Part:BBa_K3576001

PETase expression system

It is the key part that is responsible for expressing PETase. The PETase can hydrolyze PET to MHET. In order to ensure that PETase can be fully in contact with extracellular PET. The sequence of coding pelB signal peptide was added before PETase.

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  COMPATIBLE WITH RFC[21]
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal NgoMIV site found at 97
  Illegal NgoMIV site found at 123
  
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal BsaI site found at 267
  

1. Protein expression test

SDS-PAGE electrophoresis was used to check the expression of PETase protein. As shown in Figure 1, compared to the blank control, the lane contained PETase (40 KDa) protein indicated that this protein has been successfully expressed.

Figure 1 Protein SDS-PAGE electrophoresis results of PETase.

2. Enzyme Activity Test of PETase

The enzyme activity of PETase was performed by p-NP assay which is a common way to quantify hydrolytic activity. We selected p-Nitrophenylbutyrate (pNPB) as the substrate, which can be hydrolyzed to p-nitrophenol (pNP) (Figure 2-A). The concentration of pNP can be measured by the characteristic absorption at 405 nm. As shown in Figure 2-B, with the extension of reaction time, the OD405 value of p-NP gradually increased, which indicates that the degradation activity of the PETase.

Figure 2 (A)The mechanism of pNPB degradation; (B) OD405 of pNPB hydrolysis by overexpressed PETase.

Introduction

This program designed a method for degradation of Polyethylene Terephthalate (PET) by an engineered bacteria which expresses PETase encoded in constructed plasmid transformed into the Escherichia coli. Whether engineered E. coli express PEATase as designed, is assessed by repeating the experimental process and comparisons of the catalytic ability.

Method

The purpose of our experiment is to produce engineered bacteria to express PET enzyme, so that the expressed PETase can catalyze the degradation of PET. The competent cells of DH5a and BL21 were first prepared in the main part of the experiment. The competent bacteria were further transformed with constructed plasmids containing resistance gene to ampicillin and coding gene for PETase. The transformed bacteria then expanded further. In order to produce adequate enzymes, we chosed the BL21 strain for PETase production, an engineering bacterium that were optimized for protein expression. In the whole experiment process, we validated all experimental procedures by a series of standard operations. To verify successful transformation of plasmid DNA, we used DH5a strain to do two related tests. The colony PCR and plasmid digestion experiments were carried out respectively. The products of PCR and digestion were then verified using DNA electrophoresis. For PETase production, we selected BL21 strain which were transformed with expression plasimid successfully. At last, SDS-PAGE verification was performed after lysis of the expressed bacteria and the untransformed negative control bacteria.

Results

The recombinant plasmid of PET enzyme uses pET21a (+) as shown in Figure 1. This series of vectors is the most commonly used prokaryotic expression system, which controls the expression of the target protein through lactose manipulation elements. The pET21 (+) vector is integrated with the protein sequence that can express Ampicillin resistance, which is convenient for us to screen positive clones. At the same time, we added a 6xHis tag to the target sequence, and added BamHI/EcoRI digestion sites at both ends to facilitate the subsequent digestion linking experiment and protein purification.

Figure 1 The recombinant plasmid of PETase-pET21a（+）

The transformed E. coli were subjected to plasmid extraction and agarose gel electrophoresis, and the bands marked in the figure appeared between 1000-1500 bp, indicating that E. coli from all six sample sources were probably successfully transformed. The PCR product size was considered as 1493bp as show in Figure2.

Figure 2 Result of colony PCR Line1: DNA marker 1; Line2 and 9: DNA marker 2; Line3, 4, 5, 6, 7 and 8: plasmid extracted from transformed Escherichia coli

The extracted plasmid was electrophoresed after enzymatic digestion, and the size of the band labeled in the figure (1000-1500bp) is consistent with the nucleic acid sequence between the two restriction endonuclease specific binding sites of EcoR1 and BamH1(1249bp) and the absence of this band in the negative control, indicating that the nucleic acid sequence expressing PETase was probably successfully inserted in the plasmid. The Enzyme section size was considered as 1249 bp as show in Figure 3.

Figure 3 Result of enzyme digestion Line 1 and 12: 1kb plus DNA marker; Line2: D2000 DNA marker; Line 3, 4, 5, 6, 7, 8, 9 and 10: plasmid samples after enzyme digestion; Line11: negative control group

The protein extracted from the successfully transformed E. coli treated with Ripa lysate and PMSF showed bands between 40 and 55 kDa in size （which is the PETase band） after SDS-Page protein electrophoresis, and there was no such band in the negative control group, which was consistent with the expected results, indicating that E. coli successfully expressed PETase as show in Figure 4 .

Figure 4 Result of SDS-PAGE testing Line1&2: Page-Ruler Prestained Protein Ladder; Line3&4: negative control; Line5&6: Protein extracted by Ripa lysate and PMSF

After three experiments to take the average value, the absorbance curve of the experimental group at 405nm wavelength showed an overall rising trend, with a fast growth rate in 0-15min, and leveled off to reach a plateau at about 45min; the curve of the control group was flat without fluctuation and at a lower value as show in Figure 5. Blue line/red line: experimental groups containing PETase-expressing bacterial broth, acetonitrile and 4-nitrophenyl butyrate to explore the enzymatic activity of PETase at different concentrations.Gray line: control group containing bacterial solution and acetonitrile to exclude the effect of absorbance of 4-nitrophenyl butyrate on the experimental results. Yellow line: control group containing saline, acetonitrile and 4-nitrophenyl butyrate to exclude the effect of absorbance of the bacterial solution on the experimental results

Figure 5 Result of pNp catalytical reaction

Conclusion

We introduced the plasmid DNA expressing PETase into E. coli, and the presence of the target sequence was verified by PCR and following agarose gel electrophoresis. We further confirmed the success of the transformation after two experiments, enzyme digestion electrophoresis of the extracted plasmid and SDS-Page electrophoresis of the protein extracted from the successfully transformed bacterial broth. After that, we used p-NP assay to verify the enzyme activity by measuring the absorbance value of p-nitrophenol, the colored product of p-Nitrophenylbutyrate hydrolyzed by PETase, at 405 nm. After the experiments results were analyzed. The dynamic curve of the experimental group showed an overall increasing trend and was positively correlated with the concentration of the bacterial solution, reaching a plateau at about 45 min, while these performances did not appear in the control group. This means that the E. coli from the experimental group successfully expressed the PETase with hydrolytic activity.

2023 Bioplus-China’s Characterization of BBa_ K3576001

In 2023, we worked on degradation PET plastics by using engineered microorganisms. Our team performed a new characterization of the existing part BBa_K3576001 (ASTWS-China, 2020), contributing some meaningful data and conclusions. In addition, we would like to share shared with you some recent relevant research, and hope the addition of new data and papers can give some assistance and guidance to the future iGEM teams.

Experience and Results

The expression of the protein and the detection of PETase activity is the key to the experiment. This year, we built the prokaryotic expression system of PETase, which successfully expressed the protein and optimized the method of detecting the enzyme activity.

Constructed PETase_pET21-a (+) Plasmid

The recombinant plasmid of PETase used pET21-a (+) backbone. PCR amplification was performed using the PETase-SpyCatcher plasmid as the template, and the product was detected by 1% agarose gel electrophoresis, which showed a specific fragment at about 900 bp, which was consistent with the expected size (Figure 1). The vector was constructed using seamless cloning technology. The recombinant expression vector was transformed into E. coli BL21 (DE3) competent recipient cells, and the correct colonies were identified by colony PCR for the following experiment-protein expression (Figure 2).

Figure 1: Amplified the target gene. 1: DNA marker; 2-4: PCR amplification bands of PETase.

Figure 2: Colony PCR of PETase_pET21-a (+). 1: DNA marker; 2-9: PCR amplification bands of PETase.

Protein Expression

The PETase expression was detected by SDS-PAGE after induction by IPTG. As shown in the Figure 3, compared to the simple without induction, PETase induction by IPTG have protein bands 35-45 kDa, and the results indicate that the protein has been successfully expressed in BL21(DE3).

图3

Figure 3: SDS-PAGE analysis of PETase-R. 1: Marker; 2: Before IPTG induction simple; 3: With IPTG induction simple; 4: Supernatant of ultrasonic crushing with induction; 5: Pellet of ultrasonic crushing with induction; 6: Flow-through solution after nickel column affinity; 7: 20 mM imidazole eluent.

Activity Test of PETase

The enzyme activity of PETase was performed by p-NP assay. p-Nitrophenyl Butyrate (p-NPB) as the substrate, which can be hydrolyzed to p-nitrophenol (p-NP), and the pNP can be measured by the characteristic absorption at 405 nm.

The bacterial supernatants after ultrasonic crushing and bacteria solutions were mixed with substrate separately, it showed that the OD405 value of supernatants was higher than that of using bacterial solutions directly. And the team ASTWS-China used the bacterial solution to do the enzyme activity experiment. With the prolongation of reaction time, the OD405 value of supernatant increased gradually, indicating that the enzyme activity was stable.

图4 Figure 4: OD405 of pNPB hydrolysis by overexpressed PETase. (A) Compare the PETase enzyme activity in bacterial solutions and ultrasonic crushing supernatants. (B) PETaes hydrolysis reaction in ultrasonic crushing supernatants.

A Few Recent Researches of Hydrolases of PET Depolymerization

In 2022, Lu et al. used a structure-based, machine learning algorithm to engineer a robust and active PET hydrolase, FAST-PETase, that can completely degrade PET plastic in short time. In the experiment, in order to improve the activity of the PET degrading enzyme (PETase), the researchers made a machine learning algorithm to predict the mutation of the enzyme, and after engineering and testing the mutants, they selected an enzyme, FAST-PETase (functional, active, stable, and tolerant PETase), containing five mutations and shows superior PET-hydrolytic activity. Compared with wild-type PETase, FAST-PETase has high activity and excellent degradation ability. The research is published in Nature.

The PETase degradation of PET is a two-step process in which the first PETase binds to the substrate PET and the second enzyme catalyzes the hydrolysis of the substrate. The PETase was found to have the ability to degrade the hcPET (high-crystallinity PET), but with low enzymatic activity. Chen et al. (2022) designed and constructed an engineered yeast whole-cell biocatalyst to simulate both the adsorption and degradation steps in the enzymatic degradation process of PETase to achieve the efficient degradation of hcPET. In experiments, the adsorption module hydrophobic protein HFBI and degradation module PETase were artificially designed and fixed to the cell surface of yeast cells by surface co-display technology, and through optimization, the efficient biodegradation of hcPET was realized. The introduction of adhesion modules is the key to the efficient degradation of hcPET in this system. This study demonstrates engineering the whole-cell catalyst is an efficient strategy for biodegradation of PET.

There are still some problems need to be solved in this field, but PETase has shown promising application prospects. Further understanding of the molecular mechanism of PETase will provide more scientific basis for future practical applications. Therefore, we have a vision for an environmentally friendly method of recycling PET through synthetic biology.

Reference

[1] Chen Zhuozhi et al. (2022) Biodegradation of Highly Crystallized Poly(ethylene terephthalate) through Cell Surface Codisplay of Bacterial PETase and Hydrophobin. Nature Communication, DOI https://doi.org/10.1038/s41467-022-34908-z [2] Lu Hongyuan, et al. (2022) Machine Learning-aided Engineering of Hydrolases for PET Depolymerization. Nature, 604: 662-667.