Our project has achieved Engineer success. At the beginning of the project, we designed the engineering cycle of the project, and continued to improve the engineering cycle as the project progressed. Engineering success was achieved by making an effort to follow our engineering design cycle:
Research(a) → Imagine(a) → Design(a) → Build(a)→ Test(a)→ Learn(a)→ Improve(a)→ Research(b)→Imagine(b) → Design(b) → Build(b)→ Test(b)→Learn(b)→Improve(b)→Success
Fig.1. Engineering design cycle.
In the early stage, we did a lot of literature research and read some literature on improving the thermal stability of proteins and enzymes. We study the methods and principles used in the literature and apply their methods to our projects.
By reading several literatures, we have got some methods that can improve the thermal stability of proteins, such as changing the rigidity, reducing the conformational stability, and changing the interaction force.We tried to perform single point mutations according to the methods given in the literature.
We have designed several methods to improve the thermal stability of proteins such as adding hydrogen bonds, salt bridges, enhancing hydrophobic interactions, proline mutations and adding disulfide bonds. And then we divided into several groups and tried to use these methods for single point mutations. It is a remarkable fact that our mutations are not random, but a rational design based on structure. We hope to improve the thermal stability of PETase by mutating these sites.
Modeling is an extremely important part of synthetic biology. A successful model can guide and promote the implementation of the project, as well as the summary and feedback of the work. In our object we used FoldX, SWISS-MODEL and other software to model mutants we designed. Through modeling, we got new pdb files helping us to test the thermal stability of these mutants.
We used some software and websites to predict the thermal stability of mutants, including FoldX, MD, Docking, PyMOL, Fireprot, CUPSAT, I-Mutant, etc. ddG is an important parameter for us to evaluate protein thermal stability. Some of these websites will give the value of ddG, and some will give the instability index of the protein. We used FoldX to test the ddG of our mutants. While MD is used to turn ddG to ΔTm, which helped us intuitively test the thermal stability of mutants.
However, after making predictions, we found that not all mutants are satisfactory. The structure of some single point mutations met our expectations, such as the successful construction of a new salt bridge, but the predicted results were not satisfactory. We were very confused about this and tried to find why.
Based on the above problems, we make reasonable guesses. We believe that the increase of mutation sites will cause the effect of single point mutations to not be shown. For example, the newly introduced salt bridge is too close to the enzyme catalytic center, so it will affect the enzyme activity. This result was not what we wanted. So we will make improvements based on the problems encountered.
In response to the above problems encountered in Learn, we have made improvements. We have created a relatively complete mutant screening process: preliminary screening of all mutants (FoldX, Fireprot, CUPSAT, I-Mutant) → visual screening (PyMOL, SWISS-MODEL). We removed some sites with unsatisfactory ddG data and unreasonable structure. Finally, we got a mutation site with better results.
Further reading the literature, we found that many literatures mutate not only one site. They made multiple mutations to better improve the thermal stability of the protein. We believe that multiple site mutations are an excellent method to improve the thermal stability of PETase. After all, the effect of a single mutation is limited.
We believe that we should not limit our project to single point mutations, because single point mutations are very limited to the improvement of thermal stability. We should also carry our multi-site combinatorial mutations, so how should we perform combinatorial mutations?
After intense group discussion, we finally chose two ways to carry out combination mutation-rational design and random combination. We divided the members into two groups. The members of the rational design group consider combining the sites with the best single point effect and combining the structure for analysis, while the members of the random combination use the computer to perform random combination, including 5-15 mutations may exist All combinations.
For the mutants in the above Design, we still use FoldX for model building. This time we used the'mutate multiple residues' in FoldX. This feature helps us to obtain new pdb files. This is a very heavy work, we need to spend a lot of time to run FoldX.
We still use the software and website mentioned above to predict the thermal stability of mutants. In addition, we have newly added PyMOL visual screening, which is also the experience we learned from the previous step. Certain properties such as increased hydrogen bonds are displayed at some sites when single mutations are performed, but we do not display them in multiple mutations, but their stability is still improved, which makes us very confused.
Our analysis may be because FoldX can only move the side chain when making a mutant, but the main chain does not move, resulting in its characteristics cannot be displayed.
In addition, we also found that when a single point mutation exists independently, ddG larger, but when combined with other single point mutations, it will not fully exert its stability in single point mutations. We suspect that the increase in mutation sites may lead to changes in the stability-enhancing factors that originally existed in the single-point mutation, which may not fully function in multiple mutations.
For the problems encountered in the test, we thought about it and gave the corresponding solutions: The mutant got by FoldX is subjected to energy minimization again, and then analyzed.
After two cycles of engineering design, we finally designed the most ideal mutant-CRUSHER. It satisfies our improvement of PETase’s thermal stability and proves that our engineering is successful.
Single point mutations will be commercially constructed with the wild-type PETase plasmid as a template.The mutants during the accumulation step will be constructed by using a QuickChange site-directed mutagenesis kit. The introduced mutations will be confirmed by sequencing
Cloning ,expression and purification
The microbial strains and plasmids used in this study are listed in Table 1. Petase and its mutants gene will be inserted into the vector pet-21b（+）. E. coli DH5α will be used for the propagation of plasmid DNA. E. coli BL21 (DE3) will be used for the expression of PETase and its mutants. The cells will be cultivated at 37 °C in Luria-Bertani medium (LB) (1 % w/v tryptone, 0.5 % w/v yeast extract, and 1 % w/v NaCl).
Protein expression will be then induced by adding 0.1 mM IPTG and further cultured at 16℃, 220 rpm for 16 h. The E. coli cells will be harvested by centrifugation (4,000 × g, 15 min, 4°C) and resuspended in lysis buffer (50 mM Tris-HCl, pH 7.5, 300 mM NaCl, 20 mM imidazole). Cells will be disrupted by sonication on ice and the lysate will be clarified by centrifugation (18,000 × g, 40 min, 4°C). Ni-NTA affinity resin (GE Healthcare, USA) will be used to capture the target proteins in lysate. After washing unbound proteins with the lysis buffer, the bound proteins will be eluted with elution buffer (50 mM Tris-HCl, pH 7.5, 300 mM NaCl, 250 mM imidazole), and the buffer will be exchanged for 50 mM Na2HPO4-HCl, pH 7.0, and 100 mM NaCl on a PD-10 gel filtration column. The purified protein concentration will be determined based on the calculated molar extinction coefficiency at 280 nm.
|Plasmid / Strains||Description|
|pET-21b (+)||E. coli expression vector for production of protein|
|Escherichia. Coli DH5α||used for the propagation of plasmid DNA|
Table 1 The plasmids and strains used in this study
Determination of apparent melting temperatures
A fluorescence-based thermal stability assay will be used to determine apparent melting temperatures. Protein (20 µL) in the buffer (50 mM Na2HPO4, 100 mM NaCl, pH 7.5) will be mixed with 5 µl 100-fold diluted SYPRO Orange dye (Molecular Probes, Life Technologies, USA) in a thin-walled 96-well PCR plate. The plate will be sealed with optical-quality sealing tape and heated in a CFX 96 real-time polymerase chain reaction (PCR) system (Roche LightCycler96) from 25 to 90 °C at a heating rate of 1.625 °C/min. Fluorescence changes will be monitored with a charge-coupled device (CCD) camera. The excitation and emission wavelengths are 490 and 575 nm, respectively. To obtain the temperature midpoint of the protein unfolding transition, a Boltzmann model will be used to fit the fluorescence data obtained by the CCD detector:)
X-ray Photoelectron Spectroscopy
X-ray Photoelectron Spectroscopy (XPS) will be used to obtain the content information of surface elements. A 20 μg/mL PETase solution and a 20 μg/mL mutant will be prepared and dropped on the surface of PET film. After leaving overnight, remove the protein solution and blow dry with pure nitrogen. Then the PET film will be washed with ultra-pure water and dried with nitrogen. X-ray photoelectron spectroscopy analysis will be conducted to detect the contents of C, N and O elements on the surface of PET film. PET film without protein modification will be used as a control.
Scanning electron microscopy（SEM）
The PET films enzymatically digested by PETase and its mutants will be taken out for use, the degradation will be observed by scanning electron microscopy. The PET films treated with PETase and its mutants will be washed with 1% SDS, distilled water, and then ethanol. The film will be air-dried, coated with Au, and subjected to FE-SEM observation with a JSM-6320F or a FEI Sirion operating at an electron beam intensity of 5 kV. PET film without enzyme treatment was used as a control.
Enzymatic Activity Assay of the Mutants
Mutants will be incubated with PET film (Good Fellow, crystallinity 45 %, thickness 0.175 mm, diameter 6 mm) in buffer containing 50 mM glycine-NaOH (pH 9.0) for 18 h at 30 °C. Then the enzymatic activity will be determined by calculating the “turnover rate” of enzyme at each experiment. The turnover rate will be calculated by normalizing the amount (moles) of mono (2-hydroxyethyl) terephthalic acid (MHET) produced in each experiment to the amount (moles) of enzyme present in each experiment, then the resultant ratio will be further divided by enzymatic degradation time (18×3600 sec). Standard MHET will be obtained from the complete hydrolysis of BHET which will be bought from Sigma. The detailed process is as follows. Four mM BHET will be incubated with 50 nM PETase and its mutants in 40 mM Na2HPO4-HCl (pH 7.0), 80 mM NaCl, and 20 % (v/v) DMSO at 30°C. After complete hydrolysis of BHET to MHET, resulting in a 4 mM MHET solution confirmed by HPLC. All the following enzymatic activities of PETase and its mutants will be determined in the same way.
To optimize the enzyme concentration, enzyme activity will be measured at 0.1 μg/mL, 0.5 μg/mL, 1 μg/mL, 5 μg/mL, 10 μg/mL, 50 μg/mL, 100μg /mL, 200 μg/mL and 400 μg/mL, respectively. To optimize pH, the reactions will be conducted in 50 mM Na2HPO4-HCl (pH 6.0-8.0), or 50 mM glycine-NaOH (pH 9.0-10). To optimize temperature, reactions will be conducted at 20 °C, 30 °C, 40 °C and 50 °C, respectively.
Stability Assays of Mutants
To detect the thermal stability of the mutants, we will measure the degradation activity of the mutants after incubation at 30°C in 50 mM glycine-NaOH buffer (pH 9.0) for 1-7 days. To detect the chemical/solvent stability of the mutants, the mutants will be hydrolyzed at 30°C for 18 h at 50 mM glycine-NaOH (pH 9.0), supplemented with methanol (final concentration, 10 % v/v), ethanol (final concentration, 10 % v/v), and Triton X-100 (final concentration, 0.1 % v/v), respectively.
Degradation of Commercial PET Bottles
Mutants will be incubated with 6 mm diameter hcPET cut out from different commercial PET bottles (brands: Coca-cola, Nestlé, and Pepsi-cola) in 50 mM glycine-NaOH buffer (pH 9.0) for 18 h at 30 °C, respectively. The production of MHET will be quantified by HPLC.
HPLC analysis of degradation product of PET.
HPLC will be performed on a Waters e2695 equipped with a HyPURITY C18 (Thermo Fisher Scientific, No 22105-254630) column (4.6 × 250 mm). The mobile phase will be methanol/18 mM phosphate buffer (pH 2.5) at a flow rate of 0.5 mL/min, and the effluent will be monitored at a wavelength of 240 nm. The typical elution condition will be as follows: 0 to 30 min, 25 % (v/v) methanol; 30 to 50 min, 25-100 % methanol linear gradient. The MHET peak area will be used to calculate the amount of MHET produced in each PET hydrolysis reaction.
All crystals will be grown by the microbatch-under-oil method otherwise specified. Mutants will be crystallized at Crystal screen 1 (HR2-110), Crystal screen 2 (HR2-112), PEGRX 1 (HR2-082), PEGRX 2 (HR2-084), Index (HR2-144), PEG/ION 1 (HR2-126) and PEG/ION 2(HR2-098) kits. X-ray diffraction data will be collected on beamline BL19U1 at the Shanghai Synchrotron Radiation Facility. PHENIX and Coot will be used to determine the structure.
Possible problems and solutions
(1) The digested DNA ran as a smear on an agarose gel
The restriction enzyme may be bound to the DNA substrate: Need to add SDS 0.1–0.5% to the loading buffer to dissociate the enzyme from the DNA.
(2) Extra bands or a smear on the gel
If larger bands are seen in the gel than expected, this may indicate binding of the enzyme to the substrate: May need to lower the number of units in the reaction and addition of SDS 0.1–0.5% to the loading buffer to dissociate the enzyme from the substrate may help.
(3) Recombinant DNA Creation Using DNA Ligase
Inefficient ligation: Confirm at least one fragment needs to be ligated containing a 5´ phosphate moiety. Purify the DNA to remove contaminants such as salt and EDTA. Remove phosphatase prior to ligation. Confirm the activity of the ligase by carrying out a ligation control may help.
(4) Insertion of Recombinant DNA into Host Organism
If cells are not viable: Need to calculate the transformation efficiency of the competent cells or use of commercially available high efficiency competent cells.
Gene of interest (GOI) may be toxic to the cells: Need to incubate plates at lower temperature or using a strain that exerts stronger transcriptional control over the DNA fragment or gene of interest.
Enzymatic Activity Assay of the Mutants
(1) The enzyme activity is not ideal (eg. lower than the wild PETase): Optimize enzymatic hydrolysis conditions (effects of temperature, pH, protein and substrate concentration, ions, organic solvents, etc.)
(2) Discrepancy in different experimental batches - a batch of large quantities of the purified, packaged and frozen (considering the effect of the repeated freeze-thaw on protein activity)
Expression and Purification
(1) Proteins are unstable-change vectors and add reductant in the buffer during purification.
(2) Target protein size is not correct - the structure of the protein has a certain influence on the determination of molecular weight. The protein size can be accurately determined by heating the denatured protein. Verify that the protein expression is complete and the protein is prematurely terminated. The formation of dimers even polymers, can be opened by heating denatured-protein disulfide bonds (adding DTT, -ME and other reducing agents) and other means, damage secondary structure, accurate determination of molecular weight size.
(1) No crystals or crystals of bad shape- Optimize the crystallizing conditions, protein concentration, temperature, etc.
 V. Tournier, C. M. Topham, A. Gilles, et al. An engineered PET depolymerase to break down and recycle plastic bottles. 2020, 580(7802):216-219.
 Rigoldi Federica, Donini Stefano, Redaelli Alberto, et al. Review: Engineering of thermostable enzymes for industrial applications.. 2018, 2(1):011501.
 Hyeoncheol Francis Son, Seongjoon Joo, Hogyun Seo, et al. Structural bioinformatics-based protein engineering of thermo-stable PETase from Ideonella sakaiensis. 2020, 141
 Austin Harry P, Allen Mark D, Donohoe Bryon S, et al. Characterization and engineering of a plastic-degrading aromatic polyesterase.. 2018, 115(19):E4350-E4357.
 A Z C , A Y W , A Y C , et al. Efficient biodegradation of highly crystallized polyethylene terephthalate through cell surface display of bacterial PETase[J]. ence of The Total Environment, 709.
 Cheng Y , Wang B , Wang Y , et al. Soluble hydrophobin mutants produced in Escherichia coli can self-assemble at various interfaces[J]. Journal of Colloid and Interface ence, 2020, 573.