Part:BBa_K3997000

IsPETase

Usage and Biology

Polyethylene terephthalate (PET) is the most widely produced polyester plastic and its accumulation in the environment has become a global concern. At the same time, the daily intake of microplastics by humans is gradually increasing, which damages human health. Therefore, researchers believe that it is important to develop an environmental-friendly plastic degradation method by using microorganisms. Recently, a novel bacterial strain called Ideonella sakaiensis 201-F6 has been discovered that produces a couple of unique enzymes, IsPETase and MHETase, enabling the bacteria to utilize PET as their sole carbon source.

Figure 1. Action and function of IsPETase in PET degradation...

The enzyme IsPETase is a hydrolase, and it is crucial for hydrolysis of PET. To verify this property, we use E. coli as the starting strain and construct an engineered strain of IsPETase to explore its biological activity of the hydrolysis of PET. To purify the protein, we also transfer the plasmid expressing IsPETase into BL21(DE3). We use pGEX as backbone and add a GST tag at its N-terminal. The enzyme is under the regulation of T7 promoter and can be induced by adding IPTG.

The T7 promoter is often used for protein overexpression. It is powerful and specific. It is completely controlled by T7 RNAP. When T7 RNAP is present in the cell, the T7 expression system occupies an absolute advantage compared to the host expression system. Its expression The speed is 5 times that of the former.

Experimental approach

Purification of IsPETase( ~35.13 kDa)_BL21(DE3) In order to present the function of the part, the IsPETase and MHETase gene were expressed in E. coli under the control of T7 promoter. Then the bacterial cells are collected and crushed. The samples of whole expression cell lysate, supernatant and pellet of cell lysate were analyzed using SDS-PAGE and Western blot (only for his-tag proteins from pET28a vector), which is found in the corresponding protein band of approximately 35 kDa (Figure 2).

IsPETase( ~35.13 kDa)_BL21(DE3)

Fig.2 SDS-PAGE (left) and western blot (right) analysis for IsPETase cloned in pET28a and expressed in BL21(DE3) strain...

Lane M1: Protein marker

Lane M2: Western blot marker

Lane PC1: BSA (1μg)

Lane PC2: BSA (2μg)

Lane NC: Cell lysate without induction

Lane 1: Cell lysate with induction for 16h at 15 oC

Lane 2: Cell lysate with induction for 4 h at 37 oC

Lane NC1: Supernatant of cell lysate without induction

Lane 3: Supernatant of cell lysate with induction for 16h at 15 oC

Lane 4: Supernatant of cell lysate with induction for 4 h at 37 oC

Lane NC2: Pellet of cell lysate without induction

Lane 5: Pellet of cell lysate with induction for 16h at 15 oC

Lane 6: Pellet of cell lysate with induction for 4 h at 37 oC

The primary antibody for western blot is anti-His antibody In this project，western blot (right) analysis for IsPETase was cloned in pET28a, the primary antibody for western blot is anti-His antibody. IsPETase-His protein was successfully expressed.

Figure3. SDS-Page of IsPETase-His...

In addition, IsPETase genes were also cloned to the expression vector pGEX-6P-1, which produce recombinant protein fusion with Glutathione-S-transferase (GST) tag.

Fig.4 SDS-PAGE analysis for IsPETase cloned in pGEX-6P-1 and expressed in BL21(DE3) strain...

Lane 6: IsPETase Cell lysate without induction for 20 h at 16oC

Lane 7: IsPETase Cell lysate with induction for 20 h at 16oC

Lane 8,9,10: GST elution fractions of purification of lane 7 by GST-affinity chromatography

References

3. Chun-Chi Chen et al. General features to enhance enzymatic activity of poly(ethylene terephthalate) hydrolysis, Nature Catalysis(2021).

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal NheI site found at 304
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal XhoI site found at 871
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal AgeI site found at 627
  
• 1000
  COMPATIBLE WITH RFC[1000]

Improvement by 2022 Beijing_United

Compared to the old part BBa_K3997000 on IsPETase, which is a biological part submitted by iGEM21_WFLA_YK_PAO in 2021, IsPETase has the characterized for the activity of degrading PET materials. However, IsPETase didn’t totally achieve degrading PET materials. So it is really important to identify other candidates for PET materials degradation.

In this project, our team carried out an efficiency enzyme SbPETase for this part in the laboratory, adding data from PET materials degradation testing to dedicate protein function. What’s more, we also developed several mutants of SbPETase that can be used for both PET and BHET, which also provides more ideas for future iGEM teams to optimize PET materials degradation enzymes. After screening SbPETase and its mutants by detecting the compounds in the reaction mixture, we identified several more active mutants, thereby achieving PET and BHET materials degradation.

Based on the activity assessment, molecular of 13 protein mutants was docking with PET and BHET molecules. Figures 8 and 9 represent the small PET and BHET molecules wrapped in 13 protein mutants.

Figure 8. Docking conformation of BHET in the active site of PETase..

Figure 9. Docking conformation of PET in the active site of PETase..

Moreover, the binding ability of PET and BHET to 13 protein mutants was determined by calculation and distance.The top 7 by binding distance of BHET to 13 protein mutants：L61T + W132H，L61T + W132H + R259A，L61T，W132H + R259A，W132H + R259A，W132H，R259A；According to the binding distance between PET and 13 protein mutants, the top 4:T212F，L61T + W132H + R259A，W132H and WT.

Table 1. Mutants and its binding energy

Figure 10. Binding energies of the PETase and mutants using BHET as substrate.

Figure 11. Binding energies of the PETase and mutants using PET as substrate.

SbPETase Mutant (L61T + W132H + R259A)

Name: SbPETase Mutant (L61T + W132H + R259A)

Base Pairs: 834 bp

Origin: Escherichia coli, synthtic

Properties: The triple SbPETase Mutant (L61T + W132H + R259A) with increased PET lysis activity

Usage and Biology

Bba_K4290036 is the triple mutant of SbPETase. SbPETase is a novel enzyme for Poly (ethylene terephthalate) (PET) hydrolysis. PET is one of the most commonly used plastics worldwide and its accumulation in the environment is a global problem [1]. PETase has been reported to exhibit higher hydrolytic activity and specificity for PET than other enzymes at ambient temperature [2]. Enzymatic degradation of PET using PETase provides an attractive approach for plastic degradation and recycling [3]. Various enzymes showing PET depolymerization activity have been identified and investigated in recent years, such as cutinases, PETase, MHETase, lipases, and esterases[4-9]. SbPETase can degrade PET into small fragments, then transport the degraded products for further "digestion", and finally convert them into two relatively simple organic compounds, ethylene glycol, and terephthalic acid [2].

Figure 1. Schematic extracellular expression and utilization of SbPETase for PET lysis.

Construct design

1. pET22b-PelB-SbPETase and pRSFdeut-1-kil construction

SbPETase was inserted into the downstream of pelB (Figure 2A) for the periplasmic expression of SbPETase and the gene for Kil was cloned into the pRSFduet1 vector (Figure 2B) with the aim to promote the release of periplasmic SbPETase into the culture medium.

Figure 2. Plasmid profiles in this project. A. pET22b-PelB-SbPETase, B. pRSFdeut-1-kil.

We amplify SbPETase was amplified from the genome of S. brevitalea sp. nov (Figure 3A.) and ligated it to the double-enzyme digestion pET22b vector, and We amplify Kil by PCR from the plasmid containing the Kil gene (Figure 3B.), ligated to the double-enzyme digestion pRSFduet1 carrier. The recombinant plasmids pET22b-PelB-Sbpetase and pRSFDeut1-Kil were 6240 bp and 3623bp in length. To verify if the plasmid is correct, we digest plasmid pET22b-PelB-SbPETase with ApaLI and pRSFdeut-1-Kil with AseI (Figure 3C, E). We send the constructed recombinant plasmid to a sequencing company for sequencing. The returned sequencing comparison results showed that there were no mutations in the ORF region (Figure 3D, F.), and the plasmids were successfully constructed. So far, we have successfully obtained the recombinant plasmids.

Figure 3. The constructions of plasmids. A. The gene fragment of SbPETase, B. The gene fragment of Kil, C. digest plasmid pET22b-PelB-SbPETase with ApaLI, D. the sequencing result of the recombinant plasmid pET22b-PelB-SbPETase, E. digest plasmid pRSFdeut-1-Kil with AseI, F. the sequencing result of the recombinant plasmid pRSFdeut-1-Kil.

2.Protein expression and purification

We transferred the plasmid pET22b-pelB-SbPETase into E. coli BL21(DE3), expanded the culture in the LB medium and added IPTG to induce protein expression when the OD600 reached 0.4. After overnight induction and culture, we collected the cells and ultrasonic fragmentation of cells to release the intracellular proteins. Next, we used nickel column (Ni-NTA) purification to purify the protein SbPETase (Figure 4).

Figure 4. SDS-PAGE result of SbPETase.

In order to test if Kil signal peptide can improve the yield of SbPETase proteins in the culture medium, we also co-transformed the recombinant plasmids pET22b-PelB-SbPETase and pRSFDeut1-Kil into E. coli BL21(DE3), expanded the culture in the LB medium and added IPTG to induce protein expression when the OD600 reached 0.4. After overnight induction and culture, we purified the protein SbPETase we wanted and collected the data (Figure 5). In Figure 5, when co_transformed pET22b-PelB-SbPETase and pRSFDeut1-Kil, the yield of protein SbPETase in the extracellular medium is higher than without it.

Figure 5. The yield of protein SbPETase both with and without Kil.

3. Biochemical characterization of SbPETase

We set up an in vitro system and confirmed the ability to use the SbPETase to degrade PET materials. As shown in Figure 4, we demonstrated that SbPETase could degrade PET and BHET into MHET and a small quantity of TPA through HPLC (Figure 6A, B), the optimum conditions showed that at 30°C, pH 7.0 (BHET as substrate) or pH 8.0 (PET film as substrate), SbPETase showed the highest activity.

Figure 6. HPLC analysis of SbPETase degrade PET and BHET materials. A. Result recorded with an HPLC system successfully identified the MHET and BHET peak when used BHET as substrate, B. Result recorded with an HPLC system successfully identified the MHET, BHET, and TPA peak when used PET as substrate.

4. Rational design of SbPETase by site-direct mutation

Due to the differences in substrate binding sites between SbPETase, and since reported mutants showed improved catalytic efficiency, we performed the following site-directed mutagenesis: Y60A, L61T, W132H, W132A, V181I, T212F, T212S, and R259A. All the SbPETase mutant proteins were obtained from the cultured medium directly and an in vitro activity assay was performed, which generated two mutants (SbPETaseW132H, SbPETaseR259A) with improved catalytic efficiency of degrading PET (Figure 7A). The activity of another mutant SbPETaseL61T was only improved towards degrading BHET (Figure 7A). We then combined these three mutants (SbPETaseW132H, SbPETaseR259A, SbPETaseL61T), to generate three double mutants and one triple mutant. An in vitro activity assay showed that the triple mutant had the highest catalytic efficiency towards PET and BHET degradation (Figure 7B)

Figure 7. Comparison of the SbPETase mutants activity of lysis of PET.A. detection of SbPETase mutants enzyme activity on BHET, B. detection of SbPETase mutants enzyme activity on PET.

We biochemically characterized a PET-hydrolyzing SbPETase from Schlegelella brevitalea sp. nov. using a high-efficiency secretion system. Overall, our study provides a foundation for accelerating the discovery of novel PETase variants screening platforms for industrial applications.

References

1.Sinha, V.; Patel, M. R.; Patel, J. V. PET waste management by chemical recycling: a review. J. Polym. Environ. 2010, 18, 8−25.

2.Shi L, Liu H, Gao S, et al. Enhanced extracellular production of is PETase in Escherichia coli via engineering of the pelB signal peptide[J]. Journal of Agricultural and Food Chemistry, 2021, 69(7): 2245-2252.

3.Kawai, F.; Kawabata, T.; Oda, M. Current state and perspectives related to the polyethylene terephthalate hydrolases available for biorecycling. ACS Sustainable Chem. Eng. 2020, 8, 8894−8908.

4. Ronkvist, Å. M.; Xie, W.; Lu, W.; Gross, R. A. Cutinase catalyzed hydrolysis of poly(ethylene terephthalate). Macromolecules. 2009, 42, 5128−5138.

5. Tournier, V.; Topham, C. M.; Gilles, A.; David, B.; Folgoas, C.; Moya-Leclair, E.; Kamionka, E.; Desrousseaux, M. L.; Texier, H.; Gavalda, S.; Cot, M.; Guémard, E.; Dalibey, M.; Nomme, J.; Cioci, G.; Barbe, S.; Chateau, M.; André, I.; Duquesne, S.; Marty, A. An engineered PET depolymerase to break down and recycle plastic bottles. Nature 2020, 580, 216−219.

6. Barth, M.; Honak, A.; Oeser, T.; Wei, R.; Belisario-Ferrari, M. R.; Then, J.; Schmidt, J.; Zimmermann, W. A dual enzyme system composed of a polyester hydrolase and a carboxylesterase enhances the biocatalytic degradation of polyethylene terephthalate films. Biotechnol. J. 2016, 11, 1082−1087.

7. Nikolaivits, E.; Kanelli, M.; Dimarogona, M.; Topakas, E. A middle-aged enzyme still in its prime: recent advances in the field of cutinases. Catalysts 2018, 8, 612.

8. Yoshida, S.; Hiraga, K.; Takehana, T.; Taniguchi, I.; Yamaji, H.; Maeda, Y.; Toyohara, K.; Miyamoto, K.; Kimura, Y.; Oda, K. A bacterium that degrades and assimilates poly(ethylene terephthalate). Science 2016, 351, 1196−1199