Introduction

In the Design Tab, the functions of the BIP Module and the ECB Module and how they work together are explained in detail.
We, Waseda-Tokyo, have constructed a new PET decomposition device: PET TWINS. PET TWINS is a new PETase utilization system that expands the on-site use of PETase.
PET TWINS consists of the BIP Module (BIND-PETase Module) and the ECB Module (Electro Count Biosafety Module). The BIP Module expresses a fusion protein of CsgA, which is a biofilm-forming factor, and PETase, which is a PET-degrading enzyme, to design a new whole-cell type catalyst. In addition, to further improve BIP, we enhanced the stability and activity of PETase based on directed evolution. On the other hand, in the ECB Module, we designed a two-step gene expression by combining the electrically inducible promoter pSoxS and the Cre/loxP system.

BIP Module

Components of the BIP Module

The BIND-PETase consists of two underlying components, CsgA and PETase. As shown in Fig. 3.1, CsgA and PETase are connected by a linker following previous studies^[1]. When it expressed, PETase, a PET-degrading enzyme, are displayed on the Carli fibres formed by CsgA on the surface of E. coli. We call this fusion protein CsgA-PETase. Thus, the entire E. coli acts as a PET degradation catalyst (Fig. 3.1). In a previous study^[1], only wild-type PETase isolated from Ideonella sakaiensis (hereafter WT, Table. 3.1) was fused with CsgA and tested for function (Fig. 3.2 top).

Fig. 3.1. diagrammatic illustration of the BIND-PETase Module

Fig. 3.2. construction of the BIND-PETase Module

The components of our BIND-PETase are summarized in Table 3.2.1. Note that all of the constructs used in the experiments, regardless of the type of PETase mutant, were introduced on the vector pMAL-c4X. pMAL-c4X has a Ptac promoter for IPTG-induced expression and is the best low copy for Curli Fiber formation vector.

Table. 3.1. Components of the gene circuit of the BIND-PETase Module^[2]

Abbreviation Name	Detailed Name	Parts No.	Explanation
IsPETase (WT)	PETase EcCO WT	BBa_J428065	This PETase originated in Ideonella sakaiensis. PETase is the enzyme that is capable of breaking ester bonds in PET structure and degrading PET. This sequence has not been optomized for E. coli.
IsPETase (E. coli optimized)	IsPETase-W159H-S238F with C-terminal Hexahistidine Tag	BBa_K2910000	This is the sequence that has two amino acid mutants in IsPETase(WT). However, we found no mutants in IsPETase (E. coli optimized) according to sequence analysis, and codon optimization for E. coli was done. Which means that the sequence of iGEM Parts BBaK2910000 is incorrect.
IsPETase (5 mut.)	IsPETase-T88F-I208Y-S238F-S245Y-R280A with C-terminal Hexahistidine Tag	BBa_K2910003	This is the sequence that has five amino acid mutants in IsPETase(WT) Codon optimization for E. coli was done.
duraPETase	Designed based on a previous study (PETase(WT) L117F-Q119Y-T140D-W159H-G165A-I168R-A180I-S188Q-S214H-R280A)	BBa_K5436030	For duraPETase, the sequence was converted to a nucleotide sequence based on a previous study by Yinglu Cui (2021). It was then codon-optimized for E. coli.
csgA	csgA	BBa_K1583000	A sequence encoding CsgA, the major extracellular protein that makes up the Curli Fiber, a fibrous structure formed by E. coli on its own membrane.
RBS for BIND-PETase	RBS	BBa_K5436005	RBS optimized for BIND-PETase expression. This RBS adjusts the appropriate expression levels of BIND-PETase in E. coli.

By linking CsgA and various PETases with a linker, various PETases are displayed on the Carli fibres formed by csgA on the membrane surface and degrade PET. The BIP Module can overcome the challenges of free PETase through the above mechanism. The plasmid construction process is described in the Experiments tab. The actual functional analysis was divided into three main categories: activity experiments, recycling experiments, and stability experiments. The activity experiments were performed to evaluate the enzyme activity of BIND-PETase. Reutilization and stability experiments were performed to quantitatively compare the mutants in terms of reutilization and stability, which are the advantages of BIND-PETase compared to PETase in a previous study^[1]. A detailed analysis of the results is given on the Results page.

Improvements of WT

In Silico Evolution and Integrate Rational Design

There are two types of methods to improve the function of proteins by evolutionary engineering: directed evolution and rational design.

In directed evolution, mutations are randomly introduced into a gene, and mutants with improved specific functions are selected to obtain a protein with the desired function. Rational design, on the other hand, is a method to understand the relationship between protein structure and function and improve the function based on this understanding. Through these evolutionary engineering approaches, various PETase mutants were proposed (Fig. 3.3).

Fig. 3.3. Comparison of existing PET Hydrolases^[2]

We set our goal to improve the catalytic activity of PETase for industrial use (Fig. 3.4).

Fig. 3.4. Improvement of PETase activity through evolutionary engineering

We proposed the "In Silico Evolution Pipeline" (Fig. 3.5), which utilizes the knowledge of evolutionary engineering, such as directed evolution, to reproduce part of the process on a computer. We also proposed "Integrate Rational Design" (Fig. 3.6), which combines information on similar enzymes and existing mutants, and designed a total of 14 PETase mutants using these methods. By fusing them with csgA, multiple BIND-PETase mutants will be constructed and validated in Wet experiments.

Fig. 3.5. Flow diagram of In Silico Evolution Pipeline

Fig. 3.6. Flow diagram of Integrate Rational Design Pipeline

How to improve PETase based on directed evolution and rational design

Directed evolution is a time and cost challenge due to the huge search space. We have therefore developed a pipeline to reproduce directed evolution in In Silico without resorting to experiments in the Wet lab (Fig. 3.5). In this pipeline, mutagenesis and screening in directed evolution are performed in In Silico, and machine learning is used to first narrow down PETase mutants that are expected to improve enzyme activity to the $10^3$ scale. Then, In Silico screening, which corresponds to a pseudo-selection pressure, is used to narrow down to the $10^2$ scale, and finally, docking poses are confirmed by human hands to increase reliability. This cycle was repeated multiple times to design five iePETases (In Silico Evolution PETase).

Rational design requires expertise in protein engineering, an aspect that is difficult to implement. Therefore, we proposed the "Integrate Rational Design Pipeline" (Fig. 3.6) to perform rational design by combining useful sites from other proteins and mutants. This method utilizes information on more active LCCs similar to PETase and the most active known PETase mutants, DuraPETase and DepoPETase, and combines them to design new PETase mutants. Finally, we designed 9 iePETases (Integrate Rational Design PETase) by this method, confirming docking poses and eliminating unnatural structures.

Through the above process (see Model page for details), we obtain modified PETases with the desired properties of increased enzymatic activity. We will then incorporate these into the BIND-PETase gene circuit (Fig. 3.2 bottom) to improve the overall function of BIND-PETase.

Superiority and Novelty

In previous studies, the function of BIND-PETase was evaluated by fusing only PETase (WT) with csgA. Our novelty is that we aimed to improve the function of BIND-PETase by replacing the PETase(WT) part of the previous study with PETase mutants improved by various In Silico Evolution Pipeline and Integrate Rational Design Pipeline (Fig. 3.2 bottom).

ECB Module

Components of the ECB Module

In this section, the genetic circuit of the ECB Module is described. The ECB Module consists of two foundational technologies: a pSoxS Promoter, called an Electro inducible promoter, and a Cre/loxP system.

Electrically inducible promoter (pSoxS Promoter) is a specific promoter in which the expression of downstream genes is promoted by electrical stimulation. Although it originally functioned as part of the oxidative stress response operon in E. coli, it is used to achieve electrochemical control of expression. First, the application of voltage causes oxidation of pyocyanin (a redox inducible substance) at the electrode. The oxidized pyocyanin then enters the cell and oxidizes the SoxR. SoxR is permanently expressed immediately after expression. The oxidized SoxR acts on the pSoxS to promote transcription of its downstream genes. This mechanism increases the expression of downstream genes (Fig. 3.7).

Fig. 3.7. Mechanism of pSoxS promoter^[3]

The Cre/loxP system is a genetic recombination experiment that uses a site-specific recombination reaction which occurs when the DNA recombinase Cre acts on a DNA sequence called a loxP sequence. The loxP sequence is a DNA sequence consisting of 34 bp. When the Cre Recombinase recognizes the loxP sequence, Cre cuts out the region between the two loxP sequences. This is an irreversible reaction, and this mechanism can be used to remove specific DNA sequences (Fig. 3.8).

Fig. 3.8. Mechanism of Cre/loxP system

The components of the ECB Module are summarized in Table. 3.2.

Table. 3.2. List of parts used in plasmids^[4]^[5]

Parts	Explanation	References
PlacIq	Promoter which initiates the expression of lacI protein	BBa_K3257003
lacI	Gene encoding Lac repressor	Sheets M. et al. 2020
PlacUV5	Promoter activated by IPTG induction	Sheets M. et al. 2020
Pcon	Constant expression promoter	BBa_J23100
SoxR	Transcriptional activators of pSoxS	BBa_K554003
pSoxS	Electrically inducible promoter	BBa_K4216006
Cre	Protein that cuts out the sequence between two loxP sequence	BBa_I718007
PW4-loxP-TT-loxP-mRFP1	Parts that express red fluorescence when the Cre-loxP system is activated	Sheets M. et al. 2020
sfGFP	Improved green fluorescent protein	Amplified from Addgene (Plasmid #133872, Silverman et al. 2020)

Design of the ECB Module

The gene circuit created in our project is shown in Fig. 3.9 below. In the ECB Module, the combination of these techniques results in different genes being expressed at the first and second application of electrical input. The permanently expressed SoxR (reduced state) is oxidized by pyocyanin at the first voltage application. The oxidized SoxR then acts on the pSoxS promoter to promote the expression of the downstream gene, Cre Recombinase (Fig. 3.9). Then, the Cre recognizes the loxP sequence and removes the terminator, resulting in the constant expression of gene A. In the second application of voltage, SoxR is oxidized again, which promotes transcription by the pSoxS, resulting in the expression of the downstream gene B with the terminator removed (Fig. 3.10).

Fig. 3.9. Gene expression of the ECB Module after the first voltage application

Fig. 3.10. Gene expression of the ECB Module after the second voltage application

Any gene can be incorporated in gene A and gene B. By this mechanism, two different genes can be selectively expressed depending on the number of electrical inputs.

Superiority and Novelty

Previous studies have confirmed terminator truncation by Cre, light-dependent activation of Cre, as well as the electoral-responsiveness of pSoxS. Our team achieved electrically responsive Cre expression by expressing Cre downstream of pSoxS. In addition, the ability to perform electro responsive activation allows it to be applied in a wider range of environments. Furthermore, by applying the Cre/loxP System to two plasmids with different expression conditions, a two-step electrical response can be achieved, which enables applications in expression control by simple input and in biosafety.

PET TWINS: How they work together

Introduction

By incorporating the BIP and ECB module being proposed, we developed a system called PET TWINS. This system uses the positive attributes of both modules, as will be explained. The development of this new module to degrade PET offers a solution to many of the problems we face today. This promotes the industrial use of PETase, which has yet to be widely adopted.

The benefit of utilizing the ECB module is the ability to manipulate gene expression by electrical stimulation. By implementing this gene regulation system to the BIP module which degrades PET, the expression of fusion protein: BIND-PETase will be controllable. In addition, in order to prevent E. coli from proliferating to the clean environment after the role of PET degradation is completed, a toxin gene called mazF will be expressed, where its function is to kill E. coli itself, contributing to biosafety. Its mechanism can be explained as follows.

Fig. 3.11. Overview of PET TWINS

Full Mechanism

State of no voltage stimulation

In the absence of electrical input, SoxR is expressed. However, the pSoxS promoter remains inactive, preventing the expression of Cre Recombinase, subsequently halting the expression of downstream genes.

Fig. 3.12. Gene expression in the absence of electrical stimulation

First Electrical Stimulation

By adding the first electrical input, pSoxS gets activated. This leads to the expression of Cre recombinase. As mentioned earlier, Cre recombinase binds to the loxP site of both the sequences, excising the section surrounded with loxP. Since the double-terminator is surrounded by two loxP sites, the addition of Cre recombinase removes it. This subsequently expresses BIND-PETase, a fusion protein composed of csgA and PETase. At this stage, mazF will not be expressed since the pSoxS promoter found in the third sequence is immediately inactivated by reverse voltage (Details are mentioned in the Modeling Section). From the enzymic activity of PETase, the degradation of PET nearby the BIND-PETase expressing E. coli will be catalyzed.

Fig. 3.13. Gene expression after first electrical stimulation: Cre Expression

Fig. 3.14. Gene expression after first electrical stimulation: Cre binding to loxP

Fig. 3.15. Gene expression after first electrical stimulation: BIND-PETase expression

Second Electrical Stimulation

Although the activated BIP Module has advantages where purification is not required to use PETase, there are risks in using genetically engineered E. coli. This issue brings limitations to the practical use of this module, related to biosafety concerns. Therefore, the second electrical input is introduced to help solve this problem.
By adding the second electrical input, pSoxS on the third sequence will be activated. This expresses mazF, subsequently killing the E. coli itself. The E. coli will be destroyed; thus, biosafety issues can be resolved.

Fig. 3.16. Gene expression after second electrical stimulation

Conclusion

The BIP and ECB Modules can work together with this mechanism, which can control the expression of BIND-PETase using electro stimulations, at the same time considering potential biosafety issues. The application of this system can enhance the feasibility of utilizing PETase in an industrial setting.

References

Zhu B. et al. (2022), Enzymatic Degradation of Polyethylene Terephthalate Plastics by Bacterial Curli Display PETase, Environ. Sci. Technol. Lett. 9(7), 650-657.
https://doi.org/10.1021/acs.estlett.2c00332 ↩︎ ↩︎ ↩︎
Yinglu C. et al. (2021), Computational redesign of a PETase for plastic biodegradation by the GRAPE strategy, ACS Catal, 1340–1350. https://doi.org/10.1101/787069 ↩︎ ↩︎
Joshua L. et al. (2022), Synthetic biology and bioelectrochemical tools for electrogenetic system engineering,Science Advances, https://doi.org/10.1126/sciadv.abm5091 ↩︎
Sheets M. et al. (2020), Light-Inducible Recombinases for Bacterial Optogenetics, ACS Synth. Biol. 9, 2, 227–235, https://doi.org/10.1021/acssynbio.9b00395 ↩︎
Silverman A. et al. (2020), Design and Optimization of a Cell-Free Atrazine Biosensor, ACS Synth. Biol. 9, 3, 671–677, https://doi.org/10.1021/acssynbio.9b00388 ↩︎