Introduction

Our project was motivated by an awareness of environmental issues, and the combination of human practice, wet lab experiments, and dry lab simulations provided an innovative solution. The overall flow of the project is shown in the figure below.

Fig. 2.1. Overall flow of the project

Shortcomings of Existing Methods; PET as a Barrier to Recycling

Plastic recycling is one of the most important international tasks. We identified the increasing amount of microplastics in Japan's oceans as a problem. We then learned about the challenges in the plastic recycling process through research of the literature on plastic waste and interviews with relevant stakeholders. Through interviewing with esa Inc., a company involved in the material recycling of plastics, PET is a major barrier to recycling because of its robust chemical structure among plastics. For more detail, refer to Integrated Human Practice(IHP). For example, PET in composites with other plastics in waste plastics can prevent them from being recycled as pure materials. Composite plastics account for about 25% of all discarded plastics, and the ability to recycle them would go a long way toward solving the problems associated with plastics.

Fig. 2.2. PET as a barrier to plastic recycling

PET Specific Degradation by PETase

PETase has attracted attention as a biological approach to PET recycling. The hydrolysis reaction using PETase has higher specificity than chemical methods and can be used under mild conditions. PETase also has the advantage of reacting with fine resins that physical filtration systems would otherwise ignore. For these reasons, PETase is expected to play an active role in bioremediation and recycling.^[1]
For instance, if PET can be selectively removed from composite plastics by PETase, material recycling of the remaining plastics will become possible, leading to more efficient resource recycling.

Fig. 2.3. PET degradation by PETase contributes to material recycle

We found that the social implementation of such PETase is hindered by three factors, as shown in Fig. 2.4. After a meeting with OpenPETase, a NPO dedicated to tackling the waste problem using PETase, it became clear that the social implementation of PETase has not progressed as anticipated (refer to IHP). The purification process is very costly, and the protein's activity significantly decreases after purification, hindering the practical application of PETase.

Fig. 2.4. Three drawbacks of existing PETase

Although numerous papers related to PETase have been published, most of these studies have not reached the stage of social implementation. Since the discovery of PETase in 2016^[1], nearly 3,000 papers about PETase have been published to date, with 689 of them published in 2024 (as of September 29th, 2024). Of those 3,000 papers, approximately 60% focused on improving the functionality of PETase, but there are very few examples where these efforts have led to the industrial implementation of PETase.

Therefore, as the first step to expand the practical use of the extensively researched PETase, we introduced PET TWINS.

Our Solution: PET TWINS

PET TWINS (PETase The Way to ImplementatioN Success), a revolutionary PET digestion system using E. coli, offers a solution for the practical use of PETase. PET TWINS consists of two components: the BIND-PETase Module(BIP Module) and the ElectroCount Biosafety Module(ECB module).

BIND-PETase Module(BIP Module)

The limitations of conventional PETase were overcome by creating fusion proteins called BIND-PETases and improving their functionality.^[2]

Fig. 2.5. BIND-PETase Module graphical abstract

The BIND-system, a type of surface display, is a biotechnology that uses polymerization of the extra-membrane fiber-forming factor CsgA on the membrane to immobilize specific enzymes fiber-bound on cell surface fibers^[3]. This allows the enzyme to access the substrate directly without purification, thereby further stabilizing the enzyme activity. The detailed structure is shown in the Design tab (refer to Design). This system enables PETase to be displayed on the surface of the E. coli membrane, transforming E. coli into a whole-cell catalyst with PET-degrading activity. Furthermore, immobilizing PETase on the membrane surface enhances its long-term stability and reusability.^[2]

Waseda-Tokyo worked on integrating PETase into the BIND-system while improving its PET-degrading activity. To achieve this, we developed an In Silico Evolution Pipeline, and we also designed iePETase (In Silico Evolution PETase) and rdPETase (Rational Design PETase) (refer to Simulation). We loaded the 14 designed iePETases and rdPETases into the BIND-system and compared their functions with BIND-PETase(WT), which uses wild-type PETase.

Among them, we were able to characterize BIND-bearPETase (BBa_K5436124) as having an exceptionally high capability through wet experiments, energy simulations, and mathematical modeling (refer to Model). The details are thoroughly documented in BBa_K5436124. By replacing the PETase with any enzymes of interest, this part has the potential to enable purification cost reduction and improve their stability and reusability in future iGEM projects.

Fig. 2.6. Our new composite part ”Optimized RBS + BIND-bearPETase” BBa_K5436124

ElectroCount Biosafety Module(ECB Module)

The BIND-PETase Module is an excellent PET degradation system, but the use of E. coli as a whole-cell biocatalyst presents two unavoidable challenges. In our interview with Yaegaki Biotechnology regarding their fermentation technology, we identified the control of bacteria as a new challenge. To prevent leakage of E. coli, we needed a reliable method to remove them from the reaction solution after PET digestion (refer to IHP).

We also considered that, given the burden on E. coli from the constitutive expression of the introduced gene and the time required for the enzyme-mediated degradation reaction, it is necessary to regulate the expression of BIND-PETase at the appropriate timing.

Depending on the first and second electrical input, we have addressed two challenges by the ElectroCount Biosafety Module which activates the expression of two genes in order. The ElectroCount Biosafety Module combines pSoxS promoter, the electrically inducible promoter^[4]^[5], with the Cre/loxP system^[6], a sequence-specific nucleic acid cleavage technology, thereby enabling gene expression to be controlled by electrical stimulation. The detailed gene circuit is shown in the Design tab (refer to Design).

Fig. 2.7. ElectroCount Biosafety Module graphical abstract

This system acts as a kill switch by inducing expression of the suicide gene MazF^[7] and disrupting the bacterial membrane by electrical stimulation after the BIND-PETase Module has completed its role^[8], ensuring the necessary biosafety for practical whole-cell catalysis. This dual approach to cell termination is based on advice from microbiologist Prof. Tsuneda regarding the limitations of MazF sterilization (refer to IHP). Furthermore, the ElectroCount Biosafety Module induces the expression of BIND-PETase at optimal times, which is expected to alleviate the burden on E. coli associated with biological material production. The ability to remotely and easily control gene expression will facilitate more flexible social implementation in lab automation contexts.

The workings of this system were evaluated in modeling and wet lab experiments(refer to Engineering). In the wet lab experiments, we also performed electrical stimulation of gene circuits in E. coli with the advice of Prof. Nakanishi, an expert in electrochemistry (refer to IHP).

Fig. 2.8. Contribution of ElectroCount Biosafety Module to lab automation

PET TWINS －The Integrated System－

By combining these two modules, we have constructed a PET degradation system for the industrial application of PETase, called PET TWINS. In this system, the expression of BIND-PETase in E. coli  is induced by the first electrical input, allowing the modified BIND-PETase to rapidly and directly degrades PET. After PET degradation is completed, a second input triggers the expression of the suicide gene MazF in E. coli, resulting in cell death and ensuring the safety of the treated waste solution.

Instead of conducting complex wet experiments, we verified its integrated functionality using a mathematical model. To validate the operation of the ECB module, we constructed a time-dependent model where all downstream genes controlled by the pSoxS and Cre-recombination system, regulated by pSoxS, are connected through variable transmission. Additionally, to predict the operation of BIND-PETase expressed by the BIP module, we developed a time-evolution model linking the expression levels of Csg proteins inside E. coli  with their translocation to the extracellular space, the formation of curli fibers by the translocated CsgA proteins, and the surface reactions on the fibers (refer to Model). The modeling demonstrated the potential success of our project.

Fig. 2.9. How to work PET TWINS

Implementation

Our PET TWINS could be applied to plants designed to degrade discarded composite plastics. An opinion survey conducted with the general public via Mr. Takatoh, a well-known science Vtuber, revealed that installing a PET decomposition plant at the household level would be challenging. Therefore, we provisionally considered industrial-scale use as the primary application (refer to IHP).

We estimate that the plant would require roughly three tanks: the first tank for culturing E. coli, the second for degrading PET followed by sterilization of E. coli, and the third for processing the waste plastic after PET removal. In the first tank, E. coli  would grow efficiently in the culture tank because PET TWINS would not be activated there, while PET TWINS would primarily function in the second reaction tank. This hypothetical example is a useful basis for evaluating whether our project can replace existing methods. Based on this scenario, we evaluated the advantages of electric kill switches in a dry-lab environment and the concept of scaling up through modeling (refer to Model).

Model Award

Fig. 2.10. Conceptual diagram of PET decomposition plant

Fig. 2.11. Diffusion simulation for scale up

Conclusion

Concerned about the microplastic problem and uncovering various issues in plastic recycling through numerous interviews, Waseda-Tokyo has proposed PET TWINS as a new approach to realize the practical application of PETase.

The BIND-PETase Module is expected to promote plastic recycling in the future, contributing actively to waste disposal problems and the resolution of the microplastic issue. Additionally, based on evolutionary engineering insights, Waseda-Tokyo designed BIND-bearPETase and verified its superior functionality through wet experiments, energy simulations, and mathematical models.

Furthermore, we introduced the concept of the ElectroCount Biosafety Module, which induces the expression of two genes through electrical input. This system is expected to address barriers to the introduction of BIND-PETase, and its feasibility has been validated through mathematical modeling.

The integrated system, PET TWINS, will drive the social implementation of PETase.

Special Award Requirements

Integrated Human Practices Award

New Composite Part Award

Model Award

References

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