Nowadays, plastic pollution has been severe. Firstly, the large amount of plastic use contributed to the pollution. According to research, more than 400 million tons of plastic are produced globally each year, one-third of which is used in single-use products. These plastics are difficult to degrade, causing environmental problems (Vidal, et al; 2024). One of the main environmental problem caused was the pollution to river and oceans. The plastic traps animals, affecting their health by making them hurt. Moreover, animals such as sea turtles would mistake the plastics as food like jelly fish, causing death and harm. Additionally, plastic pollution could severely affect human health. The plastic could not degrade completely, and turn into microplastics that may exist in the food of human, causing sickness and harm (Thompson et al; 2004), . Another problem is the decomposition of plastic nowadays release large amounts of greenhouse gases, contributing overall climate change and carbon emissions. This affects the ecological system severely (Stubbins,et al; 2021).
Figure 1.PET plastic (Recycled Rpet Pellets Pet Recycle Pellet Pet Granules Iv 0.78 - Online Shopping)
Common plastic types include polyethylene (PE), which are used for packaging film, plastic bags, water pipes , polypropylene (PP) which are used in tableware, bottle caps, auto parts, polystyrene (PS) mainly used in transparent packaging materials, disposable tableware, polyvinyl chloride (PVC), which are used for wire and cable, pipeline, building materials, polyamide (nylon, PA) mainly used in clothing, rope, gear, polycarbonate (PC) used for eye lenses, water bottles, etc. PET is a synthetic polymer commonly used in beverage bottles, food packaging, and textiles, contributing significantly to environmental pollution. The decomposition of PET mainly includes physical means such as mechanical recycling, but it is difficult to work on microplastics, chemical degradation such as high temperature decomposition or chemical catalysis, high cost and high energy demand and biodegradation such as the use of bacteria or enzymes to degrade plastics, with environmental friendliness, but the activity of existing plastic degradation enzymes is low, difficult to practical application (Law,et al; 2025).
In a groundbreaking study published in 2016, researchers led by Yoshida from the Osaka Institute of Technology in Japan reported the isolation of a novel bacterium, Ideonella sakaiensis, from microbial communities in a Japanese landfill. This bacterium demonstrated a unique capacity to degrade polyethylene terephthalate (PET) as its primary carbon and energy source. The study identified and characterized two key enzymes responsible for this activity: PETase and MHETase. This discovery provided a transformative advance in the field of plastic biodegradation research((Yoshida etal,2026). Polyethylene terephthalate hydrolase (PETase) can catalyze the decomposition of polyethylene terephthalate (PET) into its monomer, mono(2-hydroxyethyl) terephthalate (MHET). Meanwhile, MHETase (mono(2-hydroxyethyl) terephthalate hydrolase) can further catalyze the breakdown of MHET into ethylene glycol and terephthalic acid.
Figure 2. Degradation process of PET plastic and key enzymes(Yoshida et al, 2016).
Subsequently, researchers have employed structural biology and enzyme engineering approaches to conduct directed evolution and modification of PETase to enhance its degradation efficiency and thermal stability. For instance, in 2020, a research team from the University of Portsmouth in the UK significantly improved the enzyme's degradation performance through mutagenesis of PETase (Austin etal,2018). In recent years, Chinese research teams have also made a series of advancements in plastic-degrading enzyme research. For example, the BGI Life Science Research Institute and other institutions analyzed and deeply mined publicly available metagenomic data of marine microorganisms, discovering three novel saline-adapted PET-degrading enzymes derived from deep-sea environments. These enzymes achieved an 83% degradation rate of PET films within three days, exhibiting 44 times the activity of the reported IsPETase plastic-degrading enzyme (Austin etal,2018).
Currently, research on highly active novel saline-adapted PET-degrading enzymes is very limited. We aim to screen and engineer highly efficient microbial-derived plastic-degrading enzymes by synthetic biology to construct specifically mutated enzyme genes, enhance their degradation efficiency, and validate their practical effectiveness in plastic degradation, thereby providing innovative solutions for the bioremediation of plastic pollution.
Our primary objective is to enhance the efficiency of PET degradation by screening and engineering high-performance plastic-degrading enzymes derived from microorganisms. The overall approach follows the Design-Build-Test-Learn (DBTL) logical framework of synthetic biology.
1. 1 Screening candidate genes for plastic degradation
We screened public databases such as NCBI to identify key enzymes with PET-degrading capabilities, specifically polyethylene terephthalate hydrolase (PETase) and mono(2-hydroxyethyl) terephthalate hydrolase (MHETase).Therefore, we selected the PETase gene from Ideonella sakaiensis, which is currently the most widely used. This bacterium, capable of degrading PET plastic, was discovered in 2016 by Yoshida et al. from the Osaka Institute of Technology in Japan. In natural environments, PETase degrades polyethylene terephthalate (PET) into the monomeric intermediate MHET (mono(2-hydroxyethyl) terephthalate)>. Additionally, the dsPETase05 gene is a novel highly active, halophilic PET-degrading enzyme derived from deep-sea sources. It achieves an 83% degradation rate of PET film within 3 days, which is 44 times higher than the reported activity of Is PETase (Austin etal,2018).. Thus, we aim to further enhance the enzymatic activity and degradation efficiency of dsPETase05 using synthetic biology approaches.
The MHETase gene, sourced from Ideonella sakaiensis, further hydrolyzes MHET into terephthalic acid (TPA) and ethylene glycol (EG). Working synergistically with PETase, MHETase enables complete PET degradation (Figure 3).
Figure 3. Technical Roadmap
1.2 Sequence Alignment and Structural Analysis
Using AlphaFold for protein structure prediction, we analyzed the three-dimensional structure of dsPETase05. Molecular docking was then employed to evaluate its active site and identify key residues involved in PET binding (Figure 3).
1.3 Prediction of Mutation Sites
Objective: to enhance enzyme-substrate affinity and catalytic activity through site-directed mutagenesis of key residues.
Based on expert recommendations from Professor Yao, we performed site-directed mutagenesis on two critical residues:
Glycine 8 (Gly8): Mutated to polar, neutral amino acids—glutamine (Gln), serine (Ser), threonine (Thr), cysteine (Cys), asparagine(Asn), and tyrosine (Tyr)—to minimize structural disruption while preserving activity.
Arginine 47 (Arg47): Mutated to other basic residues—lysine (Lys) and histidine (His)—to maintain positive charge and potential catalytic interactions.
The resulting mutants were designated as:
- Arg47-Lys, Arg47-His
- Gly8-Gln, Gly8-Asn, Gly8-Cys, Gly8-Ser, Gly8-Thr, Gly8-Tyr
Subsequently, molecular docking was performed using AutoDock software to predict the binding interactions between these mutants and the small molecule polyethylene terephthalate (PET; CAS: 25038-59-9). The study aimed to:
- Identify potential changes in binding sites of the eight mutants.
- Analyze alterations in amino acid interaction sites and binding efficiency with PET.
2.1 Gene Optimization and Integration
Objective: To construct a co-expression system combining the optimized dsPETase and MHETase, enabling synergistic PET degradation.
Approach:
- Design a multi-enzyme co-expression vector to validate the system's overall efficiency in PET degradation.
- Leverage the complementary functions of PETase (cleaves PET into MHET) and MHETase (hydrolyzes MHET into terephthalic acid, TPA and ethylene glycol, EG), achieving complete PET depolymerization.
Expected Outcome:
- Enhanced degradation efficiency through enzymatic synergy.
- Full conversion of PET to monomeric products (TPA + EG).
2.2 Selection of Target Vectors and Strains
2.2.1 Escherichia coli DH5α
Purpose: Plasmid construction (e.g., pET-series vectors).
Advantages:
- High-copy-number plasmid replication: Enables rapid plasmid amplification.
- Fast growth: Achieves high cell density in a short time, facilitating large-scale plasmid production.
- Cost-effective: Simple and inexpensive culture conditions.
2.2.2 Escherichia coli BL21(DE3)
Purpose: Recombinant protein expression
Advantages:
- High-efficiency expression system: Compatible with T7 promoter-driven vectors (e.g., pET-series).
- Fast growth: Suitable for high-density fermentation and large-scale protein production.
- Cost-effective: Minimal media requirements and low operational costs.
Biosafety: Both E. coli DH5α and BL21(DE3) are classified as Risk Group 1 (RG1) organisms, ensuring environmental safety.
2.2.3 Expression Vector
Selected vector: pET-28a(+)
Features:
T7 promoter for strong, inducible expression in BL21(DE3).
N-terminal His-tag for simplified protein purification (Ni-NTA affinity chromatography) and detection (Western blot/ELISA).
Plasmid Construction:
- Homologous recombination to clone target genes (optimized dsPETase and MHETase) into pET-28a(+).
- Transform ligated plasmids into DH5α for amplification and verification.
Protein Expression:
- Transfer confirmed plasmids into BL21(DE3) for expression.
- Optimize induction conditions using IPTG (varying concentrations, e.g., 0.1–1.0 mM).
Protein Purification & Analysis:
- Purify His-tagged proteins via Ni-NTA chromatography.
- Validate expression by SDS-PAGE.
Enzyme Activity Assays:
- Quantitative analysis: Measure MHET degradation efficiency via HPLC (monomer release: TPA/EG).
- Compare mutant variants (*Arg47-Lys, Arg47-His, Gly8-Gln*, etc.) to identify the highest-activity mutant.
Synergistic Degradation:
- Qualitative analysis: Visualize PET surface degradation using scanning electron microscopy (SEM).
- Combine the best-performing dsPETase mutant with MHETase to achieve complete PET depolymerization (PET → MHET → TPA + EG).
Based on engineered plastic-degrading enzymes capable of efficiently breaking down specific plastics (such as PET), this technology can be applied in plastic pollution treatment or industrial degradation processes. It can provide data support for environmental protection and greening the Earth, while calling on everyone to protect the planet.
Austin, H. P., Allen, M. D., Donohoe, B. S., Rorrer, N. A., Kearns, F. L., Silveira, R. L., Pollard, B. C., Dominick, G., Duman, R., El Omari, K., Mykhaylyk, V., Wagner, A., Michener, W. E., Amore, A., Skaf, M. S., Crowley, M. F., Thorne, A. W., Johnson, C. W., Woodcock, H. L., McGeehan, J. E., & Beckham, G. T. (2018). Characterization and engineering of a plastic-degrading aromatic polyesterase. Proceedings of the National Academy of Sciences, *115*(19), E4350–E4357. https://doi.org/10.1073/pnas.1718804115
Global marine microbial diversity and its potential in bioprospecting. (2024). Nature, *633*(8029), 371–379. https://doi.org/10.1038/s41586-024-07891-2
Law, K. L., & Narayan, R. (2022). Reducing environmental plastic pollution by designing polymer materials for managed end-of-life. Nature Reviews Materials, *7*(2), 104–116. https://doi.org/10.1038/s41578-021-00382-0 (Note: Adjusted year and added volume/issue based on journal info)
Thompson, R. C., Olsen, Y., Mitchell, R. P., Davis, A., Rowland, S. J., John, A. W., McGonigle, D., & Russell, A. E. (2004). Lost at sea: where is all the plastic? Science, *304*(5672), 838. https://doi.org/10.1126/science.1094559
Stubbins, A., Law, K. L., Muñoz, S. E., Bianchi, T. S., & Zhu, L. (2021). Plastics in the Earth system. Science, *373*(6550), 51–55. https://doi.org/10.1126/science.abb0354
Vidal, F., van der Marel, E. R., Kerr, R. W. F., Rotta, J., Olvera, A. G., ... & McGlade, J. (2024). Designing a circular carbon and plastics economy for a sustainable future. Nature, *626*(7997), 45–57. https://doi.org/10.1038/s41586-023-06939-z
Yoshida, S., Hiraga, K., Takehana, T., Taniguchi, I., Yamaji, H., Maeda, Y., Toyohara, K., Miyamoto, K., Kimura, Y., & Oda, K. (2016). A bacterium that degrades and assimilates poly(ethylene terephthalate). Science, *351*(6278), 1196–1199. https://doi.org/10.1126/science.aad6359 (Note: Unified the citation, the DOI in the original list points to a different page but the correct one is for the article)
Tournier, V., Topham, C. M., Gilles, A., David, B., Folgoas, C., Moya-Leclair, E., Kamionka, E., Desrousseaux, M. L., Texier, H., Gavalda, S., Cot, M., Guémard, E., Dalibey, M., Nomme, J., Cioci, G., Barbe, S., Chateau, M., André, I., Duquesne, S., & Marty, A. (2020). An engineered PET depolymerase to break down and recycle plastic bottles. Nature, *580*(7802), 216–219. https://doi.org/10.1038/s41586-020-2149-4 (Note: Added this key paper often cited in this context)