Results #

Highlights #

Compared with last year's project , this year's project introduced the Tag-Catcher system, which allows simultaneously displaying of PETase and MHETase, whereas in last year's project we respectively displayed PETase and MHETase on the surface of the strain and verified their plastic degradation capacity.

Based on last year’s project, PETase and MHETase can be displayed simultaneously on the same strain with good orthogonality.

Part 1 Selection of a robust host #

1.1 Introduction of Candida tropicalis #

Strong viability #

Candide tropicalis can survive in environments with acids and phenolic compounds by absorbing alkanes and fatty acids as carbon sources.

Good safety #

Candida tropicalis is a strain with class one biosafety. To prevent uracil from leaking into the environment, we employed CRISPR-Cas9 technology to knock out the essential genes for uracil synthesis in wild strains. As a result, these bacteria are now uracil nutrition-deficient. On this plate are various uracil concentrations for the strains. It was discovered that the uracil nutrition-deficient strain could not develop on a solid medium without uracil and that the number of colonies grew as the uracil concentration rose.

High industrial value #

The Candida tropicalis surface display technology is not as advanced as other surface display systems. Consequently, it is crucial to set up the system for Candida tropicalis. Additionally, Candida tropicalis has a long history of use in the manufacture of long-chain acids, which provides a solid basis for its industrial uses.

Part 2 Design of scaffold for surface display #

2.1 Build a more controllable display system #

In order to better demonstrate PETase and MHETase on the yeast cell surface, we focused on building a more controllable display system. To achieve this aim, we applied a scaffold protein to construct the Tag-Catcher system, which carried two catchers, Snoopcatcher and Spycatcher. The tag, connected to the enzyme secreted by the yeast cell, could combine with the catcher. Therefore, we would be in charge of the terminal of PETase and MHETase to the cell surface. Before displaying PETase and MHETase, we respectively used GFP and RFP to detect the function of the surface display system.

2.2 Selection of Spy/Snoop Tag and Catcher system #

In this study, we utilized both Spycatcher/Spytag and Snoopcatcher/Snooptag systems to create a controllable assembly system for autocrine multienzymes.

A domain of a protein involved in binding to human cells, from an invasive strain of Streptococcus pyogenes (Spy), was genetically dissected to generate a protein partner (SpyCatcher) and a peptide tag (SpyTag) (cartoon based on the crystal structure 2X5P). Upon mixing, SpyCatcher and SpyTag reacted rapidly and specifically to form a spontaneous amide bond, which was not reversed by boiling or mechanical stress. Fusion to SpyTag should provide a simple tool for irreversibly grasping proteins inside or outside cells. The principle of Snoopcatcher/Snooptag system is similar to Spycatcher/Spytag system. The sequence information of these two Catcher/Tag systems is as follows:

• Spytag (13 aa): AHIVMVDAYKPTK
• Spycatcher (115 aa): AMVDTLSGLSSEQGQSGDMTIEEDSATHIKFSKRDEDGKELAGATMELRDSSGKTISTWISDGQVKDFYLYPGKYTFVETAAPDGYEVATAITFTVNEQGQVTVNGKATKGDAHI
• Snooptag (14 aa): ASKLGDIEFIKVNK
• Snoopcatcher (114 aa): ASKPLRGAVFSLQKQHPDYPDIYGAIDQNGTYQNVRTGEDGKLTFKNLSDGKYRLFENSEPAGYKPVQNKPIVAFQIVNGEVRDVTSIVPQDIPATYEFTNGKHYITNEPIPPK

The specific and covalent SpyTag/SpyCatcher interaction provides a powerful way to build and link proteins into such assemblies. SpyTag (13 residue peptide) and SpyCatcher (116 residue complementary domain) spontaneously recombine to form an isopeptide bond under a range of temperatures (4–37℃), pH values (5–8). SpyTag and SpyCatcher has few limitations that they function well when fused at either the N-terminus or C-terminus.

2.3 Construction of Spy/Snoop Tag and Catcher system #

The integrated cassette contains the scaffold, GFP and RFP. The scaffold contains the signal peptide, carbohydrate-binding domain (abbreviated as CBM), Spy/Snoop catchers, v5-tag for detection of immunofluorescence, and anchor protein that can be anchored on the cell membrane. The N-terminus and C-terminus of secreted GFP and RFP are fused with signal peptides and their respective tags for specific binding to the catcher on the scaffold. Specifically, we did the following steps to construct the display system. First, the genome of Candida tropicalis is extracted. By using software Snapgene, primer is designed. Through PCR (Polymerase Chain Reaction), we obtain and amplify the genetic sequences that need to be knockout. In order to verify the PCR outcomes, the DNA sequence go through Agarose Gel Electrophoresis to compare the molecular number with designed one. If the outcome is correct and with single band, DNA is purified. On the other hand, with multiple strands, gel extraction is used to wipe off impurities in PCR system, including dNTP, enzymes, and buffers, which remarkably optimize the efficiency of further steps. Then, ligase combines linear T vector and PCR product (by Ex-Taq) to form a circular plasmid, which transforms into multiple Escherichia coli (E.coli). To ensure T vector and PCR product has been successfully fused, culture plates contain ampicillin to separate one with plasmid because of Ampr in T vector. The single colony of transformed E.coli is selected and cultivated. The restriction enzyme digests the plasmid to be linear.

To integrate this segment of the gene into the genome. We designed other primers and inverse PCR process, we combine the plasmid with marker gene URA3. After filtration, the plasmid is extracted (still verified by Gel Electrophoresis) and the strand added with marker gene, which is transformed into yeasts. The marker gene is then automatically knocked out. To validate the display function of scaffold, the gene insertion cassette, which was composed of scaffold gene (SP-CBM-SC-SC-SNC-SC-V5-GPI anchor), RFP-tag fusion protein secretory expression sequence (PFBA1 promoter, TPGK1 terminator), GFP-tag fusion protein secretory expression sequence (PFBA1 promoter, TADH2 terminator)and DLD-24 Genome homologous sequence. After PCR and DNA purification, the linear gene insertion cassette with DLD-24 border sequence at both sides, was transformed into Candida tropicalis.

2.4 Identification of spy/snoop tag and catcher system #

We use GFP and RFP to identify the tag-catcher displaying system. From left to right, the bright field is green fluorescent field and red fluorescent field. The results displayed that for strains with scaffold, the green or red fluorescence display on the surface, while the fluorescence was dispersed within the control cell.

Part 3 Surface display of PETase and MHETase #

3.1 Construction of surface display system for displaying both PETase and MHETase #

To complete this year’s goal, we mainly focused on building a double-enzyme display system of PETase and MHETase. Respectively, Snooptag was connected to MHETase while Spytag was connected to PETase. Then, each tag carried its enzyme to form strong isopeptide bonds between corresponding catcher on the scaffold and therefore displayed the two enzymes on the yeast cell surface. It’s worth noting that the ratio of PETase and MHETase is controlled to be 3:1 to achieve a higher breakdown efficiency.

3.2 Identification of surface display system for displaying both PETase and MHETase #

However, when GFP and RFP were replaced with PETase and MHETase, neither enzyme activity nor immunofluorescence was detected. We analyzed the possible reasons for this result, and since no immunofluorescence was detected, we first thought of whether it was because the v5-tag that was bound to the primary antibody detected by immunofluorescence was not exposed to the protein surface. As for why the enzyme activity was not detected, we considered whether it was due to the fused enzyme cannot fold properly. To save time, we firstly analyzed it by combining molecular modeling and molecular dynamics simulations.

Part 4 Optimization of cell surface display system #

4.1 Fused protein and scaffold protein prediction #

To analyze whether PETase-spytag and MHETase-snooptag fused protein folded correctly, we constructed a model of the fusion protein, we used prediction software such as trRosetta and ITASSER to construct the structure. The evaluation results of the two models shows the structure is convincing. So, no enzyme activity could be a steric hindrance between the fusion protein and the scaffold (See Modeling for details, https://2022.igem.wiki/ivymaker-china/model.html).

Similarly, we used I-TASSER to model our “CBM-SC-SC-SNC-SC-V5-7813” scaffold (See Modeling for details). When the display system is constructed, immunofluorescence cannot be detected, presumably as the V5 tag has been obstructed. To verify the theory, we predicted the model of the overall protein using the I-TASSER server and discovered that the V5 tag is truly embedded by other proteins.

It can be seen from the figure that the red component (V5 tag) is blocked by other components, meaning the V5 tag cannot function ideally as designed. We presumed the V5 tag would be available if it was located at the sequence's beginning, as the catchers may have a larger size that blocks the V5 tag if it is located at the end of the sequence.

4.2 Optimization of the scaffold by exchanging tag and catcher #

Through the structural analysis of the modeling results, we found the problem is likely caused by V5 tag being embedded in other transcribed proteins of the scaffold. What’s more, the entire scaffold protein is folded together, and the catcher is difficult to be all exposed. There are two potential solutions in response to this question. One, we could move the V5 tag forward to be transcribed earlier in the sequence. Two, we could reduce the molecular mass of the scaffold protein. One approach to do so is change the catchers in our original scaffold to tags belonging to its corresponding system, so change the spy catcher to spy tag and snoop catcher to snoop tag, and move the V5 tag forward.

The end result of our optimized scaffold is SP-CBM-V5-ST-ST-SNT-ST-7813.

All in all, our scaffold sequence changed from "SP-CBM-SC-SC-SNC-SC- V5-7813" to "SP-CBM-V5-ST-ST-SNT-ST-7813", and predicting protein models allowed us to analyze and solve problems with greater efficiency as it requires less time and more details than constructing the system in reality. Our predicted model revealed it was feasible and actual wet experiment proved its viability.

By observing with fluorescence microscope, we successfully detect the immunofluorescence (FITC-Fluorescein isothiocyanate isomer) outside the yeast showing the functionality of our optimized system.

4.3 Optimization of the scaffold by using newly synthesized Fast-PETase #

According to the latest report, we have synthesized Fast-PETase. The results showed that the activity of Fast-PETase was indeed higher than that of wild-type PETase.

We also measured the effectiveness of FAST-PETase more directly by testing its effect with degrading PET powder. Specifically, we took the following steps. First, we collected an appropriate amount of cultivated strains and washed it three times with 50 mM glycine-NaOH (pH 9.0-10) buffer. Second, the bacteria were incubated with 1 mL buffer containing 50 mM glycine-NaOH (pH 9.0) and 10 mg PET powder at 30℃ with a shaking speed of 900 r /min. Third, the reaction was terminated by diluting the aqueous solution with 18 mM phosphate buffer (pH 2.5) containing 10% (v/v) DMSO followed by heat treatment (85°C, 10 min). Fourth, the supernatant obtained by centrifugation (15,000 × g, 10 min) was analyzed by HPLC. The result shown in the figure below reflect a significantly larger concentration of degraded PET, MHET with FAST-PETase than wild PETase, consistent under different OD codition. Using strains displaying different ratios of FAST-PETase and PETase to degrade PET powder respectively, we found that the best degradation effect was achieved when the spytag:snooptag ratio on the scaffold of the strain was 2:1.

4.4 Optimization of the scaffold with different ratios #

We started with a ratio of snooptag: spytag=1:3. In order to obtain better catalytic effect, we optimized its proportion and successfully constructed scaffolds with different proportions.

We tested the effect of different ratios by HPLC, and found that the ratio of 2:1 performed the best among all the groups.

Part 5 Application #

5.1 Production of PET film #

To simulate real PET film, a PET film was constructed with low crystallinity, which was used to detect the degradation effect of the PET and MHET 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. The method is the same as we did last year.

Results:

5.2 Degradation of PET film #

First, Depolymerization of PET film by FAS1-PETase at 50 °C.

Second, PET monomers released from hydrolysing pretreated PET films (6 mm in diameter, roughly 25 mg) with FAS1-PElase, WI PETase(WI), ThermoPElase(Thermo), DuraPETase (Dura), LCC and ICCM at reaction temperature ranging from 50 to 72°C.

Third, Complete degradation of a pretreated water bottle (roughly 9 g) with FAST-PETase at 50 °C. 200 ml of fresh enzyme solution was replenished every 24 h to avoid product inhibition and maximize enzymatic depolymerizationrate.

Fourth, Depolymerization of five commercial polyester products (fibre fill, insulating fabric, thread, mosquito netting, batting) with various PHEs at 50 °C.

Fifth, Schematic of the closed-loop PET recycling process incorporating postconsumer coloured plastic waste depolymerization by FAS1-PElase and chemical polymerization. The crystallinity of theregenerated PET was determined as 58% by DSC. The molecular weights (Mn, Mw), polydispersity indices (D) of the regenerated PET were determined as Mn =16.4 kg mol', Mw=45.9 kg mol', D =2.80 by GPC. KH, PO, -NaOH (pH8) buffer was used for all enzymes shown in this figure. Compared with the control bacteria, the effect of FM2:1 degrading plastic film is remarkable (Fig. 15).

Sources (MLA) #

A. Host selection – Canada tropicalis #

1. Li, Yujie et al. “Development of a gRNA Expression and Processing Platform for Efficient CRISPR-Cas9-Based Gene Editing and Gene Silencing in Candida tropicalis.” Microbiology spectrum vol. 10,3 (2022): e0005922. doi:10.1128/spectrum.00059-22
2. Torkko, Juha M et al. “Candida tropicalis expresses two mitochondrial 2-enoyl thioester reductases that are able to form both homodimers and heterodimers.” The Journal of biological chemistry vol. 278,42 (2003): 41213-20. doi:10.1074/jbc.M307664200
3. Eirich, L Dudley et al. “Cloning and characterization of three fatty alcohol oxidase genes from Candida tropicalis strain ATCC 20336.” Applied and environmental microbiology vol. 70,8 (2004): 4872-9. doi:10.1128/AEM.70.8.4872-4879.2004
4. Kanai, T et al. “An n-alkane-responsive promoter element found in the gene encoding the peroxisomal protein of Candida tropicalis does not contain a C(6) zinc cluster DNA-binding motif.” Journal of bacteriology vol. 182,9 (2000): 2492-7. doi:10.1128/JB.182.9.2492-2497.2000
5. Kato, M et al. “Phylogenetic relationship and mode of evolution of yeast DNA topoisomerase II gene in the pathogenic Candida species.” Gene vol. 272,1-2 (2001): 275-81. doi:10.1016/s0378-1119(01)00526-1

B. Spy and Snoop tag and catcher system #

6. van den Berg van Saparoea, H Bart et al. “Combining Protein Ligation Systems to Expand the Functionality of Semi-Synthetic Outer Membrane Vesicle Nanoparticles.” Frontiers in microbiology vol. 11 890. 12 May. 2020, doi:10.3389/fmicb.2020.00890
7. Lang, Martina et al. “Tagging and catching: rapid isolation and efficient labeling of organelles using the covalent Spy-System in planta.” Plant methods vol. 16 122. 1 Sep. 2020, doi:10.1186/s13007-020-00663-9

C. The PCR method #

8. Waters, Daniel L E, and Frances M Shapter. “The polymerase chain reaction (PCR): general methods.” Methods in molecular biology (Clifton, N.J.) vol. 1099 (2014): 65-75. doi:10.1007/978-1-62703-715-0_7
9. Green, Michael R, and Joseph Sambrook. “The Basic Polymerase Chain Reaction (PCR).” Cold Spring Harbor protocols vol. 2018,5 10.1101/pdb.prot095117. 1 May. 2018, doi:10.1101/pdb.prot095117

D. E.coli Plasmid display system #

10. Muhamadali, Howbeer et al. “Metabolomic analysis of riboswitch containing E.coli recombinant expression system.” Molecular bioSystems vol. 12,2 (2016): 350-61. doi:10.1039/c5mb00624d
11. Kim, Chakhee et al. “Inducible plasmid display system for high-throughput selection of proteins with improved solubility.” Journal of biotechnology vol. 329 (2021): 143-150. doi:10.1016/j.jbiotec.2020.12.013
12. Lu, Hongyuan et al. “Machine learning-aided engineering of hydrolases for PET depolymerization.” Nature vol. 604,7907 (2022): 662-667. doi:10.1038/s41586-022-04599-z

E. Modeling #

13. Yang, Jianyi et al. “The I-TASSER Suite: protein structure and function prediction.” Nature methods vol. 12,1 (2015): 7-8. doi:10.1038/nmeth.3213
14. Zheng, Wei et al. “I-TASSER gateway: A protein structure and function prediction server powered by XSEDE.” Future generations computer systems : FGCS vol. 99 (2019): 73-85. doi:10.1016/j.future.2019.04.011