Background
Marine Microplastics and the Current Situation of Plastic Pollution
Microplastic pollution (MP) has emerged as one of the most pressing global environmental challenges. Microplastics—defined as plastic particles less than 5 mm in diameter—originate primarily from the fragmentation of larger plastic debris (macro-plastics), the shedding of synthetic fibers during textile washing, and the intentional inclusion of microbeads in cosmetics and personal care products. As human activities continue to intensify, microplastics have become ubiquitously distributed throughout marine ecosystems worldwide, from surface waters and coastal zones to deep-sea sediments, and even in remote regions such as the polar seas and isolated island environments.
Research has demonstrated that microplastics can exert adverse effects on terrestrial plants, including the induction of oxidative stress, disruption of chlorophyll structure, and photosynthetic inhibition. Given the complexity of seawater chemistry and the diverse physicochemical properties of microplastics, their interactions with co-existing pollutants may be significantly altered under high-salinity conditions. In particular, the adsorption behavior of microplastics toward various hazardous substances—such as heavy metals, persistent organic pollutants (POPs), antibiotics, and phenolic compounds—can be enhanced or modified, potentially facilitating the transport and bioaccumulation of these contaminants. This poses unforeseen risks to marine ecosystem integrity and, ultimately, to human health.
According to a 2023 study published in Nature, approximately 11 million metric tons of plastic enter the world’s oceans annually, with microplastics accounting for over 30% of this load.Plastic pollution not only poses severe threats to marine ecosystems—including physical harm to organisms and habitat degradation—but also has significant implications for global climate and the biogeochemical carbon cycle. For instance, the photodegradation and microbial breakdown of plastics in marine environments release greenhouse gases, thereby contributing to climate change. Furthermore, the financial costs associated with plastic waste cleanup and management are substantial, placing a growing burden on public resources and hindering sustainable economic development.
Overview of PET and PET-degrading enzymes
Polyethylene terephthalate (PET) is one of the most widely used plastic materials due to its excellent mechanical strength, chemical resistance, and versatility in industrial applications, especially in the production of beverage bottles, textile fibers, and packaging films. However, its persistence in natural environments, where it can take hundreds to thousands of years to degrade, has led towidespread accumulation i in marine and terrestrial ecosystems, becominga major contributor to global plastic pollution..Conventional recycling methods are limited by energy consumption, economic feasibility, and the inability to fully reclaim the polymer’s original quality, while incineration releases harmful greenhouse gases and toxic byproducts.
In recent years, researchers have discovered that certain microorganisms, such as Ideonella sakaiensis and Thermobifida fusca, can secrete specific enzymes to catalyze the breakdown of PET. Among these, PETase (PET hydrolase) and MHETase (mono(2-hydroxyethyl) terephthalate hydrolase) emerged as the most well-characterized. PETase catalyzes the hydrolysis of polyethylene terephthalate (PET), primarily yielding mono(2-hydroxyethyl) terephthalate (MHET) , while MHETase further degrades MHET into its monomeric components, terephthalic acid (TPA) and ethylene glycol (EG), which can be assimilated by the microorganisms for growth.
The identification of these enzymes has triggered a surge of interest in PET degradation enzyme research. Scientists have since undertaken extensive efforts to enhance the catalytic efficiency, substrate specificity, and environmental robustness of these enzymes.
Rationale for Selecting PET05 as a "Seed" Sequence
Problems and Goals
The current problems faced:
Despite recent advances in the development of PET (polyethylene terephthalate) degrading enzymes, the majority of these enzymes—both naturally occurring wild-type variants and engineered high-activity versions such as IsPETase and ICCG—are sourced from terrestrial environments. These enzymes have been optimized to function efficiently under relatively low salt concentration conditions, typically found in soil or freshwater habitats. However, many real-world applications, such as the degradation of plastic waste in marine environments or industrial settings with high salinity, require enzymes that can operate effectively in saline conditions. The sensitivity of these land-derived enzymes to high salt levels significantly restricts their utility in such scenarios. As a result, there is an urgent need to explore and develop PET degrading enzymes from marine or saline-adapted microorganisms that can maintain high catalytic activity in the presence of elevated salt concentrations. This would not only broaden the range of application environments but also improve the efficiency and feasibility of enzymatic plastic degradation in diverse ecological and industrial contexts.
Our train of thought：
PSI-BLAST (Position-Specific Iterated-BLAST) is a bioinformatics tool used for protein sequence alignment. It improves the identification of evolutionarily related distantly homologous proteins by iteratively searching and constructing position-specific scoring matrices (PSSMs). Compared to basic BLAST, PSI-BLAST offers higher sensitivity in functional annotation, structural prediction, and evolutionary analysis, especially in identifying proteins with low sequence similarity but similar function or structure.
We selected the Global Ocean Microbiome Catalog (GOMC) as the search database for PSI-BLAST. The GOMC integrates genomes of marine bacteria and archaea from the National Center for Biotechnology Information (NCBI), the Ocean Microbiomics Database (OMD), and OceanDNA4 and OceanDNA6. It contains 43,191 metagenome-assembled genomes (MAGs) derived from the analysis of marine metagenomic data made publicly available by NCBI, the European Bioinformatics Institute (EBI), and the Joint Genome Institute (JGI) between August 2009 and July 2020, spanning 3,470 microbial genera and 138 phyla.
We chose PET05 as the "seed" sequence for the PSI-BLAST analysis. In bioinformatics mining, selecting an appropriate "seed" sequence is crucial for BLAST analysis. PET05 is widely regarded as one of the most representative and valuable candidate enzymes among marine-sourced PET-degrading enzymes：
First, its structure has been elucidated through homology modeling and crystal structure prediction.
Key amino acid residues at the active site (e.g., His57, Asp149) have been clearly identified, providing a reference for subsequent functional annotation.
Second, PET05 exhibits moderate conservation within the cutinase family, meaning it can represent typical features of the family while retaining sufficient variability information, making it suitable for mining new variants or homologous sequences.
Finally, PET05 is the first enzyme to be mined from marine metagenomes and experimentally confirmed to have PET-degrading activity.
Its function has been validated through in vitro enzyme activity assays and molecular dynamics simulations, making it a reliable "seed" sequence.
our target：
For 2025 iGEM competition, our team aims to investigate and engineer plastic-degrading enzymes with high salt tolerance using synthetic biology approaches. This project is dedicated to developing effective molecular tools for in situ remediation of plastic pollution in high-salinity water bodies. By integrating bioinformatic analysis methods—including PSI-BLAST-based distant sequence mining, cluster visualization tools, and phylogenetic tree construction—we will identify a set of "PET-degrading enzymes" from the GOMC database. We will comparatively evaluate all enzymes' performance, select candidates for optimization, and conduct multiple rounds of engineering modifications to enhance enzymatic activity and thermostability, thereby increasing their practical application value.
project design
Step 1: Experimental part - Sequence mining and hierarchical sampling
We selected the GOPC database belonging to GOMC and divided it into 19 sub-databases. Using PET05 as the seed sequence, we employed 5 rounds of psi-blast to mine a large number of distant sequences of PET05. These protein sequences may all have potential PET degrading enzyme activity. We then selected an appropriate threshold to perform cluster analysis on all the sequences, and used the sequences in each cluster to construct an evolutionary tree. Based on the number of sequences in each cluster, we conducted uniform hierarchical sampling from the branches of the evolutionary tree. Finally, we obtained the most representative few sequences, and we will conduct gene synthesis and enzyme activity tests on these sequences.
Step 2: Orthogonal experiments to explore the properties of PET05 and 40 newly mined sequences
First, we constructed PET05 and 40 newly mined sequences on the pET-32a(+) plasmid. The plasmids were transformed into Escherichia coli BL21(DE3), and single colonies were picked from the plates and cultured in test tubes containing 5 mL LB medium. Then, the plasmids were transferred to 50 mL flasks for cultivation and induction of protein expression. We will purify these enzymes. We will design multiple rounds of orthogonal experiments to test whether these enzymes have enzymatic activity and the optimal conditions for exerting enzymatic activity.
Step 3: Engineering modification
We will select the enzymes with excellent properties from the second step for engineering modification. We plan to carry out the modification through two approaches. One involves the modification of enzyme thermal stability based on deep learning and physical-chemical algorithms. The other one is the mutation of the enzyme active pocket based on structural homology and molecular docking results.
Result
We conducted orthogonal experiments to test the enzyme activity of 40 mined protein sequences with potential PET-degrading activity, and found that four of them are capable of degrading low-crystallinity PET under certain conditions. We carried out a series of investigations into the properties of PET05 and discovered several of its favorable characteristics. Furthermore, significant progress has been made in the engineering modification of PET05, resulting in the development of multiple superior mutants with markedly enhanced enzymatic activity and thermal stability.
References
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