sector while also posing a serious threat to the environment due to improper littering. Recently, enzymatic PET degradation has been shown to be a viable option for a circular plastic economy, which can mitigate the plastic pollution. While protein engineering studies on specific PET degradation enzymes such as leaf-branch compost cutinase (LCC), Thermobifida sp. cutinases and Ideonella sakaiensis PETase (IsPETase) have been extensively published, other homologous PET degrading enzymes have received less attention. Ple629 is a polyester hydrolase identified from marine microbial consortium having activity on PET and the bioplastic polybutylene adipate terephthalate (PBAT). In order to explore its catalytic mechanism and improve its potential for PET hydrolysis, we solved its crystal structure in complex with a PET monomer analogue, and validated its structural and mechanistic similarity to known PET hydrolases. By structural comparisons, we identified some hot spot positions described in previous research on protein engineering of PET hydrolases. We substitute these amino acid residues in Ple629, and obtained variants with improved activity and thermo-stability. The most promising variant D226A/S279A exhibited a more than 5.5-fold improved activity on PET nanoparticles than the wild-type enzyme, suggesting its potential applicability in the biotechnological plastic recycling.

Sequentially co-immobilized PET and MHET hydrolases via Spy chemistry in calcium phosphate nanocrystals present high-performance PET degradation

Accumulation of polyethylene terephthalate (PET) has brought an enormous threat to the ecosystem. The recently reported PET hydrolase (DuraPETase) and MHET hydrolase (MHETase) can synergistically catalyze the complete PET degradation. Hence, this work was designed to develop a bienzymatic cascade catalysis by co-immobilizing the two enzymes for PET biodegradation. DuraPETase and MHETase were sequentially co-immobilized in calcium phosphate nanocrystals (CaP) through SpyTag/SpyCatcher system. MHETase-SpyCatcher was first embedded inside the nanocrystals via biomimetic mineralization, and DuraPETase-SpyTag was then conjugated on the outlayer (~1.5 µm). The bienzyme compartmentalization facilitated DuraPETase interaction with the solid substrate, and the layered structures of the nanocrystals protected the enzymes, thus enhancing their stability. The high specific surface area of the nanocrystals and the proximity effects from the bienzymatic cascade were beneficial to the improved enzyme activity. Experimental data and molecular dynamics simulations revealed the activation effect of Ca2+ on DuraPETase. Taken together, the final results indicate that the PET degradation efficiency of DuraPETase-MHETase@CaP increased by 6.1 and 1.5 times over the free bienzyme system within 10 d at 40 °C and 50 °C, with weight losses at 32.2% and 50.3%, respectively. The bienzymatic cascade with DuraPETase-MHETase@CaP can completely degrade PET, contributing to the recycling of PET.

Discovery and Genetic Code Expansion of a Polyethylene Terephthalate (PET) Hydrolase from the Human Saliva Metagenome for the Degradation and Bio‐Functionalization of PET

AbstractWe report a bioinformatic workflow and subsequent discovery of a new polyethylene terephthalate (PET) hydrolase, which we named MG8, from the human saliva metagenome. MG8 has robust PET plastic degradation activities under different temperature and salinity conditions, outperforming several naturally occurring and engineered hydrolases in degrading PET. Moreover, we genetically encoded 2,3‐diaminopropionic acid (DAP) in place of the catalytic serine residue of MG8, thereby converting a PET hydrolase into a covalent binder for bio‐functionalization of PET. We show that MG8(DAP), in conjunction with a split green fluorescent protein system, can be used to attach protein cargos to PET as well as other polyester plastics. The discovery of a highly active PET hydrolase from the human metagenome—currently an underexplored resource for industrial enzyme discovery—as well as the repurposing of such an enzyme into a plastic functionalization tool, should facilitate ongoing efforts to degrade and maximize reusability of PET.

Discovery and Genetic Code Expansion of a Polyethylene Terephthalate (PET) Hydrolase from the Human Saliva Metagenome for the Degradation and Bio-Functionalization of PET.

We report a bioinformatic workflow and subsequent discovery of a new polyethylene terephthalate (PET) hydrolase, which we named MG8, from the human saliva metagenome. MG8 has robust PET plastic degradation activities under different temperature and salinity conditions, outperforming several naturally occurring and engineered hydrolases in degrading PET. Moreover, we genetically encoded 2,3-diaminopropionic acid (DAP) in place of the catalytic serine residue of MG8, thereby converting a PET hydrolase into a covalent binder for bio-functionalization of PET. We show that MG8(DAP), in conjunction with a split green fluorescent protein system, can be used to attach protein cargos to PET as well as other polyester plastics. The discovery of a highly active PET hydrolase from the human metagenome-currently an underexplored resource for industrial enzyme discovery-as well as the repurposing of such an enzyme into a plastic functionalization tool, should facilitate ongoing efforts to degrade and maximize reusability of PET.

Towards synthetic PETtrophy: Engineering Pseudomonas putida for concurrent polyethylene terephthalate (PET) monomer metabolism and PET hydrolase expression

BackgroundBiocatalysis offers a promising path for plastic waste management and valorization, especially for hydrolysable plastics such as polyethylene terephthalate (PET). Microbial whole-cell biocatalysts for simultaneous PET degradation and growth on PET monomers would offer a one-step solution toward PET recycling or upcycling. We set out to engineer the industry-proven bacterium Pseudomonas putida for (i) metabolism of PET monomers as sole carbon sources, and (ii) efficient extracellular expression of PET hydrolases. We pursued this approach for both PET and the related polyester polybutylene adipate co-terephthalate (PBAT), aiming to learn about the determinants and potential applications of bacterial polyester-degrading biocatalysts.ResultsP. putida was engineered to metabolize the PET and PBAT monomer terephthalic acid (TA) through genomic integration of four tphII operon genes from Comamonas sp. E6. Efficient cellular TA uptake was enabled by a point mutation in the native P. putida membrane transporter MhpT. Metabolism of the PET and PBAT monomers ethylene glycol and 1,4-butanediol was achieved through adaptive laboratory evolution. We then used fast design-build-test-learn cycles to engineer extracellular PET hydrolase expression, including tests of (i) the three PET hydrolases LCC, HiC, and IsPETase; (ii) genomic versus plasmid-based expression, using expression plasmids with high, medium, and low cellular copy number; (iii) three different promoter systems; (iv) three membrane anchor proteins for PET hydrolase cell surface display; and (v) a 30-mer signal peptide library for PET hydrolase secretion. PET hydrolase surface display and secretion was successfully engineered but often resulted in host cell fitness costs, which could be mitigated by promoter choice and altering construct copy number. Plastic biodegradation assays with the best PET hydrolase expression constructs genomically integrated into our monomer-metabolizing P. putida strains resulted in various degrees of plastic depolymerization, although self-sustaining bacterial growth remained elusive.ConclusionOur results show that balancing extracellular PET hydrolase expression with cellular fitness under nutrient-limiting conditions is a challenge. The precise knowledge of such bottlenecks, together with the vast array of PET hydrolase expression tools generated and tested here, may serve as a baseline for future efforts to engineer P. putida or other bacterial hosts towards becoming efficient whole-cell polyester-degrading biocatalysts.

Polyesterase activities in bacterial isolates from seaweed and sponges, with potential utility in polyethylene terephthalate plastic and nanoparticle hydrolysis

Many marine bacteria have evolved to produce a range of extracellular enzymes which facilitate their growth and survival in the harsh, oligotrophic conditions often present in marine environments. Marine sponge derived Streptomycesstrains have previously been reported to produce polyesterase enzymes, which are of interest for several biotechnological applications, including polyethylene terephthalate (PET) plastic hydrolysis. Bacteria isolated from sponges and seaweed were screened for polyester hydrolysis activities using plate-clearing assays. Lipolytic and polyesterolytic activities were initially identified by employing tributyrin and polycaprolactone diol agar-based assay systems, respectively. Polyesterase activity was subsequently confirmed on both polycaprolactone and on PET-nanoparticle agar plates, resulting in the prioritisation of six isolates for Illumina next-generation genome sequencing. These include three Bacillusspp., isolated from the brown seaweed Ascophyllum nodosum,and from marine lake sponges Stelligera stuposaand Eurypon major, together with a Maribacterstrain again isolated from S. stuposa, and Brachybacteriumsp. and Micrococcussp. isolates of deep-sea sponges Pheronemasp. and Inflatella pellicula, that were sampled at depths of 2129m and 2900m, respectively. Genome mining and comparative genomic analysis of these isolates is currently underway to identify genes encoding the observed activities and to assess homology with known PET hydrolases. Microbes found living in association with filter-feeding sponges may have increased exposure to the plastics and microplastics that widely contaminate our marine ecosystems, thus representing a promising source of degradative activities towards synthetic polymers that could contribute to new plastic waste management strategies.