Overview
Polyester plastics such as PET, PBAT, and PLA are produced on a massive scale, with China alone exceeding 100 million tons in 2024. Once released into the environment—especially the ocean—these materials are highly resistant to degradation and eventually fragment into persistent microplastics, posing serious risks to ecosystems and human health.
To address this challenge, POLYGONE, aims to develop a marine-derived ester hydrolase that is naturally adapted to the harsh oceanic environment of low temperature, high salinity, and high pressure. Such enzymes provide the catalytic activity and stability required for efficient degradation in marine polluted environments.
With synthetic biology, we designed a two-way strategy that integrates source-level biodegradability with end-of-pipeline microplastic management.
This enzyme was further immobilized on nano-calcium carbonate-silica composites, enabling direct incorporation into polyester production to create plastics that degrade naturally in marine conditions—addressing pollution at its source.
At the end-of-life stage, we designed an E. coli-based co-surface display system capable of enriching and degrading microplastics while detecting their degradation product TPA. This provides a practical platform for long-term monitoring and remediation.
At the source, the optimized marine enzyme is immobilized on nano-calcium carbonate-silica composites, enabling incorporation into polyester production and conferring natural degradability to plastics. At the end-of-life stage, we constructed an E. coli-based co-surface display system to enrich and degrade microplastics while detecting the degradation product terephthalic acid (TPA).
By integrating source-level design with downstream management, our project delivers a scalable and sustainable solution for the full life cycle of polyester plastics, contributing to cleaner oceans and a healthier planet.
Chassis strain
For our project, we mainly employed two commonly used E. coli laboratory strains: BL21 (DE3) and DH5 α, each fulfilling complementary roles.
BL21 (DE3) was chosen as the primary strain for protein expression. This strain is one of the most widely used hosts for heterologous protein expression due to its high efficiency, genetic stability, and well-defined physiological properties. Another key factor in our selection of this strain was the maturity of E. coli cell surface display technology. Many anchoring motifs, such as INPNC and Lpp-OmpA, used in this project, have been extensively studied and validated, enabling reliable fusion and presentation of target proteins on the cell membrane for efficient construction of whole-cell catalysts.
DH5 α on the other hand, was employed for molecular cloning and plasmid amplification. This strain is optimized for high transformation efficiency, stable plasmid maintenance, and reduced recombination, making it the standard choice for DNA assembly and storage.
By relying on DH5 α for cloning tasks and BL21 (DE3) for protein expression, we ensured a clear division of labor between chassis strains that enhanced both workflow efficiency and experimental reproducibility.
Strategy 1: Enzyme Mining
To discover highly active and stable polyester-degrading enzymes in marine environments, we planned to establish a multi-step de novo enzyme discovery process:
1. Template Selection and BLAST Search
In the enzyme mining stage, we selected Thermobifida fusca cutinase (TfCut) as our reference template. TfCut is one of the earliest and most extensively studied polyester hydrolases, with proven activity against aliphatic–aromatic polyesters such as PBAT and PET. Structurally, it adopts the canonical α/β-hydrolase fold with a conserved Ser–His–Asp catalytic triad, and its high-resolution crystal structure provides reliable information for homology modeling and rational engineering. These characteristics make TfCut not only a historically important enzyme but also an ideal benchmark for guiding our mining pipeline.
Building on this benchmark, we carried out homology searches using NCBI BLAST to identify novel polyester hydrolases with potential for marine application. To ensure the reliability of our dataset, we applied strict thresholds for sequence identity (≥35%), query coverage (≥50%), and statistical significance (E-value ≤1.0E-58). To further refine the pool, we focused on sequences with identity ≥40% and moderate coverage (35–60%), effectively removing incomplete or irrelevant entries while retaining candidates most likely to share both structural and functional features with TfCut.
2. Sequence Filtering
After obtaining initial BLAST hits, we designed a multi-layered screening strategy to progressively refine the candidate set. Multiple sequence alignment (MSA) using MAFFT L-INS-i ensured high alignment accuracy among candidate sequences and confirmed the conservation of the canonical Ser-His-Asp catalytic triad and other motifs critical for α/β-hydrolase activity. Next, we constructed a phylogenetic tree to prioritize marine-derived enzymes that naturally adapt to conditions such as low temperature, high salinity, and high pressure. Solubility predictions were performed using multiple independent online tools, and the results were cross-referenced to minimize prediction bias and retain sequences with high soluble expression potential. The results were further subjected to structural modeling, substrate docking, and molecular dynamics (MD) simulations to identify high-performing hydrolases.
This multi-step computational filtering process allowed us to whittle the large initial BLAST pool down to a few high-confidence candidate sequences with broad substrate specificity, high predicted stability, and expression feasibility.
3. Experimental Verification and Heterologous Expression
To validate the functionality of our mined enzyme candidates, we cloned the selected sequences into the pET-28a(+) vector with an N-terminal His-Tag and expressed them in E. coli BL21(DE3), a well-established expression host with a clear genetic background. The recombinant proteins were purified using nickel affinity chromatography and their polyester hydrolysis activity was tested using PET nanosheets of different crystallinities (e.g. LC-PET and HC-PET). Enzyme performance was evaluated by monitoring the accumulation of terephthalic acid (TPA), detected at 240 nm (A240).
For enzymes that showed promising activity in these assays, we conducted systematic biochemical characterizations, including determination of optimal temperature, Tm, and salt tolerance. These results provided the foundation for further protein engineering and application-oriented design.
4. Computer-Assisted Protein Redesign to Improve Solubility
Heterologous expression of heterologous proteins in E. coli often results in low expression levels, so we anticipated challenges in their soluble expression in E. coli. This is often due to factors such as discrepancies in codon usage, mRNA secondary structure that hinders translation, and inefficient folding.
To address this bottleneck, we employed ProteinMPNN, a deep learning–based sequence design tool. By preserving the enzyme’s three-dimensional backbone and catalytic center conformation, ProteinMPNN enables global amino acid sequence optimization aimed at improving folding efficiency and solubility.
Predicted variants were evaluated through structural alignment and solubility prediction, and the best candidates were cloned into pET-28a(+) and expressed in BL21(DE3). Variants with improved solubility were further characterized for reaction temperature, Tm, and salt tolerance, ensuring they remained functional while gaining better expression properties.
5. Consensus-Based Rational Design to Enhance Stability
Beyond solubility, stability is another critical characteristic of industrially immobilized enzymes. Immobilization often exposes enzymes to environments such as high temperatures, which place higher demands on their intrinsic stability.
To meet this requirement, we designed a consensus-based rational design strategy, integrating three different stability prediction tools: PROSS (global scaffold optimization), FireProt (integration of evolutionary and energetic calculations), and PoPMuSiC (machine learning and force field evaluation).
By intersecting the results from these tools, we identified high-confidence mutations and tested them via single-point mutagenesis. The resulting variants were experimentally evaluated for activity and stability. This strategy enabled us to efficiently and systematically screen for engineered variants that are more stable and suitable for application.
Strategy 2: Enzyme immobilization and activity testing
To ensure that our engineered polyester hydrolase can be directly applied under industrial and environmental conditions, we developed a strategy based on enzyme immobilization and functional validation:
1. Composite Carrier Immobilization
Free enzymes are easy to denaturation under heat or in the presence of organic solvents, which severely limits their industrial applications. To overcome this limitation, we employed immobilization techniques to provide a stable microenvironment for the enzymes. Among various methods (adsorption, covalent binding, entrapment), we selected a sol–gel entrapment strategy, which confines enzymes within the pores of an inorganic network. This configuration prevents enzyme leakage while maintaining sufficient diffusion channels for substrates and products.
For carrier design, we proposed a CaCO₃–SiO₂ composite. Calcium carbonate offers low cost, wide availability, and excellent biocompatibility, making it an ideal additive for polyester plastics. However, single-component carriers often fail to meet all requirements. By combining CaCO₃ with silica—known for its high surface area and and chemical stability—we created a composite carrier that not only provides a mild microenvironment for enzymes but also withstands the shear stress encountered during polymer processing. This design leverages the complementary properties of both materials, achieving a synergistic effect for improved immobilized enzyme stability.
2. Film Preparation and Distribution Analysis
To test the performance of the immobilized enzymes in functional materials, we incorporated them into different polyester matrices, including PBAT and PBAT/PLA (1:1 blend), and processed them into thin films.
The distribution of immobilized enzymes within the films was evaluated using confocal laser scanning microscopy (CLSM), with enzymes pre-labeled by FITC fluorescence. This allowed us to visually and quantitatively assess the uniformity of enzyme dispersion in the polymer matrix.
To confirm that enzyme incorporation did not compromise the integrity of the material, we conducted mechanical strength tests (tensile strength and elongation at break). These ensured that the films retained sufficient mechanical performance while gaining biodegradation capability, thereby demonstrating the feasibility of our material design.
3. Marine Degradation Simulation
Laboratory degradation tests often fail to capture the complexity of marine environments, which involve varying salinity, microbial communities, temperature fluctuations, and water currents. To provide more realistic validation, we established a marine-simulating degradation system.
We use real seawater collected 10 km offshore as the degradation medium, preserving the natural microbial and chemical composition. Thin-film samples were incubated in aquaria under gentle shaking to mimic ocean currents and maintained at ambient temperature.
Degradation was evaluated through both macroscopic observations (surface cracks, fragmentation) and microscopic analysis using scanning electron microscopy (SEM), which revealed erosion patterns and pore formation at the polymer surface. Films without enzymes served as controls, ensuring that observed effects could be attributed to enzymatic activity rather than abiotic or microbial factors.
Strategy 3: A monitoring platform derive from an enzyme-derived product
Strategy 3: Integrated enzymatic degradation and monitoring platform
To achieve targeted removal and quantitative evaluation of polyester microplastics in aquatic environments, we designed an integrated enzymatic degradation and monitoring platform.
1. Surface Co-display System
Traditional enzyme-based degradation often faces limitations such as high purification cost, rapid inactivation, and difficulty in recovery. To overcome these issues, we employed surface display technology, enabling enzymatic degradation and monitoring platform that are stable, affordable, and recyclable.
However, single-anchor surface display systems often suffer from low loading efficiency and enzyme detachment in complex aquatic environments. To address this, we want to design a dual-anchoring system:
INPNC–mfp-3: Displays adhesion protein mfp-3 , enabling specific polyester microplastic binding and local enrichment of substrates.
Lpp-OmpA–SpyCatcher: Displays SpyCatcher on the cell surface, which covalently binds SpyTag-fused polyester hydrolases, ensuring stable and efficient enzyme loading.
This dual anchoring strategy minimizes spatial competition,enables efficient co-display, enhances local enzyme density, and significantly improves microplastic degradation efficiency compared with single-display systems.
Plasmid & Promoter Design
To ensure robust co-expression of both anchors while avoiding competition, we adopted a dual-plasmid system:
INPNC–mfp-3 was cloned into pETlac, with the original lac promoter replaced by the strong constitutive promoter lpp1.2, achieving spontaneous, high-level expression without induction.
Lpp-OmpA–SpyCatcher was cloned into pET-28a(+), allowing independent expression in E. coli BL21(DE3).
SpyTag–hydrolase was cloned into pET-28a(+) with an N-terminal His-tag, enabling soluble expression, purification, and subsequent covalent binding to surface SpyCatcher.
This design provides dual antibiotic markers for reliable screening, ensures balanced expression, aviods competition between two enzymes expression, and improves both stability and efficiency of the surface display system.
2. Surface Display and Functional Validation
INPNC-mCherry and sfGFP-SpyTag plasmids were constructed and expressed as fusion proteins. Successful co-display was determined by observing the colocalization of red and green signals on the cell surface using fluorescence microscopy and confocal imaging.
The mfp-3 adhesion activity assay can be evaluated by quantifying bacterial binding to microplastic particles. The synergistic effect of the dual-anchor system can be assessed by comparing TPA release between strains carrying only the SpyCatcher-SpyTag hydrolase and those carrying the complete dual-anchor system.
3. Microplastic Degradation and Product-Based Monitoring
Terephthalic acid (TPA) is a characteristic degradation product of PET and PBAT. It exhibits very low background levels in natural environments and possesses a unique aromatic structure that reacts with hydrogen peroxide to generate a measurable fluorescent signal. Leveraging this property, we could establish a calibration curve correlating TPA concentration with fluorescence intensity, thereby developing a fluorescence-based chemical detection method, which provides an indirect yet reliable indicator linking TPA concentration to the progress of polyester degradation, and consequently to microplastic levels.
Building on this chemical analysis, we further envision the development of an engineered biosensor strain capable of responding specifically to TPA. Such a strain would allow real-time, in situ monitoring of microplastic degradation through fluorescence output, enabling continuous and dynamic assessment of microplastic levels directly in aquatic environments.