PETase-G4S-50-5-C-R287V

IsPETase, a PET hydrolase discovered from the bacterium Ideonella sakaiensis 201-F6, has become a research favorite for its ability to efficiently degrade PET at low temperature ^[1]. It breaks PET into bis(2-hydroxyethyl) terephthalate (BHET) and 2-hydroxyethyl methyl terephthalate (MHET) by hydrolyzing the ester bond, followed by hydrolysis to the end products EG and TPA with the help of MHETase (Fig. 1). However, due to the surface hydrophobicity of PET, it is difficult for PETase to directly bind to it, which negatively affects the hydrolysis rate of PETase on PET ^[2].

Fig. 1. Schematic diagram of the PETase pathway for PET degradation.

Plastic-binding peptides are a class of short-chain peptides that can bind to PET plastic surfaces through hydrophobic binding and other forces, and are a potential solution to improve the degradability of PETase towards PET. However, the sources of PET-binding peptides are currently limited, and they are usually unearthed in a single-point trial-and-error manner, which is inefficient.

In this regard, firstly, we performed PET-binding peptide prediction with the help of deep learning, and, then, fused PETase with the efficient binding peptide obtained from the prediction and screening through linker to construct a fusion protein, so as to improve the binding ability of PETase to PET microplastics with the help of PET-binding peptide, thus improving the degradation efficiency.

We obtained short peptides with improved PET plastic binding ability, but there is still room for improvement in the expression level of the fusion proteins and the microplastic substrate binding ability. Therefore, we used Mutcompute-super to introduce beneficial mutations at specific sites, to further improve the expression level of the fusion protein and the degradation ability of the microplastic substrate by molecular modification ^[4].

Our fusion proteins consist of four basic components:
1) PETase
IsPETase-derived modified mutant with high catalytic performance and stability.

2) G4S linker
A flexible linker, consisting of 4 glycine and 1 serine in a single unit repeated three times, is used to link two structural domains of a fusion protein to achieve proper separation of functional domains to reduce their unfavorable interactions ^[3].

3) PET-binding peptide
We used both LSTM and graph neural network (GCN) models to optimise the prediction of high-affinity to obtain efficient PET-binding peptides. For further improvement, we used Mutcompute-super to introduce beneficial mutations at specific sites of PET-binding peptide, and then used the extracted bioinformatics features as the input for the 3D deconvolutional neural network of Mutcompute-super. We then combined a machine learning classifier to predict the optimal amino acid mutation combination to optimize the protein's folding conformation, and further improved the degradation ability of the fusion protein towards PET substrate ^[4].

4) His tag
The his tag added to the C-terminus of the fusion protein allows specific binding to immobilised metal ions such as Ni²⁺ by forming stable complexes, facilitating purification and testing of the fusion protein.

Fig. 2. Schematic diagram of the fusion protein gene expression cassette.

Molecular construction

The fusion protein gene was synthesised and cloned into plasmid pET-21b（with T7 promotor BBa_K4790075, lac operator BBa_K4790078, RBS BBa_K4790079, and T7 terminator BBa_K4790076). Based on the mutation site R287V predicted by the dry lab, we successfully constructed recombinant plasmid pET21b-PETase-G4S-50-5-C-R287V-His tag using MEGAWHOP cloning method.

Protein expression and purification

The recombinant plasmid was transformed into Escherichia coli BL21 (DE3) for expression and cultured in a 50 mL flask to express sufficient fusion protein.

After fermentation optimization with the fusion protein 50-4-N-G4S-PETase, we determined the final concentration of IPTG to be 0.05 mmol/L. After induction, the culture was incubated at 16°C and fermented for 48 h using TB medium. After culture, the fermentation broth was centrifuged, wall-broken by high-pressure homogenization, and then centrifuged again. The fermentation supernatant and supernatant of cell fragmentation were collected as the crude enzyme.

The crude enzyme activity was measured by continuous spectrophotometry method. The reaction system was 1500 μL including 30 μL 50 mmol/L pNPB, 30 μL crude enzyme liquid and 1440 μL 100 mmol/L pH 8.0 phosphate buffer. The generation rate of p-nitrophenol during 60 s was recorded at the wavelength of 405 nm. One enzyme activity unit (U) was defined as the amount of enzyme required to hydrolyze pNPB producing 1 μmol p-nitrophenol during 60 s at 60°C.

The crude enzyme liquid of the supernatant of cell fragmentation with higher enzyme activity was subjected to Ni²⁺ column affinity chromatography. After chromatography, the collected liquid was dialyzed, freeze-dried and re-solubilized. Then the obtained liquid is the pure enzyme solution. SDS-PAGE analysis showed that there was a clear band near 35 kDa (Fig. 3), indicating that the target fusion protein was successfully obtained after Ni²⁺ column affinity chromatography.

Fig. 3. SDS-PAGE analysis. M: Marker; FS: Fermentation supernatant; PC: Precipitation of cell fragmentation; SC: Supernatant of cell fragmentation; P: Purified enzyme.

Enzyme property characterization

1) Optimal pH
Enzyme activity was determined using phosphate buffer at 60°C 100 mmol/L pH 6.0-9.0 (gradient of 1.0). The relative enzyme activity at each pH was calculated using the highest enzyme activity measured as 100%. The highest enzyme activity of the fusion protein was at pH 8.0 (Fig. 4).

Fig. 4. Optimum pH of PETase-G4S-50-5-C-R287V.

2) Optimal temperature
The enzyme activity was determined by preheating the pH 8.0 phosphate buffer for 10 min at different temperatures (30, 40, 50, 60, 70°C). The highest enzyme activity measured was taken as 100% and the relative enzyme activity at each temperature was calculated. The enzyme activity of the fusion protein increased gradually at 30-60°C and decreased slightly at 70°C, with the highest enzyme activity at 60°C. (Fig. 5).

Fig. 5. Optimum temperature for PETase-G4S-50-5-C-R287V.

3) Thermostability
The pure enzyme solution was placed in a 50°C water bath and sampled at 24 h intervals to determine the residual enzyme activity. The enzyme activity at 0 h of reaction was defined as 100%. The fusion protein possessed good stability at 50°C reaction temperature (Fig. 6).

Fig. 6. Temperature stability for PETase-G4S-50-5-C-R287V at 50°C.

Characterization of PET degradation property

PET microplastics (final concentration 2.9 g/L) were degraded by 0.01 mg/mL fusion protein under the condition of 100 mM glycine-NaOH buffer (pH 9.0) at 50°C. A total of 50 μL reaction liquid was taken mixing with 450 μL methanol after 24 h PET substrate degradation. After centrifugation, the supernatant was collected and filtered by aqueous phase filtration membrane, then the filtrate was analyzed by high performance liquid chromatography (HPLC).

Since BHET can be categorized as the incomplete degradation product of PET, and TPA and MHET can be categorized as the final degradation product of PET, we selected TPA and MHET as the indicators to calculate the product release. Compared with the original enzyme, the product release amount of PETase-G4S-50-5-C-R287V was 207.49% of that of the PETase, and 164.38% of that of the wild type (BBa_K5157056)(Fig. 7).

Fig. 7. HPLC analysis of PET degradation of PETase and PETase-G4S-50-5-C-R287V.

Introduction of point mutation to PET-binding peptide 50-5 effectively affected PETase-G4S-50-C for PET degradation, so it was chosen to superimpose the mutations in the next step.

Future usage

We have fused PET-binding peptides obtained through model prediction with PETase through linkers, providing new insights to enhance the degradation capability of PETase. Meanwhile, these PET-binding peptides may also improve the degradation capabilities of other PET-degrading enzymes in the future. Furthermore, our model-predicted single-point mutations have achieved some success. Our team will conduct combinatorial mutations on the base of exceptional single-point mutations to futher enhance PET microplastic degradation effects. We aspire for our efforts to support and guide other iGEM teams while providing inspiration for more effective solutions to the PET microplastic degradation challenge in the future.

References

[1] Yoshida S, Hiraga K, Takehana T, et al. A bacterium that degrades and assimilates poly(ethylene terephthalate) [J]. Science, 2016, 351(6278): 1196-1199.
[2] Puspitasari N, Tsai S L, Lee C K. Fungal hydrophobin RolA enhanced PETase hydrolysis of polyethylene terephthalate [J]. Applied Biochemistry and Biotechnology, 2021, 193(5): 1284-1295.
[3] Chen X, Zaro J L, Shen W C. Fusion protein linkers: property, design and functionality [J]. Advanced Drug Delivery Reviews, 2013, 65(10): 1357-1369.
[4] Deng Z H, Cai C, Wang S T, et al. A protein design method based on amino acid microenvironment and EMO neural network, CN118136092A [P/OL].

Sequence and Features

Assembly Compatibility:

10
INCOMPATIBLE WITH RFC[10]
Illegal SpeI site found at 751
12
INCOMPATIBLE WITH RFC[12]
Illegal SpeI site found at 751
21
INCOMPATIBLE WITH RFC[21]
Illegal BglII site found at 614
23
INCOMPATIBLE WITH RFC[23]
Illegal SpeI site found at 751
25
INCOMPATIBLE WITH RFC[25]
Illegal SpeI site found at 751
Illegal NgoMIV site found at 142
1000
COMPATIBLE WITH RFC[1000]

Part:BBa_K5157103