Overview

Based on the topic of addressing marine plastic pollution and the treatment of microplastics in high-salinity wastewater using synthetic biology approaches, we conducted an in-depth investigation and mining of the Global Ocean Microbial Community (GOMC) metagenomic database. Utilizing the PET05 sequence derived from GOMC, we performed a psi-blast search to identify potential protein sequences with PET-degrading capabilities. To support this analysis, we developed a clustering visualization tool that assists in the selection of clustering parameters and the visualization of results. In parallel, we implemented two strategies for the engineering of PET05, conducting a total of four rounds of optimization, and made significant contributions to characterizing the enzyme activity and thermal stability of PET05.

 PET05

We have conducted a comprehensive study on PET05, a highly salt-tolerant PET-degrading enzyme that is currently under-researched. Through orthogonal experimental design, we determined the optimal reaction conditions for PET05 and measured its melting temperature (Tm). We further performed a long-term enzymatic degradation experiment lasting 312 hours, with periodic sampling and product quantification. The results showed that PET05 maintains enzymatic activity for nearly 200 hours under conditions of 55°C, 500 mM glycine, and 5 M NaCl. Moreover, the degradation products TPA and MHET continued to increase steadily over the 200-hour period. This sustained activity under such extreme conditions is a unique and valuable property not previously observed in any known PET-degrading enzyme.

 Enzymes from Marine

1. Database Partitioning and psi-blast

Using PET05 as a seed sequence, we conducted a psi-blast search in the GOPC (Global Ocean Prokaryotic Community) sub-database of GOMC to identify distantly related protein sequences with potential PET-degrading functions. To improve search efficiency and optimize the use of computational resources, we innovatively partitioned the large GOPC database into 14 sub-databases. For each sub-database, we set specific E-value thresholds based on the sequence composition and functional background. This partitioning strategy not only significantly enhanced the speed of psi-blast but also reduced redundant results and improved the accuracy of distantly related sequence mining. The combination of database partitioning and psi-blast parameter optimization has not only improved the efficiency and accuracy of protein function mining but also provided a scalable and customizable solution for identifying functionally relevant genes from complex environmental samples.

2. Development of Clustering Visualization Software

We developed a clustering visualization software tailored to the needs of distantly related protein sequence analysis in synthetic biology and environmental biotechnology research. This tool is efficient, intuitive, and user-friendly, and has undergone multiple rounds of testing and optimization to ensure its stability and practicality. It significantly enhances researchers’ efficiency and accuracy in handling large-scale protein sequence datasets.

3. Four Novel Marine PET-Degrading Enzymes

We successfully identified four novel enzymes with potential for PET degradation, all originating from marine environments. This discovery not only expands the diversity and sources of known PET-degrading enzymes but also provides more candidate genes for further engineering in synthetic biology. Given the complexity and biodiversity of marine ecosystems, these newly discovered enzymes serve as a model for the exploration of novel degrading enzymes from extreme or specialized environments.

 Engineering Transformation

Through four rounds of engineering optimization using two distinct strategies, we significantly improved the enzymatic activity and thermal stability of PET05, laying a solid foundation for its application in complex environments. The engineered PET05 retains its original catalytic activity while demonstrating enhanced thermal stability, enabling it to effectively degrade PET materials even under elevated temperatures or unstable environmental conditions. These performance improvements greatly enhance its applicability in challenging natural environments such as marine and high-salinity waters, making in-situ bioremediation more feasible.

The improved PET05 mutants obtained through engineering can now serve as new seed sequences for re-injection into psi-blast or other homology-based search tools. The data and experience accumulated during the engineering process not only provide theoretical support for further optimization of PET05 but also attract the attention of researchers in the fields of synthetic biology, environmental engineering, and industrial microbiology. The enhanced functional properties encourage collaborative efforts to advance the study and engineering of this enzyme, accelerating its transition from basic research to industrial application.

 Bioremediation

By thoroughly mining the Global Ocean Microbial Community (GOMC) database and screening for protein sequences with potential PET-degrading functions, we have identified key molecular tools for the development of engineered microorganisms capable of in-situ biodegradation in polluted environments. This approach eliminates the high costs and low efficiency associated with traditional methods such as sampling, transportation, and centralized processing. Instead, it enables the degradation of pollutants directly at the source, offering superior environmental adaptability and sustainability.

Microplastic pollution has become a global environmental challenge, and our project offers a green, efficient, and sustainable solution using synthetic biology. This strategy can help mitigate long-term damage to marine ecosystems and contribute to the protection of marine biodiversity.

 Discussion and Inspiration

Our project provides the following insights and inspirations for researchers in the field of synthetic biology:

1. The investigation into the properties of PET05 offers new ideas for the engineering of PET-degrading enzymes, such as extending reaction duration and enhancing salt tolerance.
2. The method of database partitioning before psi-blast search serves as an innovative and helpful approach for efficient database mining.
3. The clustering visualization software we developed has undergone multiple rounds of testing and comes with a detailed user manual. It provides a practical and accessible tool for researchers who need to perform clustering-based sampling of large, distantly related protein sequence datasets.
4. Among the 40 proteins selected through clustering analysis and phylogenetic tree construction, four were confirmed to possess PET-degrading activity. This finding suggests a strong correlation between the enzyme activity and their phylogenetic proximity, defining a promising scope for future mining and testing of related sequences.
5. The successful engineering of PET05 demonstrates its potential as a candidate for large-scale production and application in commercial and bioremediation contexts. Although the current activity level of PET05 and its mutants is still relatively low, significantly lower than that of widely used enzymes such as ICCG, its unique properties make it suitable for extreme environments such as high-salinity waters, enabling in-situ bioremediation in such conditions.