Part:BBa_K5436124

Optimized RBS for BIND-System+BIND-bearPETase+6xHisTag

Sequence and Features

Molecular weight: 46.6 kDa

Codon optimized for E.coli BL21(DE3) cells.

Assembly Compatibility:

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  Illegal PstI site found at 395
  Illegal NotI site found at 550
  
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  Illegal BamHI site found at 478
  
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  Illegal PstI site found at 395
  
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Abstract

BIND-bearPETse Graphical 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
   • Functional Characterization
   • Curli Fiber Formation Assay
   • pNPB Hydrolysis Assay
   • Storage Activity Assay
   • Reusability Assay
   • PET Bottle Powder Degradation Assay
   • Plastic Pellet Degradation Assay
4. In Silico Energy Simulation
   • AutoDock
   • PyRosetta
   • FoldX
   • MACE
5. Mathematical Modeling
   • Membrane transport model
   • PET degradation efficiency model
6. Conclusion

Overview

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

Fig 1. The advantages of BIND-bearPETase over free-PETase

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.

Fig 2. BIND-bearPETase docking to PET polymer

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

Fig. 3. Components of RBS+BIND-bearPETase+6xHis

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

Molecular Cloning

We used NEBuilder HiFi DNA Assembly [5] 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.

Fig. 4. Electrophoresis and Plasmid map of the pMAL-c4X-RBS+BIND-bearPETase

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 tab.

Fig. 5. Confirmation of BIND-bearPETase expression (picked up 3 colonies).

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 the 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. 6. 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.

Fig. 6. Robust pellet of BIND-bearPETase

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. 7. It can be observed that Curli fibers are formed and stained in a manner dependent on the presence of BIND-bearPETase.

Fig. 7. Curli Fiber Staining of BIND-bearPETase

Next, the absorbance of the supernatant was measured and compared between BIND-bearPETase and other variants (Fig. 8).

Fig. 8. Intensity of Curli Fiber Formation

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. 9). 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 later section of PET Bottle Powder Degradation Assay, BIND-bearPETase demonstrated the highest practical degradation of PET among these variants.)

Fig. 9. pNPB Hydrolysis Assay of BIND-PETase variants, including BIND-bearPETase

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.

Fig. 10. The advantages of BIND-bearPETase over free-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. 11). Additionally, we assessed the increase in activity when the storage temperature was changed to either 4°C or room temperature.

Fig. 11. Storage Activity Assay on different condition; **(A)**4°C, (B) RT

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. 12).

Fig. 12. Reusability of BIND-PETase variants including BIND-bearPETase (Cycle1-3)

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.

PET Bottle Powder Degradation Assay

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. 13).

Fig. 13. Enzymatic hydrolysis of PET by PETases and MHETases[6]

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[^7]. The results were measured 1 day and 3 days after the reaction.

Fig. 14. HPLC chromatogram for the degradation products of PET bottle powder by BIND-bearPETase

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. 15). 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.

Fig. 15. Degradation products of PET by BIND-bearPETase under different pH conditions.

Plastic Pellet Degradation Assay

さらに、Waseda-Tokyo2024は、BIND-bearPETaseがどこまで実用的であるかを考察するために、リサイクル工場に存在するプラスチックペレットに対し、BIND-bearPETaseを作用させた。ここでは、ペレットの重量を測定する事で、その分解を確認する事ができた。
挿入予定

Fig. 15. Degradation of Plastic Pellets

分解は確認出来たが、その効率は、私たちの求めている効率には達していなかった事が分かった。この実験は、1回限りのデモ検証だった。その為、反応条件の再検討が出来ておらず、今回の反応では、酵素の量を少なめにしていたり、攪拌しながら反応を進めることができず静置だった。その為、少ない量のBIND-PETaseは沈殿し、基質と酵素の接触があまり期待できなかった。

今回は最低限BIND-PETaseがペレットを分解可能であることを示す事ができたので、次回更なる検証をする際には、ペレットとBIND-PETase懸濁液が常に混ぜ合わせられるような系かつ、より高密度でで反応を行う事でBIND-PETaseの真価を見ることができるだろう。
なお、このペレットは、実際にプラスチックリサイクルを行う企業esaに尋ね、譲渡していただくことができた。 この場を借りて感謝申し上げます。

Simulation

私たちは、Wet実験に加えてコンピュータを用いたbearPETaseの特性検証を行った。私たちが用いたツールは以下の通りである。

• AutoDock VIna [8]
• PyRosetta [9]
• MACE
• FoldX [10]

AutoDock Vinaが出力するエネルギーの値から結合親和性を評価することができる。エネルギーが低いほど結合親和性が高く、結合親和性が高ければ実際のWet実験で活性が高くなることが期待できる。PyRosettaが出力する自由エネルギーの値からPETaseの構造の安定性を評価することができる。MACEは私たちが構築した機械学習モデルであり、○○。FoldXは...

AutoDock VIna

Method

AutoDock Vinaを用いた検証ではPET2量体のPDBQTファイルと PETase(WT)、 duraPETase、PETase(ID23)、bearPETase(ID24)のPDBQTファイルを用意して分子ドッキングを実行した。BIND-PETaseではなくPETaseに対して検証を行った理由は、 BIND-PETaseに対して検証すると計算量が増えるから、実際に酵素活性と関係がある部分はPETaseの部分であるからである。PET2量体のPDBQTファイルとした理由の一つはPETaseはPETを分解するタンパク質であるのでPET分子が2量体以上でないと現実に即していないからである。もう一つはエネルギーの比較であれば2量体で十分であるからである。AutoDock Vinaが出力したエネルギーの値を用いてbearPETaseの結合親和性を評価した。

Results

各PETaseに対して分子ドッキングを行った結果AutoDock Vinaが出力したエネルギーの値を以下の**Table. 1.に示す。グラフをFig. 17.**に示す。 **Fig. 17.**は上がマイナスの値になっていることに注意する。

Table. 1. The result of molecular docking with AutoDock Vina

PETase variants	Affinity (kJ/mol)
PETase(WT)	-5.3
duraPETase	-3.7
PETase(ID23)	-5.4
BearPETase(ID24)	-5.3

Fig. 17. The result of molecular docking with AutoDock Vina

**Table. 1.とFig. 17.**より、bearPETaseは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. 15.**に示されているようにbearPETaseは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(WT)エネルギーの値と同じでPETase(ID23)のエネルギーの値より高くなっており **Fig. 15.**の結果に反する。これはAutoDock Vinaのアルゴリズムによるシミュレーションの運に影響されていると考えられる。

最後に、bearPETaseとPET分子が結合している様子を以下に示す。

Fig. 18A. bearPETase docking to PET polymer

Fig. 18B. bearPETase docking to PET polymer (Displayed using electronic density map)

Fig. 18A.とFig. 18B.の中で赤い点で示されているのがbearPETaseの結合部位である。PET分子はbearPETaseの結合部位にうまく結合している。したがってbearPETaseの結合親和性が視覚的にも示された。

これらの結果より**bearPETaseは祖先であるduraPETaseよりも結合親和性が高いことがコンピュータシミュレーションの観点から示された。**そしてWet実験でもduraPETaseより活性が高いことが大いに期待できる。コンピュータシミュレーション上ではPETase(WT)とbearPETaseの結合親和性は等しいという結果となったが、実際にはduraPETaseはPETase(WT)より活性が高いことが示されているのでbearPETaseはPETase(WT)よりも活性が高くなるであろうことがこの結果から予想できる。

PyRosetta

Method

PyRosettaを用いた検証ではBIND-PETase(WT)、 BIND-duraPETase、 BIND-PETase(ID23)、 BIND-bearPETase(ID24)のPDBファイルを入力し、PyRosettaが出力する自由エネルギーの値を用いてbearPETaseの構造の安定性を評価した。AutoDock Vinaの場合と異なり、構造の安定性はBIND-PETase全体で評価する必要があることからBIND-PETaseを用いて検証を行った。

Results

各BIND-PETaseに対してPyRosettaが出力したエネルギーの値を以下の**Table. 2.に示す。グラフをFig. 19.**に示す。 **Fig. 19.**は上がマイナスの値になっていることに注意する。とにおいて

MACEを用いた検証では...
FoldXを用いた検証では...

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

Waseda-Tokyo iGEM 2024チームは、「BIND-bearPETase」という新たな酵素システムを開発し、PET（ポリエチレンテレフタレート）分解を効率化しました。また、この技術は、他の酵素にも応用でき、BIND-Systemを活用することで酵素の精製コストを削減し、利便性を向上できることを示唆しました。

Wet Experimentsでは、精製をせずとも、PETを加水分解する活性が利用可能であることを示した。さらにそれが、日常に存在する身近なPETボトル由来のPETに適用可能であることを示し、このパーツの実用可能性を示した。

また、他のBIND-PETase変異体よりも、BIND-bearPETaseが高い加水分解活性を持つことを確認できた。さらにそれは、長期間保存しても、活性が維持されることや、酵素の再利用が可能であることを、実験的に示した。

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[7] F Kawai et al. (2022) Efficient depolymerization of polyethylene terephthalate (PET) and polyethylene furanoate by engineered PET hydrolase Cut190. AMB Expr 12(134) doi: 10.1186/s13568-022-01474-y