Part:BBa_K1921022

PETase+linker.b+GCW61

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
  COMPATIBLE WITH RFC[25]
• 1000
  COMPATIBLE WITH RFC[1000]

Usage

As a cell wall protein, GCW61 is often used as an anchor protein in Pichia pastoris to display some protein on the surface. By fusing GCW61 with the PETase, it can be displayed on the outer of the yeast cell wall.
By expressing the fusion protein , PETase ,which can degrade macromolecular polymers into monomerswas, expressed on the surface of Pichia pastoris,. And the whole cell catalyst for the degradation of PET was obtained. Have the PETase fixed on the cell wall, on the one hand can improve the stability of PETase, on the other hand, it is easy to control the degradation reaction of PET and PETase recycling.

Biology

GCW61 was gained from Pichia pastoris GS115. As one of the Glycosylphosphatidylinositoled cell wall proteins (GPI-CWPs), GCW61 is located in the outer layer of yeast cell wall, its C terminal is oligo mannose glycosylated. Subsequently, the mannose chain of GCW61 connect with the β-1,6 dextranomer of inner cell wall layer by forming covalent connection, thus, the GCW61 is fixed in the outer layer of the cell wall protein.
PETase was found from a kind of microorganism living on PET as the main carbon source. It can degrade macromolecular polymers into monomers. Surface display can reveal the protein whose gene code is coalescing the gene code of target protein or polypeptide with the counterpart of ankyrin on the surface of the host cell wall to harvest the whole cell catalyst.

Reference

[1] Kinoshita T, Fujiata M. Overview of GPI biosynthesis [J]. The enzymes. 2009;26:1-30.
[2] Orlean P, Mennon AK. Thematic review series: lipid posttranslational modifications. GPI anchoring of protein in yeast and mammalian cells, or: how we learned to stop worrying and love glycophospholipids [J]. Journal of lipid research.2007;48(5):993-1011.
[3] Mouyna I, Fontaine T, Vai M, et al. Glycosylphosphatidy linositol-anchored glucanosyltransferases play an active role in the biosynthesis of the fungal cell wall[J]. Journal of Biological Chemistry.2000;275(20):14882-14889.
[4]Shosuke Yoshida, Kazumi Hiraga, Toshihiko Takehana, Ikuo Taniguchi,Hironao Yamaji, Yasuhito Maeda, Kiyotsuna Toyohara,Kenji Miyamoto, Yoshiharu Kimura, Kohei Oda. A bacterium that degrades and assimilates poly(ethylene terephthalate) [J].science，2016（351）：1196-1199.
[5] DongHeng Guo, YanShan Xu, YaJun Kang et al (2016). Synthesis of octyl-β- d -glucopyranoside catalyzed by Thai rosewood β-glucosidase - displaying Pichia pastoris, in an aqueous/organic two-phase system[J]. Enzyme & Microbial Technology, 2016, 85:90–97.

Surface display HPLC Results

Surface display in Pichia Pastoris:

Figure 1. The activity of P. pastoris PETase-GCW61. a&b used the first group of yeast; c&d used the third of yeast; a&c:the activity in different yeasts'concentration under the best hour; b&d: the activity in different hours under the best concentration.

Co-display in Pichia Pastoris:

Figure 2. The activity of the first group of ppic9-PETase-GCW51 & ppiczaA-sJanus-GCW61 co-display transformants in different hours and amount of yeast.

Figure 3. The activity of the second group of ppic9-PETase-GCW51 & ppiczaA-sJanus-GCW61 co-display transformants in different hours and amount of yeast.

Figure 4. The activity of the first group of ppic9-PETase-GCW51 & ppiczaA-inJanus-GCW61 co-display transformants in different hours and amount of yeast.

Figure 5. The activity of the second group of ppic9-PETase-GCW51 & ppiczaA-inJanus-GCW61 co-display transformants in different hours and amount of yeast.

Figure 6. The activity of ppic9-PETase-GCW51 & ppiczaA-inJanus-GCW61 co-display transformant and ppic9-PETase-GCW51 & ppiczaA-sJanus-GCW61 co-display transformant in best condition.

CONTRIBUTED BY 2024 IGEM TEAM MINGDAO

In 2016, iGEM Team TJUSLS-China made significant progress in developing PET-degrading enzymes, specifically PETase from *Ideonella sakaiensis* 201-F6. They anchored PETase to the surface of *Pichia pastoris* using the GPI-related cell wall protein GCW61. Inspired by their work, we explored whether adding anchor proteins enhances PETase functionality and identified potentially better anchors, such as Pir1 from *Saccharomyces cerevisiae*. We conducted 3D protein structure modeling to compare wild-type PETase, PETase-GCW61, and PETase-Pir1 in terms of 3D images, ligand binding site residues, active site residues, and stability (free energy). These analyses determined the benefits of using anchor proteins and suggested potential improvements in anchor selection.

3D Protein Structure Modeling Tools

• **TASSER123** was utilized for predicting and scoring ligand binding site residues and active site residues. Each residue set includes a C-SCORE, indicating the confidence of the predicted interactions and site specifications.
• **YASARA4/FoldX5** was used for calculating the stability of each variant expressed in kcal/mol, which helps in understanding the structural integrity and potential functional efficiency of each variant under different conditions.
• **I-TASSER & PyMOL6** were employed for generating and visualizing the 3D structure images of each variant, allowing detailed observation of molecular architecture and potential functional sites.

Amino Acid Sequences of PETase Variants with Anchor Proteins

• **PETase-WT**
• **PETase-GCW61**
• **PETase-Pir1**

Result

Table 1 | Comparison of PETase Variants in Terms of Ligand Binding Sites, Active Sites, Stability, and 3D Structures

• Ligand binding site residues and active site residues predicted by I-TASSER, with C-SCORE representing a confidence score for estimating the quality of predicted models.
• Stability in terms of free energy (kcal/mol) predicted by YASARA with the FoldX plugin using models from I-TASSER.
• Protein 3D structure output generated by PyMOL using models from I-TASSER.

• • Figure 1** | (A) PETase (B) PETase-GCW61 (C) PETase-Pir1. The 3D protein models were generated by I-TASSER and imported into PyMOL for visualization. The models are colored by secondary structures: turquoise for alpha-helices, purple for beta-sheets, and pink for unstructured or flexible loops. Sphere colors: blue for GS linkers and red for either GCW61 or Pir1 anchor proteins. Glowing residues highlight: yellow for the predicted active sites and green for the original catalytic triad.

• (Fake photo placeholder)*

PETase from *Ideonella sakaiensis* 201-F6 has a conserved catalytic triad (S160-H237-D206)7. In the 2016 TJUSLS-China project9, PETase was displayed on the surface of *P. pastoris* by attaching a C-terminal GCW61 anchor protein through a GS-linker. We modeled PETase using I-TASSER. The predicted active site in wild-type PETase (PETase-WT) matched published data7 with the corresponding catalytic triad (S134-H211-D180)—the amino acid residue shift is due to removing the signal peptide at the N-terminus of PETase.

For the fusion with an anchor protein, PETase-GCW61, the predicted ligand binding sites changed, and the predicted active site residues also moved to S99-D124. However, the catalytic triad remained visually intact, although not predicted as active sites by I-TASSER. Therefore, PETase-GCW61 might maintain enzyme effectiveness, as verified in the TJUSLS-China project.

To find a better anchor protein, we modeled PETase with the GS-linker Pir1 (PETase-Pir1). The predicted ligand sites of PETase-Pir1 were more similar to PETase-GCW61 than PETase-WT. The predicted active site of PETase-Pir1 included S134, one of the conserved catalytic triad residues. The 3D structure of PETase-Pir1 differed from PETase-GCW61, but the catalytic triad remained intact, suggesting that PETase-Pir1 could maintain desired biological activity and should be experimentally tested.

To express enzymes in a yeast system, the free energy calculated by YASARA with the FoldX plugin was used to determine protein stability. Compared to wild-type PETase (60.5 kcal/mol), PETase-GCW61 and PETase-Pir1 showed worse stability with 411.28 kcal/mol and 603.72 kcal/mol, respectively. This raises concerns about protein expression levels and stability as a product, which should be verified experimentally.

Conclusion

Our 3D protein structure modeling indicates that adding anchor proteins like GCW61 and Pir1 to PETase maintains the enzyme’s functional integrity, with both variants showing potential for enhanced PET degradation. However, the decreased stability of PETase-GCW61 and PETase-Pir1 is a concern that needs further experimental validation. These findings suggest that selecting appropriate anchor proteins can significantly enhance the efficiency of enzyme display systems in *Pichia pastoris*. Future work will focus on optimizing these anchor systems, addressing stability issues, and validating their practical applications in environmental biotechnology.

Reference

1. Wei Zheng, Chengxin Zhang, Yang Li, Robin Pearce, Eric W. Bell, Yang Zhang. Folding non-homology proteins by coupling deep-learning contact maps with I-TASSER assembly simulations. Cell Reports Methods, 1: 100014 (2021). 2. Chengxin Zhang, Peter L. Freddolino, and Yang Zhang. COFACTOR: improved protein function prediction by combining structure, sequence, and protein-protein interaction information. Nucleic Acids Research, 45: W291-299 (2017). 3. Jianyi Yang, Yang Zhang. I-TASSER server: new development for protein structure and function predictions. Nucleic Acids Research, 43: W174-W181, 2015. 4. Krieger E., Dunbrack R. L., Hooft R. W. W., & Vriend G. (2012). YASARA View—molecular graphics for all devices—from smartphones to workstations. Bioinformatics, 28(3), 253-254. YASARA 5. Schymkowitz J., Borg J., Stricher F., Nys R., Rousseau F., & Serrano L. (2005). The FoldX web server: An online force field. Nucleic Acids Research, 33(suppl_2), W382-W388. FoldX 6. Schrödinger LLC. (2015). The PyMOL Molecular Graphics System, Version 1.8. 7. Austin HP, Allen MD, Donohoe BS, et al. Characterization and engineering of a plastic-degrading aromatic polyesterase. Proc Natl Acad Sci U S A. 2018 May 8;115(19):E4350-E4357. doi: 10.1073/pnas.1718804115. Epub 2018 Apr 17. PMID: 29666242; PMCID: PMC5948967. 8. Berselli A., Ramos MJ., Menziani MC. Novel Pet-Degrading Enzymes: Structure-Function from a Computational Perspective. Chembiochem. 2021 Jun 15;22(12):2032-2050. doi: 10.1002/cbic.202000841. Epub 2021 Mar 23. PMID: 33470503. 9. https://2016.igem.org/Team:TJUSLS_China