1. Basic Changes to Original Design #

1.1 Adding MHETase directly onto original PETase Plasmid #

The simplest solution would be to add a new promoter after the terminator of PETase sequence and attach MHETase sequence (Figure 1). A similar idea would be to delete the terminator for the PETase sequence and attach a MHETase sequence directly after the anchor protein sequence (Figure 2). Eventually, yeasts contain this plasmid may display PETase and MHETase on its surface. In the method, the proportion of PETase and MHETase activated will be 1:1 since they exist in the same plasmid. However, just simply adding the MHETase would result in an abnormally large plasmid, which could lead to problems in gene transcription.

1.2 Adding two signal peptides #

Another method considered in the beginning of our research was attaching one enzyme to the C terminus of the anchor protein and attaching the other on the N terminus. The use of signal peptides was intended to help guide both enzymes to the anchor protein. This would allow for dual display of both enzymes (Figure 3)

A similar method for dual display of proteins on the yeast cell surface was outlined in 2017. (See Figure 4) From this image from the study, Aga1p and Aga2p are used as the anchor proteins for the proteins they use (although for our study, we’ll have to change that to a protein that works with PETase and MHETase). One protein is fused to the N terminal of Aga2p and the other is fused to the C terminal. They also included 3 tags, linkers and spacers for the optimization of protein function. Aga2p naturally forms disulfide bonds with Aga1P which would be then anchored to the yeast cell. However, a problem with this model is that the bonds holding the Aga1p and Aga2p could be easily broken and render the enzymes useless.

1.3 Adding Spacers #

Target protein accessibility can also be increased by optimizing the length of the spacer between the target and anchor proteins. So spacer can be added to the sequence in order to increase the target protein accessibility.

1.4 RBS #

The difference between test and control is the presence of SS-signal peptide before the enzyme. I placed the enzymes in this order because it fits the order of the chemical process when degrading polyethylene terephthalate or plastic. Furthermore, PETase is closer to the promoter so it has a higher expression than MHETase corresponds with past experimental result and literature shown in figure 2.

2. Different systems to consider #

2.1 Peptide Ligase #

After further research, we discovered natural peptide ligases. Peptide ligases can enzymatically cross-link natural polymeric chains for a wide range of biomedical applications. We hoped to utilize one to connect the MHETase and PETase on the cell surface. The peptide ester forms an acylase intermediate with the serine of a peptide-ligase, which is then aminolysed to form a peptide bond with the n-terminal residue of another polymerization chain (see Figure 5). It was also found that the usage of peptide ligases is more efficient than the hydrolysis of naturally occurring peptide bonds alone. In addition, peptide-ligases have the potential to bind to cellular adhesion ECM proteins growth factors into polymeric networks to enhance cell attachment, growth, and differentiation.

However, a notable problem with this method is that as an enzyme, peptide ligase’s gene transcription might also make the plasmid too big and render it unusable. In addition, Also, different peptide ligases, such as butelase and peptiligase, aren’t a mature technology in the synthetic biological field.

2.2 Chimeric Enzymes #

Another method we considered upon further research was creating a chimeric enzyme of PETase and MHETase. In a study we referenced, scientists created chimeric proteins with various lengths of linkers to bind MHETase and PETase into one enzyme. MHETase has a core domain similar to that of PETase, making it possible to develop a chimeric enzyme. The study compared the degradation of substrate by PETase alone, MHETase alone, a mix of PETase and MHETase, and the newly engineered chimeric protein. They found that the chimeric enzyme outperformed all other groups, demonstrating a potential for increased catalytic activity. However, the chimeric enzyme combined one of PETase and MHETase, so the proportion and number of enzymes are limited. Moreover, the length of the sequence of the two enzymes is quite large, which may result in difficulty in expressing the enzymes. However, the chimeric enzyme combined one of PETase and MHETase, so the proportion and number of enzymes are limited. Moreover, the length of the sequence of the two enzymes is quite large, which may result in difficulty in expressing the enzymes.

2.3 Scaffold proteins #

Based on research, it is also plausible to construct cell surface display by interactions between proteins. Via quantitatively controlling the display system, we can use scaffold proteins to support the expressed enzymes. Similar to our lab method, E.coli cells are used to insert target DNA of the enzymes into plasmid and then transformed into the yeast cell, followed by PCR operation to tremendously increase copies of the DNA as well as inflorescence analysis. Therefore, two pairs of enzymes are connected by peptide bonds and presented on the cell surface, leading to final test by enzyme activities. However, this method has a disadvantage that the peptide bond formed between proteins may be fragile and easily broken down by other proteases. Therefore, it is not adopted by us.

3. Our official Way #

This method is popular in recent years. SpyTag is a peptide, and SpyCatcher is a protein corresponding to it. They can recombine and spontaneously form isopeptide bond coupling. This combines protein assembly with chemical reactions, genetically coded chemical reactions. We developed a chemical reaction library based on SpyTag and SpyCatcher by editing SpyTag and SpyCatcher with the method of protein engineering.

In our experiment, we have engineered a Spy Tag-Catcher pair to carry PETase and MHETase to the surface of Candida tropicalis and therefore break down plastic wastes. During the procedure, we first created a scaffold protein by adding a specific length of sequence to present on the Candida tropicalis’s surface. Therefore, the catcher was bound to the scaffold and also the tag was bound to the catcher via the connection of isopeptide bonds. Eventually, both the enzymes can be connected to the tag.

The advantage of this method is that we can use the concentration of tag and catcher to adjust and find the most efficient ratio of PETase and MHETase. Also, kinetic control can be applied to make Tag-Catcher ligations for directed assembly, which makes the procedure better approach to the ideal results.

4. References #

System design displaying both MHETase and PETase

1. Lim, Sungwon et al. "Dual Display Of Proteins On The Yeast Cell Surface Simplifies Quantification Of Binding Interactions And Enzymatic Bioconjugation Reactions". Biotechnology Journal, vol 12, no. 5, 2017, p. 1600696. Wiley, doi:10.1002/biot.201600696.
2. Tanaka, T., Yamada, R., Ogino, C., & Kondo, A. (2012). Recent developments in yeast cell surface display toward extended applications in biotechnology. Applied microbiology and biotechnology, 95(3), 577–591. https://doi.org/10.1007/s00253-012-4175-0
3. Knott, Brandon C et al. “Characterization and engineering of a two-enzyme system for plastics depolymerization.” Proceedings of the National Academy of Sciences of the United States of America vol. 117,41 (2020): 25476-25485. doi:10.1073/pnas.2006753117

Evaluating which system to use:

4. Brandon C. Knott, Erika Erickson https://orcid.org/0000-0001-7806-9348, Mark D. Allen, +16 , Japheth E. Gado, Rosie Graham https://orcid.org/0000-0001-5371-3942, Fiona L. Kearns, Isabel Pardo, Ece Topuzlu, Jared J. Anderson, Harry P. Austin, Graham Domin. “Characterization and Engineering of a Two-Enzyme System for Plastics Depolymerization.” PNAS, 28 Sept. 2020,
5. Narayanan, Kannan Badri, and Sung Soo Han. “Peptide Ligases: A Novel and Potential Enzyme Toolbox for Catalytic Cross-Linking of Protein/Peptide-Based Biomaterial Scaffolds for Tissue Engineering.” Enzyme and Microbial Technology, Elsevier, 7 Jan. 2022
6. Ito J, Kosugi A, Tanaka T, Kuroda K, Shibasaki S, Ogino C, Ueda M, Fukuda H, Doi RH, Kondo A. Regulation of the display ratio of enzymes on the Saccharomyces cerevisiae cell surface by the immunoglobulin G and cellulosomal enzyme binding domains. Appl Environ Microbiol. 2009 Jun;75(12):4149-54. doi: 10.1128/AEM.00318-09. Epub 2009 May 1. PMID: 19411409; PMCID: PMC2698344.
7. Tan LL, Hoon SS, Wong FT (2016) Kinetic Controlled Tag-Catcher Interactions for Directed Covalent Protein Assembly. PLoS ONE 11(10): e0165074. doi:10.1371/journal.pone.0165074