Abstract

This part was designed for the construction of Whole-cell Biocatalysts "BIND-bearPETase." Waseda-Tokyo2024 thoroughly investigated its functionality through wet lab experiments, mathematical modeling, and energetic simulations. Additionally, this part holds great value for the iGEM community by addressing the urgent need for better plastic waste management and expanding any enzyme availability.

Agenda

1. Overview
2. Components
3. Cloning & Expression
4. Functional Characterization
   • Curli Fiber Formation Assay
   • pNPB Hydrolysis Assay
   • Storage Activity Assay
   • Reusability Assay
   • PET Bottle Powder Degradation Assay
   • Plastic Pellet Degradation Assay
5. In Silico Energy Simulation
   • Affinity Simulation using AutoDock Vina
   • Stability Simulation using PyRosetta
   • Stability Simulation using FoldX
6. Mathematical Modeling
   • Membrane transport model
   • PET degradation efficiency model
7. Conclusion

Overview

This "BIND-bearPETase" offers benefits that address the shortcomings of conventional free PETase shown below.

BearPETase has two meanings. The first comes from the verb “bear,” as BearPETase demonstrates strength in the stability of enzymes and can “bear” burdens. The second meaning relates to the cute animal mascot “Waseda Bear” of our school! ʕ•ᴥ•ʔ ʕ•ᴥ•ʔ ʕ•ᴥ•ʔʕ•ᴥ•ʔ ʕ•ᴥ•ʔ ʕ•ᴥ•ʔ

This part encodes the CsgA-bearPETase fusion protein. CsgA is an extracellular fibrous structure-forming factor that constructs Curli Fibers on the surface of the E. coli membrane. By fusing bearPETase to CsgA, we enabled the presentation of bearPETase on the cell membrane surface in a fiber-linked manner.

This enables direct access to substrates without the need for purification, as well as the stabilization of enzyme activity and the reuse of enzymes. This is a technique referred to as the BIND-System ^[1], and whole-cell biocatalysts equipped with PETase are called BIND-PETase ^[2].

The key effort in this part was creating “bearPETase” ,the optimal PETase for the BIND-System. BearPETase, uniquely developed by Waseda-Tokyo 2024, combines mutations from depoPETase (Shi et al., 2023) ^[3] and duraPETase (Cui et al., 2021) ^[4] developed through directed evolution. We generated several variant groups and identified the optimal one through functional comparisons in wet experiments.

Furthermore, this part significantly contributes to the iGEM community by expanding enzyme availability. As mentioned above, the BIND-System reduces concerns about purification costs and quality, making them negligible. It also allows for maintaining and reusing proteins with unstable activity. By replacing the bearPETase portion with other BioBricks, any enzyme's use can be simplified.

Components

I. Optimized RBS for BIND-System (Waseda-Tokyo2024, BBa_K5436005)
This RBS is designed to efficiently drive the BIND-System. In some existing BioBricks, inappropriate RBS strength can either overload E. coli with excessive expression or result in no expression. We've designed an RBS to optimize the amount of CsgA displayed on E. coli’s surface as components of curli fibers, which will aid future iGEMers using the BIND-System.
II. csgA-taa(Waseda-Tokyo2024, BBa_K5436006)
CsgA-taa is a modified version of BBa_K1583000 from iGEM15_TU_Delft, with the stop codon removed, enabling the expression of the desired protein in a fused state after the Curli fiber formation factor CsgA.
III. BamHI_Linker (Waseda-Tokyo2024, BBa_K5436020)
This uses the BamHI recognition site, which consists of 6 nucleotides, directly as a linker. The BamHI recognition site encodes glycine and serine, which are commonly used amino acids in linker sequences.
IV. bearPETase (Waseda-Tokyo2024, BBa_K5436015)
BearPETase was rationally designed by Waseda-Tokyo 2024 to enhance its enzymatic activity. As shown below, we confirmed that its enzymatic activity surpassed that of existing variants. The existing PETase variants include depoPETase and duraPETase, and combining both was expected to improve enzymatic activity. Based on that consideration, we created 81 combinations, excluding the overlapping mutations Q119Y and Q119R, and generated 3D structures using AlphaFold 2, selecting those with stable structures.
V. 6x HisTag (Waseda-Tokyo2024, BBa_K5436021)
It is useful in protein purification and also beneficial for Western blotting, where anti-His Tag antibodies are used as primary antibodies.

Cloning & Expression

Designing RBS for BIND-System

The "Optimized RBS for BIND-System (BBa_K5436005)" included in this part was carefully designed by the RBS Calculator from Salis Lab^[5], rather than reusing an existing RBS.

Existing RBS used in previous CsgA overexpression experiments did not meet our criteria. The RBS included in the pRha + CsgA (BBa_K1583100) developed by iGEM15_TU_Delft had a transcriptional rate of 40.80, which was insufficient for the expression levels we required.

On the other hand, the transcriptional rate of the RBS in Rec-PhoA/CsgA (Addgene #170787)^[6] was approximately 700, and it appeared to meet our requirements. Referring to that order of magnitude, we newly designed an RBS for BIND-PETase (WT) with a transcriptional rate of 800 using the RBS Calculator.

As mentioned later, this optimized RBS was sufficient to induce the expression of CsgA-bearPETase.

Molecular Cloning

We used NEBuilder HiFi DNA Assembly ^[7] to obtain plasmids encoding BIND-bearPETase. The DNA fragments encoding bearPETase were prepared with Gene Fragments Synthesis Service (Twist Bioscience).

After culturing and miniprepping, we ran electrophoresis, observing bands near the expected size. Sequence analysis confirmed the correct plasmid sequences.

Western Blotting

Samples induced for the expression of CsgA-bearPETase by IPTG were lysed, and when subjected to Western Blotting using His-Tag as the primary antibody, a clear band was observed around 45 kDa, confirming the overexpression of the target protein. For detailed protocols of the lysis, refer to our wiki, Experiments.

Functional Characterization

A total of 7 wet experiments were conducted to thoroughly investigate the function of BIND-bearPETase. During this process, we compared BIND-bearPETase with its ancestor sequence BIND-PETase (WT) (BBa_K5436130), BIND-duraPETase (BBa_K5436133), and BIND-PETase (ID23) (BBa_K5436123), which is created with a similar design strategy. The results are documented below.

On the Wiki, BIND-bearPETase was evaluated by comparing it with numerous variants not shown here. The process is detailed in the Engineering Success section of our wiki.

Curli Fiber Formation Assay

The formation of Curli fibers of BIND-bearPETase was quantitatively measured. Whether Curli fibers are formed correctly is crucial for the enzyme's stability and reusability.

After centrifuging the BIND-bearPETase suspension, the resulting pellet exhibited a robust structure that did not break apart even after multiple pipetting, as shown in Fig. 8. This suggests that the formation of Curli fibers due to the overexpression of CsgA-bearPETase led to the development of a biofilm structure in E. coli.

In the Curli Fiber Formation Assay, Congo Red dye is used to stain Curli fibers, followed by centrifugation to form a pellet. Subsequently, the absorbance of the supernatant is measured to quantify the formation of Curli fibers. If the Congo Red dye is incorporated into the pellet and the supernatant appears pale, it can be confirmed that Curli fibers have been properly formed.

The results of Congo Red staining for BIND-bearPETase are shown in Fig. 9. It can be observed that Curli fibers are formed and stained in a manner dependent on the presence of BIND-bearPETase.

Next, the absorbance of the supernatant was measured and compared between BIND-bearPETase and other variants (Fig. 10).

Although BIND-bearPETase exhibited lower Curli fiber formation ability compared to BIND-PETase (WT),it had a higher Curli fiber formation ability than BIND-duraPETase, which is ancient of BIND-bearPETase. Additionally, it was found that BIND-bearPETase and BIND-PETase (ID23) possess a similar level of Curli fiber formation ability.

Based on these results, it can be concluded that bearPETase is more suited for the BIND-System in terms of Curli fiber formation ability among the many improved PETases.

pNPB Hydrolysis Assay

The activity of BIND-bearPETase was investigated in an easy way(Fig. 11). Para-nitrophenyl butyrate (pNPB) produces yellow para-nitrophenol (pNP) upon hydrolysis, and we measured this product. However, the magnitude of hydrolytic activity against pNPB does not necessarily correspond to the activity against PET polymers.
Therefore, it is important to note that the pNPB Hydrolysis Assay only provides a simplified assessment of activity. (As will be discussed in PET Bottle Powder Degradation Assay section, BIND-bearPETase demonstrated the highest practical degradation of PET among these variants.)

It was confirmed that the activities of BIND-bearPETase and BIND-PETase (ID23) increased compared to their ancestor sequences, BIND-PETase (WT) and BIND-duraPETase. BIND-bearPETase and BIND-PETase (ID23) designed by Waseda-Tokyo demonstrated superior performance, suggesting they possess more advantageous features for the practical application of PETase.

Storage Activity Assay & Reusability Assay

Here, we document the experimental results that verify the strengths of BIND-bearPETase regarding the stability and reusability of the enzyme in the social implementation of PETase.

Storage Activity Assay

Since various BIND-PETases are whole-cell biocatalysts utilizing live E. coli, proper storage conditions allow for protein expression and bacterial growth, which can maintain or enhance their activity.
The activities of BIND-bearPETase were evaluated on days 0, 5, and 11 after expression using the pNPB Hydrolysis Assay (Fig. 13). Additionally, we assessed the increase in activity when the storage temperature was changed to either 4°C or room temperature.

During storage, both BIND-bearPETase and BIND-PETase (ID23) exhibited a greater increase in activity over time compared to BIND-PETase (WT) and BIND-duraPETase.
When stored at room temperature, BIND-bearPETase showed the highest increase in activity. These results suggest that BIND-bearPETase has greater convenience in storage compared to other BIND-PETases, making it advantageous for practical applications."

Reusability Assay

BIND-bearPETase could be reused three times after a single reaction, with the presence of activity confirmed through the pNPB Hydrolysis Assay. The activity after reuse was also observed for BIND-PETase (WT) and other variants (Fig. 14).

It was observed that the activity increased after reuse. This may be due to the contamination of the reaction product, pNP, during the collecting stage of BIND-PETases. In this measurement, it was inevitably difficult to accurately assess the reusability because pNP contaminated the reaction system.

However, we attempted to conduct washing operations as thoroughly as possible to achieve the most accurate measurements. Additionally, the promotion of PETase enzyme folding due to the initial reaction may also contribute to the observed increase in activity.

BIND-duraPETase, BIND-PETase (ID23), and BIND-bearPETase exhibited an increase in activity during reuse. While the exact reasons for the activity increase upon reuse could not be identified, it was confirmed that at least BIND-bearPETase does not significantly lose activity even after reuse, indicating its advantage for practical applications.

Plastic Pellet Degradation Assay

Waseda-Tokyo 2024 evaluated the practical degradation activity of BIND-bearPETase. In this process, composite plastic pellets (PETPEPP) used in actual recycling plants and single-material pellets (PET(N)) were utilized as substrates.

After adding BIND-bearPETase suspension to the reaction system at pH 7.0 and pH 9.0 and allowing it to act for five days, mass reduction was confirmed in both types of pellets. The negative control did not show any weight loss (data not shown). For comparison, BIND-PETase (ID23) was also included.

It was demonstrated that BIND-bearPETase and BIND-PETase(ID23) are capable of degrading the pellets. However, due to the pellets' heterogeneity, quantitative experiments are needed for accurate activity comparisons between variants.

The pellets were provided by the recycling company esa Inc., and we would like to take this opportunity to express our gratitude.

PET Bottle Powder Degradation Assay

Next, we conducted HPLC analysis using PET bottle powder to perform a more quantitative comparison of BIND-bearPETase activity. In the previously mentioned pellet degradation experiments, the heterogeneity of the pellets made it difficult to accurately compare enzyme activities. Therefore, quantitative validation was crucial.

It was confirmed that BIND-bearPETase possesses the highest practical activity against PET powder compared to other variants.
PETase decomposes the PET polymer, resulting in the formation of TPA, MHET, and BHET (Fig. 16).

Waseda-Tokyo 2024 quantified the products TPA, MHET, and BHET, generated by BIND-bearPETase, using High-Performance Liquid Chromatography (HPLC).
PET bottles, commonly used in everyday life, were ground with sandpaper, and BIND-bearPETase was applied.

In addition to pH 7.0, the reaction was also carried out at pH 9.0, as many PETases are reported to have optimal conditions at pH 8.5 or higher ^[9]. The results were measured 1 day and 3 days after the reaction.

In this way, it was confirmed that the products TPA, MHET, and BHET were generated by BIND-bearPETase. Additionally, it was suggested that the optimal pH for BIND-bearPETase is also pH 9.0.

Furthermore, we quantitatively compared the amounts of these degradation products (Fig. 19). Contrary to the pNPB hydrolysis assay mentioned earlier, BIND-bearPETase degraded PET bottle powder more effectively than BIND-PETase (ID23). BIND-bearPETase exhibited 10 times the activity of its ancestor BIND-duraPETase and 1.5 times that of its sibling BIND-PETase (ID23). These findings suggest that bearPETase, developed by Waseda-Tokyo, is well-suited for the BIND-System and demonstrates high practical activity.

In Silico Energy Simulation

Since the structural stability of bearPETase cannot be quantitatively measured through wet-lab experiments, we used these tools to evaluate its affinity for PET and stability more effectively:

• AutoDock Vina ^[10]
• PyRosetta ^[11]
• FoldX ^[12]

We can assess binding affinity from the energy values provided by AutoDock Vina, where lower energy indicates higher binding affinity. Higher binding affinity suggests greater activity in actual wet-lab experiments. PyRosetta outputs score values that allow us to evaluate the structural stability of bearPETase. FoldX also provides energy values that help assess bearPETase's stability.

Affinity Simulation using AutoDock Vina

Method

We performed molecular docking using AutoDock Vina, preparing the PDBQT files of PET dimer along with those of PETase (WT), duraPETase, PETase (ID23), and bearPETase. The reason we conducted the validation on PETase instead of BIND-PETase is that we focused on the PETase domain, which is directly related to enzymatic activity. Using the energy values output by AutoDock Vina, we evaluated the binding affinity of bearPETase to the PET dimer.

Results

The energy values output by AutoDock Vina from the molecular docking performed on each PETase are shown in Table 1. The graph is presented in Fig. 20. Note that in Fig. 20, the values are plotted with the negative values facing upwards.

Table. 1.とFig. 20.より、bearPETaseはPETase(WT)やduraPETaseより結合親和性が高いことがわかる。つまりWet実験でも活性が高くなることが期待できる。実際、Fig. 19.に示されているようにbearPETaseはPETase(WT)やduraPETaseより活性が高くなっており、**シミュレーション通りの結果となっている。**一方でbearPETaseのエネルギーの値はPETase(ID23)のエネルギーの値と同じとなっており Fig. 19.の結果に反する。この結果について考察する。 PETaseがPET分子を分解する過程は次のようになっている。

It was confirmed that bearPETase has higher binding affinity than PETase(WT) and duraPETase. This suggests that it is likely to exhibit higher activity in wet experiments as well. In fact, as shown in Fig. 19 of PET Powder Degradation Assay section, BIND-bearPETase demonstrates greater activity compared to BIND-duraPETase, consistent with the simulation results. However, the energy value for bearPETase is the same as that of PETase (ID23), which contradicts the results shown in Fig. 19. This discrepancy requires further discussion. The process by which PETase breaks down PET molecules is as follows ^[13].

1. PET molecules dock onto PETase.
2. PETase breaks down the PET molecules.
3. The PET molecules are released from PETase.

However, molecular docking tools like AutoDock Vina only simulate the first stage, where PET molecules dock onto PETase. Therefore, it is likely that the differences in results observed in Fig. 19 and Fig. 20 are influenced by stages 2 and 3.

Nonetheless, we can at least conclude that bearPETase is expected to exhibit higher activity than PETase (WT) and duraPETase.

Finally, we visualized the docking interaction between bearPETase and the PET molecule using PyMOL ^[14] (Fig. 21, Fig. 22).

In Fig. 21 and Fig. 22, the red dots indicate the binding site of BIND-bearPETase. The PET molecule is successfully bound to this binding site, visually confirming the high binding affinity of BIND-bearPETase.

From these results, it is demonstrated from a computational simulation perspective that BIND-bearPETase has higher binding affinity than BIND-PETase (WT) and its ancestor, BIND-duraPETase.

Stability Simulation using PyRosetta

Method

To evaluate the whole structural stability of the fusion protein BIND-PETase, we conducted validation using PyRosetta. For this, we input the PDB files of BIND-PETase (WT), BIND-duraPETase, BIND-PETase (ID23), and BIND-bearPETase. By comparing the score values output by PyRosetta, we assessed the structural stability of BIND-bearPETase in relation to the others.

Results

Among the compared variants, BIND-bearPETase was found to be the most structurally stable. The score values output by PyRosetta are shown below (Fig. 23, Table 2). Note that in Fig. 23, the negative values are plotted upwards. Additionally, the values represent the output after each BIND-PETase structure was optimized to minimize energy before being input into PyRosetta.

Table. 2.とFig. 23.から、PyRosettaの出力するスコアの値はBIND-bearPETaseが最も低いことがわかった。これはBIND-PETase(WT)、祖先であるBIND-duraPETase、兄弟であるBIND-PETase(ID23)よりもBIND-bearPETaseの構造が安定であることを示している。

Wet Experimentで検証したように、BIND-bearPETaseのStorage Activityや、Reusabilityにとってタンパク質の構造安定性は1つの要素に成り得る。
よってPyRosettaが出力するスコアの値よりBIND-bearPETaseの優位性が示された。

Stability Simulation using FoldX

Method

FoldXを用いた検証ではPyRosettaを用いた検証の時と全く同じものを用いた。それらをFoldXに入力し、出力されたギブズ自由エネルギーの値を用いてBIND-bearPETaseの構造の安定性を評価した。

Results

各BIND-PETaseに対してFoldXが出力したギブズ自由エネルギーの値を以下のTable. 3.に示す。グラフをFig. 24.に示す。Table. 3.とFig. 24.について、ギブス自由エネルギーの値を示しているので値が小さい方が構造が安定であるということに注意する。

Table. 3.とFig. 24.よりFoldXの出力するギブズ自由エネルギーの値は〇〇が最も低いことがわかった。これは〇〇の構造が最も安定であることを示している。

Conclusion

この章で私たちは以下のことを示した。

• AutoDock Vinaを使ってbearPETaseの結合親和性を示し、Wet実験での活性の高さが期待できること
• PyRosettaとFoldXを使ってBIND-bearPETaseの構造が安定であること
• これらの結果よりBIND-bearPETaseは３つのBIND-PETaseより優位性があること

Mathematical Model

Since the membrane transport of BIND-bearPETase cannot be directly examined through wet-lab experiments, we used a mathematical model to investigate how BIND-bearPETase exits the membrane.

Membrane transport model

In the modeling section, we developed a membrane transport model of BIND-bearPETase and genome-derived CsgA, which explains the process of CsgA production in the cytoplasm until the transportation to the extracellular.

In the wet lab, CsgA outside of the membrane was detected by the Congo Red Assay as shown in Curli Fiber Section(Fig. 8, Fig. 9 and Fig. 10 ). However, due to the characteristics of Congo Red Assay, it was not possible to directly measure the Curli Fiber quantitatively. Here, we used mathematical formulas to simulate the transport of CsgA, and estimate the expression level of curli fibers formed by the surface display.

The specific formulas and results are shown in our wiki(Model). Since CsgA and B are the main two factors that determine the formation of Curli Fibers, the concentration of the extracellular CsgA and B will be shown.

From this model, the concentration of extracellular CsgA and B were found to be gradually increasing. We were able to visually observe the alterations in concentrations by the transportation of CsgB and BIND-bearPETase.

PET degradation efficiency model

執筆担当者：@Yuto TORIYAMA @Joseph Yokobori 調整お願いします
膜外輸送モデル＋PET分解効率のモデル
Wetで検証できなかったサーフェスディプレイ
PETの長さ, Fiberの長さからPET分解量を計算する

本パーツを実装するにあたって、大腸菌内での物質発現からPET分解につながる過程を示す必要ある。そのため、modelingによって膜外輸送の過程からPET分解までの一連の過程が成立することを示した。
膜外輸送において、BIND-PETaseにおけるcsgAの発現だけでなく、大腸菌内にはcurli fiberを形成するために必要な分子を発現できる仕組みが存在する。この機構を介してcsgAが大腸菌外に移動するため、この流れを定量化した。次に膜外輸送されたcsgAがを形成し、そこに結合したPETaseがPET分解を行う。PETの長さとfiberの長さに応じたPET分解量を定量化した。
一連の流れの定量化により、本プロジェクトで打ち出したPET分解が十分機能することを評価できた。

Conclusion

We, Waseda-Tokyo 2024 team has developed a novel enzyme system called "BIND-bearPETase," which makes the use of PETase more accessible and efficient. This technology can also be applied to other enzymes, suggesting that the BIND-System can reduce enzyme purification costs and improve convenience.

In Wet Experiments, it was confirmed that BIND-bearPETase has higher hydrolytic activity compared to other BIND-PETase variants. Additionally, experiments verified that BIND-bearPETase does not require purification, can be stored for approximately two weeks, and can be reused up to three times. Furthermore, it was demonstrated that BIND-bearPETase can be applied to PET from everyday PET bottles, showcasing the practical potential of this part.

In Dry Experiments, energy simulations were used to verify the stability of PETase-PET docking and the structural stability, which could not be confirmed in Wet Experiments. Moreover, a mathematical model allowed for the examination of outer membrane transport.

The detailed documentation of BIND-bearPETase will serve as a crucial guide for future iGEMers who wish to use or modify and apply this system.

References