Bacterial surface display of PETase mutants and MHETase for an efficient dual-enzyme cascade catalysis.

Biological degradation of PET plastic holds great potential for plastic recycling. However, the high costs associated with preparing free enzymes for degrading PET make it unfeasible for industrial applications. Hence, we developed various cell catalysts by surface-displaying PETase mutants and MHETase using autotransporters in E. coli and P. putida. The efficiency of surface display was enhanced through modifying the host, co-expressing molecular chaperones, and evoluting the autotransporter. In strain EC9F, PET degradation rate was boosted to 3.85 mM/d, 51-fold and 23-fold increase compared to free enzyme and initial strain ED1, respectively. The reusability of cell catalyst EC9F was demonstrated with over 38 % and 30 % of its initial activity retained after 22 cycles of BHET degradation and 3 cycles of PET degradation. The highest reported PET degradation rate of 4.95 mM/d was achieved by the dual-enzyme cascade catalytic system EC9F+EM2 + R, a mixture of cell catalyst EC9F and EM2 with surfactant rhamnolipid.

A customized self-assembled synergistic biocatalyst for plastic depolymerization.

The enzymatic degradation of plastic offers a green, sustainable strategy and scalable circular carbon route for solving polyester waste. Among the earlies discovered plastic-degrading enzymes are PET hydrolase (PETase) and MHET hydrolase (MHETase), which act synergistically. To promote the adsorption of enzymes on PET surfaces, increase their robustness, and enable directly depolymerization, we designed hydrophobin HFBI fused-PETase and MHETase. A customized self-assembled synergistic biocatalyst (MC@CaZn-MOF) was further developed to promote the two-step depolymerization process. The tailored catalysts showed better adhesion to the PET surface and desirable durability, retaining over 70% relative activity after incubation at pH 8.0 and 60 °C for 120 h. Importantly, MC@CaZn-MOF could directly decompose untreated AGf-PET to generate 9.5 mM TPA with weight loss over 90%. The successful implementation of a bifunctional customized catalyst makes the large-scale biocatalytic degradation of PET feasible, contributing to polymer upcycling and environmental sustainability.

Unveiling potential PET degrading eukaryotes through in silico bioprospecting of PETases.

This study addresses the environmental problem of PET plastic through in silico bioprospecting for the identification and experimental validation of novel PET degrading eukaryotes through the in silico bioprospectingI of PETases, employing a methodology that combines Hidden Markov Models (HMMs), clustering techniques, molecular docking, and dynamic simulations. A total of 424 putative PETase sequences were identified from 219 eukaryotic organisms, highlighting six sequences with low affinity energies. The Aspergillus luchuensis sequence showed the lowest Gibbs free energy and exhibited stability at different temperatures in molecular dynamics assays. Experimental validation, through a plate clearance assay and HPLC, confirmed PETase activity in three wild-type fungal strains, with A. luchuensis showing the highest efficiency. The results obtained demonstrate the effectiveness of combining computational and experimental approaches as proof of concept to discover and validate eukaryotes with PET-degrading capabilities opening new perspectives for the sustainable management of this type of waste and contributing to its environmental mitigation.

Streamlined screening of extracellularly expressed PETase libraries for improved polyethylene terephthalate degradation.

Enzyme-mediated polyethylene terephthalate (PET) depolymerization has recently emerged as a sustainable solution for PET recycling. Towards an industrial-scale implementation of this technology, various strategies are being explored to enhance PET depolymerization (PETase) activity and improve enzyme stability, expression, and purification processes. Recently, rational engineering of a known PET hydrolase (LCC-leaf compost cutinase) has resulted in the isolation of a variant harboring four-point mutations (LCC-ICCG), presenting increased PETase activity and thermal stability. Here, we revealed the enzyme's natural extracellular expression and used it to efficiently screen error-prone genetic libraries based on LCC-ICCG for enhanced activity toward consumer-grade PET. Following multiple rounds of mutagenesis and screening, we successfully isolated variants that exhibited up to a 60% increase in PETase activity. Among other mutations, the improved variants showed a histidine to tyrosine substitution at position 218, a residue known to be involved in substrate binding and stabilization. Introducing H218Y mutation on the background of LCC-ICCG (named here LCC-ICCG/H218Y) resulted in a similar level of activity improvement. Analysis of the solved structure of LCC-ICCG/H218Y compared to other known PETases featuring different amino acids at the equivalent position suggests that H218Y substitution promotes enhanced PETase activity. The expression and screening processes developed in this study can be further used to optimize additional enzymatic parameters crucial for efficient enzymatic degradation of consumer-grade PET.

Molecular engineering of PETase for efficient PET biodegradation.

The widespread utilization of polyethylene terephthalate (PET) has caused a variety of environmental and health problems. Compared with traditional thermomechanical or chemical PET cycling, the biodegradation of PET may offer a more feasible solution. Though the PETase from Ideonalla sakaiensis (IsPETase) displays interesting PET degrading performance under mild conditions; the relatively low thermal stability of IsPETase limits its practical application. In this study, enzyme-catalysed PET degradation was investigated with the promising IsPETase mutant HotPETase (HP). On this basis, a carbohydrate-binding module from Bacillus anthracis (BaCBM) was fused to the C-terminus of HP to construct the PETase mutant (HLCB) for increased PET degradation. Furthermore, to effectively improve PET accessibility and PET-degrading activity, the truncated outer membrane hybrid protein (FadL) was used to expose PETase and BaCBM on the surface of E. coli (BL21with) to develop regenerable whole-cell biocatalysts (D-HLCB). Results showed that, among the tested small-molecular weight ester compounds (p-nitrophenyl phosphate (pNPP), p-Nitrophenyl acetate (pNPA), 4-Nitrophenyl butyrate (pNPB)), PETase displayed the highest hydrolysing activity against pNPP. HP displayed the highest catalytic activity (1.94 µM(p-NP)/min) at 50 °C and increased longevity at 40 °C. The fused BaCBM could clearly improve the catalytic performance of PETase by increasing the optimal reaction temperature and improving the thermostability. When HLCB was used for PET degradation, the yield of monomeric products (255.7 µM) was ∼25.5 % greater than that obtained after 50 h of HP-catalysed PET degradation. Moreover, the highest yield of monomeric products from the D-HLCB-mediated system reached 1.03 mM. The whole-cell catalyst D-HLCB displayed good reusability and stability and could maintain more than 54.6 % of its initial activity for nine cycles. Finally, molecular docking simulations were utilized to investigate the binding mechanism and the reaction mechanism of HLCB, which may provide theoretical evidence to further increase the PET-degrading activities of PETases through rational design. The proposed strategy and developed variants show potential for achieving complete biodegradation of PET under mild conditions.

Genetic Modifications in Bacteria for the Degradation of Synthetic Polymers: A Review.

Synthetic polymers, commonly known as plastics, are currently present in all aspects of our lives. Although they are useful, they present the problem of what to do with them after their lifespan. There are currently mechanical and chemical methods to treat plastics, but these are methods that, among other disadvantages, can be expensive in terms of energy or produce polluting gases. A more environmentally friendly alternative is recycling, although this practice is not widespread. Based on the practice of the so-called circular economy, many studies are focused on the biodegradation of these polymers by enzymes. Using enzymes is a harmless method that can also generate substances with high added value. Novel and enhanced plastic-degrading enzymes have been obtained by modifying the amino acid sequence of existing ones, especially on their active site, using a wide variety of genetic approaches. Currently, many studies focus on the common aim of achieving strains with greater hydrolytic activity toward a different range of plastic polymers. Although in most cases the depolymerization rate is improved, more research is required to develop effective biodegradation strategies for plastic recycling or upcycling. This review focuses on a compilation and discussion of the most important research outcomes carried out on microbial biotechnology to degrade and recycle plastics.

Application of an alchemical free energy method for the prediction of thermostable DuraPETase variants.

Non-equilibrium (NEQ) alchemical free energy calculations are an emerging tool for accurately predicting changes in protein folding free energy resulting from amino acid mutations. In this study, this method in combination with the Rosetta ddg monomer tool was applied to predict more thermostable variants of the polyethylene terephthalate (PET) degrading enzyme DuraPETase. The Rosetta ddg monomer tool efficiently enriched promising mutations prior to more accurate prediction by NEQ alchemical free energy calculations. The relative change in folding free energy of 96 single amino acid mutations was calculated by NEQ alchemical free energy calculation. Experimental validation of ten of the highest scoring variants identified two mutations (DuraPETaseS61M and DuraPETaseS223Y) that increased the melting temperature (Tm) of the enzyme by up to 1 °C. The calculated relative change in folding free energy showed an excellent correlation with experimentally determined Tm resulting in a Pearson's correlation coefficient of r = - 0.84. Limitations in the prediction of strongly stabilizing mutations were, however, encountered and are discussed. Despite these challenges, this study demonstrates the practical applicability of NEQ alchemical free energy calculations in prospective enzyme engineering projects. KEY POINTS: • Rosetta ddg monomer enriches stabilizing mutations in a library of DuraPETase variants • NEQ free energy calculations accurately predict changes in Tm of DuraPETase • The DuraPETase variants S223Y, S42M, and S61M have increased Tm.

Enhancing PET Degrading Enzymes: A Combinatory Approach.

Plastic waste has become a substantial environmental issue. A potential strategy to mitigate this problem is to use enzymatic hydrolysis of plastics to depolymerize post-consumer waste and allow it to be reused. Over the last few decades, the use of enzymatic PET-degrading enzymes has shown promise as a great solution for creating a circular plastic waste economy. PsPETase from Piscinibacter sakaiensis has been identified as an enzyme with tremendous potential for such applications. But to improve its efficiency, enzyme engineering has been applied aiming at enhancing its thermal stability, enzymatic activity, and ease of production. Here, we combine different strategies such as structure-based rational design, ancestral sequence reconstruction and machine learning to engineer a more highly active Combi-PETase variant with a melting temperature of 70 °C and optimal performance at 60 °C. Furthermore, this study demonstrates that these approaches, commonly used in other works of enzyme engineering, are most effective when utilized in combination, enabling the improvement of enzymes for industrial applications.

Molecular Insights into the Enhanced Activity and/or Thermostability of PET Hydrolase by D186 Mutations.

PETase exhibits a high degradation activity for polyethylene terephthalate (PET) plastic under moderate temperatures. However, the effect of non-active site residues in the second shell of PETase on the catalytic performance remains unclear. Herein, we proposed a crystal structure- and sequence-based strategy to identify the key non-active site residue. D186 in the second shell of PETase was found to be capable of modulating the enzyme activity and stability. The most active PETaseD186N improved both the activity and thermostability with an increase in Tm by 8.89 °C. The PET degradation product concentrations were 1.86 and 3.69 times higher than those obtained with PETaseWT at 30 and 40 °C, respectively. The most stable PETaseD186V showed an increase in Tm of 12.91 °C over PETaseWT. Molecular dynamics (MD) simulations revealed that the D186 mutations could elevate the substrate binding free energy and change substrate binding mode, and/or rigidify the flexible Loop 10, and lock Loop 10 and Helix 6 by hydrogen bonding, leading to the enhanced activity and/or thermostability of PETase variants. This work unraveled the contribution of the key second-shell residue in PETase in influencing the enzyme activity and stability, which would benefit in the rational design of efficient and thermostable PETase.

New combined absorption/1H NMR method for qualitative and quantitative analysis of PET degradation products.

Poly(ethylene terephthalate) (PET) is a very valuable and beneficial material for industrial purposes, with various different applications. Due to the high annual production volume of over 50 million tons worldwide and the indiscriminate disposal by consumers, the polymers accumulate in the environment, causing negative effects on various ecosystems. Biodegradation via suitable enzymes represents a promising approach to combat the plastic waste issue so validated methods are required to measure the efficiency and efficacy of these enzymes. PETase and MHETase from Ideonella sakaiensis are suitable enzymes needed in combination to completely degrade PET into its environmentally friendly monomers. In this project, we compare and combine a previously described bulk absorbance measurement method with a newly established 1H NMR analysis method of the PET degradation products mono(2-hydroxyethyl) terephthalic acid, bis(2-hydroxyethyl) terephthalic acid and terephthalic acid. Both were optimized regarding different solvents, pH values and drying processes. The accuracy of the measurements can be confirmed with sensitivity limits of 2.5-5 µM for the absorption method and 5-10 µM for the 1H NMR analysis. The combination of the described methods therefore allows a quantitative analysis by using bulk absorption coupled with a qualitative analysis through 1H NMR. The methods established in our work can potentially contribute to the development of suitable recycling strategies of PET using recombinant enzymes.

Assessment of Kaistella jeonii esterase conformational dynamics in response to poly(ethylene terephthalate) binding.

The pervasive presence of plastic in the environment has reached a concerning scale, being identified in many ecosystems. Bioremediation is the cheapest and most eco-friendly alternative to remove this polymer from affected areas. Recent work described that a novel cold-active esterase enzyme extracted from the bacteria Kaistella jeonii could promiscuously degrade PET. Compared to the well-known PETase from Ideonella sakaiensis, this novel esterase presents a low sequence identity yet has a remarkably similar folding. However, enzymatic assays demonstrated a lower catalytic efficiency. In this work, we employed a strict computational approach to investigate the binding mechanism between the esterase and PET. Understanding the underlying mechanism of binding can shed light on the evolutive mechanism of how enzymes have been evolving to degrade these artificial molecules and help develop rational engineering approaches to improve PETase-like enzymes. Our results indicate that this esterase misses a disulfide bridge, keeping the catalytic residues closer and possibly influencing its catalytic efficiency. Moreover, we describe the structural response to the interaction between enzyme and PET, indicating local and global effects. Our results aid in deepening the knowledge behind the mechanism of biological catalysis of PET degradation and as a base for the engineering of novel PETases.

Analysis of Poly(ethylene terephthalate) degradation kinetics of evolved IsPETase variants using a surface crowding model.

Poly(ethylene terephthalate) (PET) is a major plastic polymer utilized in the single-use and textile industries. The discovery of PET-degrading enzymes (PETases) has led to an increased interest in the biological recycling of PET in addition to mechanical recycling. IsPETase from Ideonella sakaiensis is a candidate catalyst, but little is understood about its structure-function relationships with regards to PET degradation. To understand the effects of mutations on IsPETase productivity, we develop a directed evolution assay to identify mutations beneficial to PET film degradation at 30 °C. IsPETase also displays enzyme concentration-dependent inhibition effects, and surface crowding has been proposed as a causal phenomenon. Based on total internal reflectance fluorescence microscopy and adsorption experiments, IsPETase is likely experiencing crowded conditions on PET films. Molecular dynamics simulations of IsPETase variants reveal a decrease in active site flexibility in free enzymes and reduced probability of productive active site formation in substrate-bound enzymes under crowding. Hence, we develop a surface crowding model to analyze the biochemical effects of three hit mutations (T116P, S238N, S290P) that enhanced ambient temperature activity and/or thermostability. We find that T116P decreases susceptibility to crowding, resulting in higher PET degradation product accumulation despite no change in intrinsic catalytic rate. In conclusion, we show that a macromolecular crowding-based biochemical model can be used to analyze the effects of mutations on properties of PETases and that crowding behavior is a major property to be targeted for enzyme engineering for improved PET degradation.

Insight on recently discovered PET polyester-degrading enzymes, thermostability and activity analyses.

The flexibility and the low production costs offered by plastics have made them crucial to society. Unfortunately, due to their resistance to biological degradation, plastics remain in the environment for an extended period of time, posing a growing risk to life on earth. Synthetic treatments of plastic waste damage the environment and may cause damage to human health. Bacterial and fungal isolates have been reported to degrade plastic polymers in a logistic safe approach with the help of their microbial cell enzymes. Recently, the bacterial strain Ideonella sakaiensis (201-F6) was discovered to break down and assimilate polyethylene terephthalate (PET) plastic via metabolic processes at 30 °C to 37 °C. PETase and MHETase enzymes help the bacterium to accomplish such tremendous action at lower temperatures than previously discovered enzymes. In addition to functioning at low temperatures, the noble bacterium's enzymes have amazing qualities over pH and PET plastic degradation, including a shorter period of degradation. It has been proven that using the enzyme PETase, this bacterium hydrolyzes the ester linkages of PET plastic, resulting in production of terephthalic acid (TPA), nontoxic compound and mono-2-hydroxyethyl (MHET), along with further depolymerization of MHET to release ethylene glycogen (EG) and terephthalic acid (TPA) by the second enzyme MHETase. Enzymatic plastic degradation has been proposed as an environmentally friendly and long-term solution to plastic waste in the environment. As a result, this review focuses on the enzymes involved in hydrolyzing PET plastic polymers, as well as some of the other microorganisms involved in plastic degradation.

Chemical synthesis of a 28 kDa full-length PET degrading enzyme ICCG by the removable backbone modification strategy.

Chemical protein synthesis offers a powerful way to access otherwise-difficult-to-obtain proteins such as mirror-image proteins. Although a large number of proteins have been chemically synthesized to date, the acquisition to proteins containing hydrophobic peptide fragments has proven challenging. Here, we describe an approach that combines the removable backbone modification strategy and the peptide hydrazide-based native chemical ligation for the chemical synthesis of a 28 kDa full-length PET degrading enzyme IGGC (a higher depolymerization efficiency of variant leaf-branch compost cutinase (LCC)) containing hydrophobic peptide segments. The synthetic ICCG exhibits the enzymatic activity and will be useful in establishing the corresponding mirror-image version of ICCG.

The effect of double (S238F/W159H) mutations on the structure and dynamics of PET degrading enzyme.

Polyethylene terephthalate (PET) is one of the highly produced synthetic polymers worldwide and had acquired attention due to its impact resistance, high clarity, and light weight. PET has become the first choice in making disposable bottles, leading to massive scales of production resulting in very high utilization across various facets of our daily life. Unfortunately, PET accumulates as waste and is highly resistant to biodegradation, thus presenting a serious threat to the ecosystem. Degradation of PET by enzymatic hydrolysis is a promising strategy to depolymerize the PET into its monomers. In recent studies, a plastic-degrading enzyme known as PETase (IsPETase) from the Ideonella sakaiensis has been identified to hydrolyze PET. The wild-type enzyme from Ideonella sp., has been engineered to improve the catalytic activity. While the IsPETase and its variants have been the subject of extensive structural and biochemical studies, the corresponding computational studies to support the improved activity of the mutant enzyme is not fully understood. In this work, we employed all-atom classical molecular dynamics simulations of the wild-type and double mutant IsPETase enzymes to investigate the underlying reason for the improved catalytic activity in the double mutant by means of structure-dynamics-function relationship. Our results show that the engineered mutations reshape the active site structure, volume, and dynamics of the protein loops which is crucial for substrate binding. We also demonstrate that addition of aromatic and hydrogen bond-forming residues near catalytic site improves binding affinity. This work will enable the rational design of mutants for enhanced PET degrading activity.Communicated by Ramaswamy H. Sarma.

Complete genome sequence of PETase type IIa-harboring Marinobacter nanhaiticus D15-8W, isolated from a South China Sea sediment.

Marinobacter nanhaiticus D15-8W is known for its ability to metabolize polycyclic aromatic hydrocarbons. Here, we report the complete circular genome sequence of this strain to be 5,336,660 bp (G + C content, 58.6%; 4,869 protein-coding sequences) with one plasmid (69,655 bp).

Expression and engineering of PET-degrading enzymes from Microbispora, Nonomuraea, and Micromonospora.

IMPORTANCE: Mismanagement of PET plastic waste significantly threatens human and environmental health. Together with the relentless increase in plastic production, plastic pollution is an issue of rising concern. In response to this challenge, scientists are investigating eco-friendly approaches, such as bioprocessing and microbial factories, to sustainably manage the growing quantity of plastic waste in our ecosystem. Industrial applicability of enzymes capable of degrading PET is limited by numerous factors, including their scarcity in nature. The objective of this study is to enhance our understanding of this group of enzymes by identifying and characterizing novel enzymes that can facilitate the breakdown of PET waste. This data will expand the enzymatic repertoire and provide valuable insights into the prerequisites for successful PET degradation.

Recent advances in the biodegradation of polyethylene terephthalate with cutinase-like enzymes.

Polyethylene terephthalate (PET) is a synthetic polymer in the polyester family. It is widely found in objects used daily, including packaging materials (such as bottles and containers), textiles (such as fibers), and even in the automotive and electronics industries. PET is known for its excellent mechanical properties, chemical resistance, and transparency. However, these features (e.g., high hydrophobicity and high molecular weight) also make PET highly resistant to degradation by wild-type microorganisms or physicochemical methods in nature, contributing to the accumulation of plastic waste in the environment. Therefore, accelerated PET recycling is becoming increasingly urgent to address the global environmental problem caused by plastic wastes and prevent plastic pollution. In addition to traditional physical cycling (e.g., pyrolysis, gasification) and chemical cycling (e.g., chemical depolymerization), biodegradation can be used, which involves breaking down organic materials into simpler compounds by microorganisms or PET-degrading enzymes. Lipases and cutinases are the two classes of enzymes that have been studied extensively for this purpose. Biodegradation of PET is an attractive approach for managing PET waste, as it can help reduce environmental pollution and promote a circular economy. During the past few years, great advances have been accomplished in PET biodegradation. In this review, current knowledge on cutinase-like PET hydrolases (such as TfCut2, Cut190, HiC, and LCC) was described in detail, including the structures, ligand-protein interactions, and rational protein engineering for improved PET-degrading performance. In particular, applications of the engineered catalysts were highlighted, such as improving the PET hydrolytic activity by constructing fusion proteins. The review is expected to provide novel insights for the biodegradation of complex polymers.

Engineering Plastic Eating Enzymes Using Structural Biology.

Plastic pollution has emerged as a significant environmental concern in recent years and has prompted the exploration of innovative biotechnological solutions to mitigate plastic's negative impact. The discovery of enzymes capable of degrading specific types of plastics holds promise as a potential solution. However, challenges with efficiency, industrial scalability, and the diverse range of the plastic waste in question, have hindered their widespread application. Structural biology provides valuable insights into the intricate interactions between enzymes and plastic materials at an atomic level, and a deeper understanding of their underlying mechanisms is essential to harness their potential to address the mounting plastic waste crisis. This review article examines the current biochemical and biophysical methods that may facilitate the development of enzymes capable of degrading polyethylene terephthalate (PET), one of the most extensively used plastics. It also discusses the challenges that must be addressed before substantial advancements can be achieved in using these enzymes as a solution to the plastic pollution problem.

Enzyme selection, optimization, and production toward biodegradation of post-consumer poly(ethylene terephthalate) at scale.

Poly(ethylene terephthalate) (PET) is one of the world's most widely used polyester plastics. Due to its chemical stability, PET is extremely difficult to hydrolyze in a natural environment. Recent discoveries in new polyester hydrolases and breakthroughs in enzyme engineering strategies have inspired enormous research on biorecycling of PET. This study summarizes our research efforts toward large-scale, efficient, and economical biodegradation of post-consumer waste PET, including PET hydrolase selection and optimization, high-yield enzyme production, and high-capacity enzymatic degradation of post-consumer waste PET. First, genes encoding PETase and MHETase from Ideonella sakaiensis and the ICCG variant of leaf-branch compost cutinase (LCCICCG ) were codon-optimized and expressed in Escherichia coli BL21(DE3) for high-yield production. To further lower the enzyme production cost, a pelB leader sequence was fused to LCCICCG so that the enzyme can be secreted into the medium to facilitate recovery. To help bind the enzyme on the hydrophobic surface of PET, a substrate-binding module in a polyhydroxyalkanoate depolymerase from Alcaligenes faecalis (PBM) was fused to the C-terminus of LCCICCG . The resulting four different LCCICCG variants (LCC, PelB-LCC, LCC-PBM, and PelB-LCC-PBM), together with PETase and MHETase, were compared for PET degradation efficiency. A fed-batch fermentation process was developed to produce the target enzymes up to 1.2 g L-1 . Finally, the best enzyme, PelB-LCC, was selected and used for the efficient degradation of 200 g L-1 recycled PET in a well-controlled, stirred-tank reactor. The results will help develop an economical and scalable biorecycling process toward a circular PET economy.