Part 1 Bioinformatics prediction and analysis of anchor proteins
1.1 Selection of a robust host
Previous researches have compared the surface display system of different hosts as shown in Table 1. Candida Tropicalis was chosen as the host strain with strong viability, good safety and high industrial value.
Table 1 Comparison of surface display enzyme hosts
[1] D. Gercke, C. Furtmann, I. E. P. Tozakidis, J. Jose, Highly Crystalline Post-Consumer PET Waste Hydrolysis by Surface Displayed PETase Using a Bacterial Whole-Cell Biocatalyst[J].ChemCatChem 2021, 13, 3479
[2] Chen Z, Wang Y, Cheng Y, et al. Efficient biodegradation of highly crystallized polyethylene terephthalate through cell surface display of bacterial PETase [J]. Science of The Total Environment, 2019, 709:136138.
Strong viability
Candida Tropicalis is capable of absorbing alkanes and fatty acids as carbon sources and surviving in environments containing acids and phenolic substances.
Good safety
Candida Tropicalisis a level one biosafety strain. (https://www.atcc.org/products/20336)
We used CRISPR-CAS9 technology to knock out the key genes of uracil synthesis in wild strains, which turned them into uracil nutrition-deficient strains to prevent leakage to the environment. Here are strains on the plate with different concentrations of uracil. It was found that the uracil nutrition-deficient strain constructed could not grow in solid medium without uracil, and the colony number increased with the increase of uracil concentration.
Fig.1 Growth of uracil nutritionally deficient strain in solid medium with different concentrations of uracil. (a) Growth medium without uracil. (Coating) (b) Growth medium with 0.006 g/L uracil. (Coating) (c) Growth medium supplement with 0.06 g/L uracil. (Coating) (d) Growth medium without uracil. (Plate streaking). (e) Growth medium with 0.006 g/L uracil. (Plate streaking). (f) Growth medium supplement with 0.06 g/L uracil. (Plate streaking)
High industrial value
Compared with other surface display systems, the surface display technology on Candida Tropicalis has not been fully developed. Therefore, it is worthy to establish the system on Candida Tropicalis. In addition, Candida Tropicalis is maturely used in the production of long-chain diacid, which lays a good foundation for its industrial applications.
1.2 Prediction of GPI-anchored proteins
Our experiment focused on selecting the best potential anchor protein for PETase surface display system. To achieve this goal, an anchor protein screening system was used to predict feasible candidates for our surface display system. The screening system included different online protein databases. For surface display systems, each polypeptide follows the same structure with a signal peptide on the N terminus to lead the protein out of the cell, a serine/threonine to support the structure, and a GPI-anchored protein on the C terminus to bind to the inner cell membrane. Figure 1 illustrated the workflow of screening process.
Fig.2 Prediction model of GPI-anchored Protein.
The link of resources for GPI-anchored protein prediction were listed below.
Secreted signal peptide prediction database:
http://www.cbs.dtu.dk/services/SignalP/
GPI modification site prediction in fungi:
http://mendel.imp.ac.at/gpi/fungi_server.html
Prediction of transmembrane helices in proteins:
http://www.cbs.dtu.dk/services/TMHMM/
Basic protein properties prediction database:
https://web.expasy.org/protparam/
Protein subcellular localization prediction tool:
https://www.genscript.com/psort.html
Website for conservative domain prediction:
https://www.ncbi.nlm.nih.gov/Structure/wrpsb.cgi
Predictions of mucin type GalNAc O-glycosylation sites in mammalian proteins:
http://www.cbs.dtu.dk/services/NetOGlyc/
Results
129 GPI-anchored proteins (alleles included) were obtained, including ALS, Zan, HYR, HYR3, PHR, PHR2 and RHD3 gene families. The main information of several potential GPI-anchored proteins is presented in Table 2
Table 2 Putative GPI-anchored proteins in C. tropicalis
Take 4609 as an example to explain the workflow.
First, the big-PI Fungal Predictor was used to predict GPI modification sites and it was found that there are potential C-terminal GPI-Modification Sites at the position of amino acid residue 438. Then use the website SignalP 5.0 to predict the protein sequence numbered 4609, and the results show that Cleavage site between pos. 18 and 19: VSA-SY. Probability: 0.8063. Finally, the transmembrane domain prediction was performed by TMHMM. Results showed that there was no transmembrane domain and other related websites were used to predict its basic properties at the same time. In summary, the 4609 protein was preliminarily inferred to be a GPI-anchored protein.
Fig.3 Prediction of GPI modification sites
Fig.4 Signal peptide prediction for 4609
Fig.5 Prediction of transmembrane domain
Part 2 Identification of biological functions
2.1 GFP was used to screen suitable anchor proteins
Fig.6 Structure of composite parts.
Promoter--GAPDH gene promoter
Terminator--GAPDH gene Terminator
yeGFP--Enhanced Green Fluorescent Protein
V5-tag: V5 tag is a short peptide tag for detection and purification of proteins. The V5 tag can be fused/cloned to a recombinant protein and detected in ELISA, flow cytometry, immunoprecipitation, immunofluorescence, and Western blotting with antibodies and Nanobodies.
The structures of control and test groups were shown in Figure 3. Both groups involve GFP and V5-tag. GFP was used to indicate protein expression and V5-tag was used for protein purification. In the test group, a signal peptide was added upstream of the GFP which will allow the polypeptide to be exposed to the outer cellular space.
2.2 Construction of plasmids
Plasmid construction was completed in E. coli, and then plasmid was extracted and verified by enzyme digestion and further confirmed by sequencing. Subsequently the purified fragments were then transformed into Candida tropicalis following a previously protocol. Finally, the transformants were selected and further seeded in the liquid medium. After 24 to 36 hours, the genome was extracted as a template to verify the purpose fragments.
Fig.7 Construction of the strain 4609. Plasmid map of Ts-CAT2-gda324-URA3-P-SS-yeGFP3-V5-4609-T (Left). Verification of recombinant plasmids with restriction enzyme digestion.
M:DL 15000 DNA Maker(left); 1：Plasmid Ts-CAT2-gda324-URA3-P-SS-yeGFP3-V5-4609-T double enzyme digestion (EcoR Ⅰ &Xba Ⅰ) (Right).
Fig.8 Construction of the strain 5105. Plasmid map of Ts-CAT2-gda324-URA3-P-SS-yeGFP3-V5-5105-T (Left). Verification of recombinant plasmids with restriction enzyme digestion. M: DL 15000 DNA Maker; 1: Plasmid Ts-CAT2-gda324-URA3-P-SS-yeGFP3-V5-5105-T digested by EcoRⅠ& XbaⅠ(Right).
Results
Green fluorescence was detected in 9 of the anchored proteins with confocal laser microscopy, indicating that they could function normally.
Fig.9 Representative images of yeGFP.
Fig.10 Representative images of 25 screened anchor proteins.
Fig.11 Representative images of 4609 (further screened anchor protein).
Part 3 Surface display of PETase and MHETase
Fig.12 Structures of the gene circuits.
Fig.13 Construction of the surface display system of PETase. Plasmid map of Ts-CAT2-gda324-URA3-P-PETase-V5-4609-T (Left). Verification of recombinant plasmids with restriction enzyme digestion. M:DL 15000 DNA Maker; 1：Plasmid Ts-CAT2-gda324-URA3-P-SS-yeGFP3-ALS3-T; 2：Plasmid Ts-CAT2-gda324-URA3-P-SS-yeGFP3-ALS3-T digested by EcoRⅠ& ApaⅠ(Right).
Part 4 Evaluation of degradation effect
4.1 Determination of enzyme activity
Fig.14 Determination of enzyme activity of PETase.
The overall enzyme activity of PETase was measured. Firstly, a crude test was carried out to show the enzyme activity at different temperatures. Then, p-nitrophenol absorption was measured because PETase could also catalyze substrates into p-nitrophenol. The results showed that PET-4609 and 5105 performed better than the wild type ATCC20336 and cytPET (Figure 14).
4.2 Determination of degradation products with HPLC
To characterize the enzyme degradation efficiency more accurately, HPLC method was adopted. Purified TPA, MHET, and BHET standards were run as control. For the treated group, we incubated yeast with PET powder and tested for its degradation. The data showed that PETase was able to degrade PET into TPA and MHET and at the same time (Figure 15). According to the HPLC detection results, the product content of the degraded PET powder of each strain were plotted (Figure 16). It was demonstrated that target products were not detected in the group of ATCC 20336. Compared with the control strain, degradation products were obviously presented in the strain PET-4609 and PET-5105, indicating PETase is displayed on the surface of Candida tropicalis with high enzyme activity. However, several products were detected in the strain cytPET which should not be detected theoretically. It was speculated that a part of PETase is released due to the lysis of cells.
In conclusion, these results demonstrated that the surface display system was indeed able to degrade PET, which was consistent with the previous results.
Fig.15 Determination of degradation products with HPLC
Fig.16 Hydrolysate content of PET powder. Content of PET powder: 10 mg, thallus: OD=5, reaction system: 1 mL, reaction time: 18 h.
4.3 Surface display of MHETase to improve the degradation effect
In order to further improve the degradation effect of PET powder, Candida tropicalis was used to display MHETase. First of all, we measured the activity of MHETase.
Methods
Conditions: 200 mg/L MHET as the substrate,1 mL reaction system, bacterial mass OD=1, buffer: 50 mM glycine-NaOH (pH 9.0), 30 °C, 900 rpm.
Then centrifuge at 15000 g for 1 min and the supernatant was taken. The product was detected by HPLC.
We set different reaction times, namely 10, 20, 30, 60, and 90 min. The HPLC results showed that the substrate decreased sharply within 60 min, and the degradation was almost complete at 60 min. Besides, MHET could not be detected at 90 minutes, indicating that the display of MHETase was successful.
Fig.17 Determination of enzyme activity of MHETase.
After confirming that Candida tropicalis showed a good degradation effect of MHETase, we used different proportions of cells (expressing PETase and MHETase, respectively) to degrade PET powder, and then respectively measured and calculated the content of different degradation products.
Reaction conditions for degrading PET powder:
10 mg PET powder-the substrate
the reaction system-1 mL
bacteria displaying PETase OD=5
bacteria displaying MHETase-different proportions
the buffer：50 mM glycine-NaOH (pH 9.0)
30 °C，for 18 h, 900 rpm.
The reaction was terminated by heating at 100°C for 10 min. The supernatant was centrifuged at 15000 g for 1 min, and the product was detected by HPLC.
The products of PETase degradation of PET are MHET, BHET and TPA. And MHETase can degrade MHET (one of the products of PETase degradation) into TPA and EG. Therefore, the content of MHET can reflect the activity of PETase, and the content of TPA reflects the result of co-catalysis.
The results showed that when the displaying MHETase cells were added, MHET could be completely degraded after 18 hours. The results showed that the amount of MHETase added was not proportional to the degradation effect. When the ratio of PETase and MHETase cells was 2:1, the degradation effect was the best.
Fig.18 HPLC determination of the degradation effect of different proportions of PETase and MHETase. Determination of MHET (Left). Determination of TPA (Right). P represents the cell displaying PETase. M represents the cell displaying MHETase. P+M represents the mixing of the two kinds of cells, and the OD600 of P is controlled at 5.
4.4 Application scenarios
To make our research closer to reality, we made a PET film with low crystallinity, which was used to quickly detect the degradation effect of PET enzyme.
Methods:
Cut the Coca-Cola bottle into PET flakes (⌀6 mm, 10 mg) and then dissolve them in 0.5 mL 1,1,1,3,3,3-hexafluoro-2-propanol solution. Amorphous PET can be generated by the volatilization of the solution.
Results:
Fig.19 The process of making PET films. (a). 10 mg PET plastic bottle with high crystallinity. (b). PET treated with hexafluoro-isopropyl alcohol. (c). PET film with low crystallinity
References
Sagong, Hye-Young, et al. "Decomposition Of The PET Film By MHETase Using Exo-PETase Function." ACS Catalysis, volume 10, issue 8, 2020, pp.4805-4812.
Knott, Brandon C., et al. "Characterization And Engineering Of A Two-enzyme System For Plastics Depolymerization." Proceedings of the National Academy of Sciences, volume 117, issue 41, 2020, pp.25476-25485.
Kim, Dong Hyun, et al. "One-Pot Chemo-bioprocess Of PET Depolymerization And Recycling Enabled By A Biocompatible Catalyst, Betaine." ACS Catalysis, volume 11, issue 7, 2021, pp.3996-4008.
Meng, Xiangxi, et al. "Protein Engineering Of Stable IsPETase For PET Plastic Degradation By Premuse." International Journal of Biological Macromolecules, volume 180, 2021, pp. 667-676.
