Part:BBa_K3576001:Experience

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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). 图1 图2

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.

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