Engineering Success

Demonstrate engineering success in a part of your project by going through at least one iteration of the engineering design cycle.

Existing Part: BBa_K1921022 (PETase+linker.b+GCW61)


CONTRIBUTED BY 2024 IGEM TEAM MINGDAO

In 2016, iGEM Team TJUSLS-China made significant progress in developing PET-degrading enzymes, 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 want to explore if adding anchor proteins enhances PETase functionality and to identify better anchors such as Pir1 from Saccharomyces cerevisiae. We performed 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 Modeling

  • TASSER1,2,3 was utilized for predicting and scoring ligand binding site residues and active site residues. Each residue set includes a C-SCORE, which indicates 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 xylanase variants with anchor proteins

Xylanase-WT

Xylanase-GCW61

Xylanase-Pir1


RESULT

Table 1 | Comparison of Xylanase 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 FoldX plugin using models from I-TASSER

*** Protein 3D structure output generated by PyMOL using models from I-TASSER

Figure 1 | (A) Xylanase (B) Xylanase-GCW61 (C) Xylanase-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.

PETase from Ideonella sakaiensis 201-F6 has a conserved catalytic triad (S160-H237-D206)7,8. 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 data7,8, with the corresponding catalytic triad (S134-H211-D180), the amino acid residues shift is due to removing the signal peptide in 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 the enzyme effectiveness, which has been 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 the 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, Rorrer NA, Kearns FL, Silveira RL, Pollard BC, Dominick G, Duman R, El Omari K, Mykhaylyk V, Wagner A, Michener WE, Amore A, Skaf MS, Crowley MF, Thorne AW, Johnson CW, Woodcock HL, McGeehan JE, Beckham GT. 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