Team:Hong Kong CityU/Poster

Plastilicious Coli: A plastic-eating bug

Presented by Team Hong_Kong_CityU

Ahn Dohyun (David), CHAN Tsz Ki (Delta), FUNG Kai Shing (Jason), FUNG Suet Ching (Annisa), Niharika PANT (Niharika), WAN Ka Ming (Ming), CHEUNG Tsz Kiu (Enoch), ZHANG Shuyuan (Harrison), SO Yung Mau (Ellis)

Dr. KONG Yuen Chong Richard, Dr. LAI Keng Po Ball, Mr. HO Cheuk Hin Jeff, Dr. Man-Kit TSE, Dr. Kai Chung LAU, Dr. CHEUNG Kwok Ming Wolf, Department of Chemistry, College of Science, City University of Hong Kong

Abstract:
The widespread use of plastics in recent decades has led to an alarming growth of plastic waste consisting of non-biodegradable and non-incinerable plastics such as polyethylene terephthalate (PET) and polyurethane (PU). Plastic waste that are disposed into the environment produce a variety of severe environmental pollution problems. This has motivated our team to look for viable research solutions to help mitigate the plastic waste problem. In this project, we have adopted an in-silico mutagenesis approach to design two mutant PU-degrading enzymes - papain and polyurethanase esterase A (PueA) - that could potentially be used to facilitate PET and PU biodegradation. Ultimately, we are aiming to create a multi-plastic-degrading E. coli superbug (Plastilicious Coli) that contains a composite “3-enzyme” biobrick made up of the two mutant enzyme genes (papain and PueA) developed in this project, and a wild-type polyethylene terephthalate hydrolase (PETase) gene. We aim to use the Plastilicious Coli to develop an effective and sustainable technology to help reduce plastic waste and better protect our environment.

Introduction

In 2018, the global plastic waste generated reached 6.9 million metric tons (Mt) (Silva et al., 2020). This increasing amount of plastic waste causes serious environmental pollution which threatens environmental and public health.

PET plastics can cause long-term adverse effects in relation to landfill saturation and ecological disturbances.
Approximately 675,000 tons of polyurethane (PU) foam were disposed in 2011, occupying 15 percent of foam processing in China alone (Yang et al., 2012).
Current management strategies of plastic wastes, such as landfills and incineration, cause harm to the environment. Additionally, most plastic waste are not recyclable.
Therefore, our project aims to construct a multi-plastic degrading bacterium with enhanced capability to biodegrade plastic waste. We employed an in-silico mutagenesis approach to bioengineer two plastic degrading enzymes - papain and polyurethane esterase (PueA). The two mutant enzymes will then be combined with a wild-type PETase gene to construct a “3-enzyme” bio-brick to create an E.coli clone (called Plastilicious Coli) that is equipped with multi-plastic degrading ability.

-Silva, A., Prata, J., Walker, T., Campos, D., Duarte, A., Soares, A., . . . Rocha-Santos, T. (2020). Rethinking and optimizing plastic waste management under COVID-19 pandemic: Policy solutions based on redesign and reduction of single-use plastics and personal protective equipment. Science of the Total Environment 742, 140565.
-Yang, W., Dong, Q., Liu, S., Xie, H., Liu, L., & Li, J. (2012). Recycling and Disposal Methods for Polyurethane Foam Wastes. Procedia Environmental Sciences, 16, 167-175. doi:10.1016/j.proenv.2012.10.023.

Problems

A dramatic increase in plastic waste from 2 million tons per year in 1950s to more than 380 million tons per year in 2015 has been reported, and the problem is predicted to worsen if effective and practical measures are not introduced in the near future to help reduce the plastic waste problem.
The plastic waste problem is mainly due to the high demands for shopping bags, bottles, disposable caps, straws, and a large variety of industrial and household products and poor public awareness about recycling practices. Bad odor, toxic leachates, and landfill gases due to accumulation of plastic wastes would result in adverse impacts to our living environment.
When plastic wastes end up in our oceans, marine life is seriously threatened. For example, we have witnessed various marine animals such as sea turtles and whales being starved to death due to ingestion of huge volumes of plastic waste in the ocean.

Geyer, R., Jambeck, J., & Law, K. (2017, July 01). Production, use, and fate of all plastics ever made. Retrieved August 25, 2020, from https://advances.sciencemag.org/content/3/7/e1700782.full

Current solutions

Current plastic management practices include landfills, incineration, and recycling.
However, these practices have various shortcomings:

Landfill disposal: This method produces bad odor, toxic leachates (aldehydes, ammonia, cyanides, nitrogen oxides), and emission of landfill gases, which harms the environment and human health.
Incineration: Produces a variety of toxic by-products.
Recycling: Recycled plastics are inferior in quality compared to the original. Some recycling methods, such as tertiary recycling, involves burning plastics which would produce toxic fumes.

In 2015, plastic waste recycling increased by only 3.5 % as compared to the previous 5 years. For industrial sectors, such as packaging, healthcare, fisheries and agriculture, plastics are in high demand because of their enhanced physicochemical properties. Inadequacies in plastic waste management practices and alarming increase in plastic waste is of global concern.

Geyer, R., Jambeck, J., & Law, K. (2017, July 01). Production, use, and fate of all plastics ever made. Retrieved August 25, 2020, from https://advances.sciencemag.org/content/3/7/e1700782.full.

Percentage of recycled plastic waste from 2010 to 2015.

Idea

Our team aims to create a multi-plastic degrading E. coli superbug, the Plastilicious Coli, as a plastic biodegradation device.

Our idea is to introduce a biobrick that consists of 3 genes that encode for PET- and PU-degrading enzymes into E. coli to construct a multi-plastic degrading superbug.

A 4-step approach for the biobrick design:

Selecting plastic-degrading enzymes as study targets.
3D-structure prediction of selected enzymes by Homology modelling.
Molecular docking analysis of Enzyme-Substrate complex and in silico mutagenesis to enhance the plastic degrading efficiency of selected enzymes
Redocking analysis of mutant Enzyme-Substrate complexes to verify the enhanced binding affinity of mutant enzymes for their respective plastic substrates.

Engineering

Engineering:

The Engineering part is divided into 5 Stages:
Stage 1: Selection of plastic-degrading enzymes and protein structure modelling;
Stage 2: In silico molecular docking analysis of enzyme proteins to plastic substrates;
Stage 3: In silico site-directed mutagenesis of papain and PueA enzyme genes;
Stage 4: Re-docking analysis of mutant papain and mutant PueA enzymes;
Stage 5: Construction of a “Papain-PueA-PETase” biobrick to create “Plastilicious Coli”.

Each of the above stages is further explained in the following sections.

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Modelling

Stage 1: Selection of plastic-degrading enzymes and protein structure modelling

3 enzymes were selected - polyethylene terephthalate hydrolase (PETase), papain (papaya proteinase 1), and polyurethanase esterase A (PueA).
For protein engineering investigations, a computational modeling approach was used.
For enzymatic activity, PETase is an extensively well studied PET-degrading enzyme and the molecular mechanism of action has been verified by crystallographic analysis (Chen et al., 2018). While papain and PueA enzymes were less well studied, their enzymatic activity on polyurethane (PU) plastic has been previously demonstrated (Phua et al., 1987; Stern and Howard, 2000).
For 3D enzyme structures, the 3D structure of PETase and papain have been previously documented by other researchers (Kim et al., 1992; Joo et al., 2018) and are available from the Protein Data Bank. However, the 3D structure of the PueA enzyme has not been elucidated and was therefore derived by homology modelling in this stage using the MODELLER and YASARA tools and refined using the PROCHECK program. The predicted 3D PueA structure was then further examined (along with the PETase and papain enzymes) by docking analysis in the next stage.

3D structure of PETase

3D structure of Papain

3D structure of PueA

-Casiraghi, C., Hartschuh, A., Lidorikis, E., Qian, H., Harutyunyan, H., Gokus, T., . . . Ferrari, A. C. (2007). Rayleigh Imaging of Graphene and -Graphene Layers. Nano Letters, 7(9), 2711-2717. doi:10.1021/nl071168m
-Joo, S., Cho, I. J., Seo, H., Son, H. F., Sagong, H., Shin, T. J., . . . Kim, K. (2018). Structural insight into molecular mechanism of poly(ethylene terephthalate) degradation. Nature Communications, 9(1). doi:10.1038/s41467-018-02881-1
-Kim, M. J., Yamamoto, D., Matsumoto, K., Inoue, M., Ishida, T., Mizuno, H., . . . Kitamura, K. (1992). Crystal structure of papain-E64-c complex. Binding diversity of E64-c to papain S2 and S3 subsites. Biochemical Journal, 287(3), 797-803. doi:10.1042/bj2870797
-Phua, S. K., Castillo, E., Anderson, J. M., & Hiltner, A. (1987). Biodegradation of a polyurethanein vitro. Journal of Biomedical Materials Research, 21(2), 231-246. doi:10.1002/jbm.820210207
-Stern, R. V., & Howard, G. T. (2000). The polyester polyurethanase gene (pueA) fromPseudomonas chlororaphisencodes a lipase. FEMS Microbiology Letters, 185(2), 163-168. doi:10.1111/j.1574-6968.2000.tb09056.x

Docking

Stage 2: In silico molecular docking analysis of enzyme proteins to plastic substrates
This was carried out in two parts.

In part 1, the performance reliability of five in silico docking programs (Autodock Vina, DockThor, EDock, SwissDock, and PatchDock) was compared using the reported PETase crystal structure of Ideonella sakaiensis as the reference model (Joo et al., 2018). The five software packages have all been previously used for protein structure studies (Duhovny et al., 2002; Schneidman-Duhovny et al., 2005; Trott & Olson, 2009; Grosdidier et al., 2011; Santos et al., 2020). However, the efficiencies of these softwares for in silico docking analysis of plastic-degrading enzymes with plastic ligands (i.e., enzyme-substrate complex) have not been tested.
The docking simulations of the PETase enzyme-PET substrate (i.e. enzyme-substrate) complex were run using these five docking programs, to identify the docking software most appropriate for our project. The docking software yielding binding conformations and free energies (ΔG in -kcal/mol) most similar to those stated by Joo et al. (2018) was then selected.
In part 2, the program chosen from Part 1 was used to conduct the docking analysis of the “papain enzyme-plastic substrate” and the “PueA enzyme-plastic substrate” complexes, where the interaction and binding affinity of the enzyme-substrate complexes (papain and pueA) were evaluated on the basis of the Gibb’s free energy (ΔG values) (Du et al., 2016).

Docking:

Table. Docking simulations of PET-PETase interactions using different docking softwares. In silico docking analysis using (a) AutoDock Vina; (b) DockThor; (c) EDock; (d) SwissDock; and (e) PatchDock.

Docking:

Table. Docking simulations of PET-PETase interactions using different docking softwares. In silico docking analysis using (a) AutoDock Vina; (b) DockThor; (c) EDock; (d) SwissDock; and (e) PatchDock.

-Du, X., Li, Y., Xia, Y., Ai, S., Liang, J., Sang, P., . . . Liu, S. (2016). Insights into Protein–Ligand Interactions: Mechanisms, Models, and Methods. International Journal of Molecular Sciences, 17(2), 144. doi:10.3390/ijms17020144
-Duhovny, D., Nussinov, R., & Wolfson, H. J. (2002). Efficient Unbound Docking of Rigid Molecules. Lecture Notes in Computer Science Algorithms in Bioinformatics, 185-200. doi:10.1007/3-540-45784-4_14
-Grosdidier, A., Zoete, V., & Michielin, O. (2011). SwissDock, a protein-small molecule docking web service based on EADock DSS. Nucleic Acids Research, 39(Suppl). doi:10.1093/nar/gkr366
-Joo, S., Cho, I. J., Seo, H., Son, H. F., Sagong, H., Shin, T. J., . . . Kim, K. (2018). Structural insight into molecular mechanism of poly(ethylene terephthalate) degradation. Nature Communications, 9(1). doi:10.1038/s41467-018-02881-1
-Santos, K. B., Guedes, I. A., Karl, A. L., & Dardenne, L. E. (2020). Highly Flexible Ligand Docking: Benchmarking of the DockThor Program on the LEADS-PEP Protein–Peptide Data Set. Journal of Chemical Information and Modeling, 60(2), 667-683. doi:10.1021/acs.jcim.9b00905
-Schneidman-Duhovny, D., Inbar, Y., Nussinov, R., & Wolfson, H. J. (2005). PatchDock and SymmDock: Servers for rigid and symmetric docking. Nucleic Acids Research, 33(Web Server). doi:10.1093/nar/gki481
-Trott, O., & Olson, A. J. (2009). AutoDock Vina: Improving the speed and accuracy of docking with a new scoring function, efficient optimization, and multithreading. Journal of Computational Chemistry. doi:10.1002/jcc.21334

Mutageneisis

In-silico Mutagenesis:

Here, we assumed that switching the residue of polar amino acid to hydrophobic at the ligand-binding site will improve the binding of the enzyme to non-polar plastics to promote the degradation of the plastic polymer. It was determined that the amino acid residues intended for mutagenesis should satisfy two eligibility criteria:

It should be positioned near the ligand-binding site;
It should be a conserved residue among similar enzymes. According to research papers, certain residues are essential to the activity of the enzyme protein.

Based on these parameters, we produced two mutated enzymes – Papain G23W and PueA R392F – in which glycine was converted to tryptophan in 23 position in papain, while arginine was converted to phenylalanine in 392 position in PueA.

After the in-silico mutagenesis, we conducted a re-docking study of the two mutant enzymes to validate their increase in binding affinities. The ΔG value of the mutant enzyme papain improved from -7.6 to -8.6 kcal/mol compared to the wild type enzyme, while the PueA value improved from -8.2 to -8.8 kcal/mol compared to the wild type. Our results showed that the in-silico mutagenesis approach has been successful, but the efficacy of the approach must be experimentally checked, as is typically the case for most other genetically engineered enzymes.

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Biobrick Creation

Biobricks:

Confer “Plastilicious Coli” the ability to degrade PET and PUR plastics
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Biobricks:

We have successfully in silico engineered two polyurethane-degrading enzymes – papain-G23W and pueA-R392F – that have demonstrated improvement in binding affinity for polyurethane plastic. These two mutant enzyme biobricks (BBa K3511000; BBa K3511001) will be combined with the wild-type PETase biobrick (BBa K2013002) to create a "3-enzyme" biobrick which will be introduced into E. coli to construct a superbug (Plastilicious Coli) to develop an effective and efficient multi-plastic biodegradation process.

-Howard, G. T. (2011). 7. Microbial biodegradation of polyurethane. Retrieved September 30, 2020, from http://www.southeastern.edu/acad_research/depts/biol/faculty/publications/pdf/2011/howard2011.pdf
-Joo, S., Cho, I. J., Seo, H., Son, H. F., Sagong, H.-Y., Shin, T. J., Choi, S. Y., Lee, S. Y., Kim, K.-J. (2018). Structural insight into molecular mechanism of poly(ethylene terephthalate) degradation. Nature Communications, 9(1). doi:10.1038/s41467-018-02881-1
-Paul, S., Stang, A., Lennartz, K., Tenbusch, M., & Überla, K. (2012, October 15). Selection of a T7 promoter mutant with enhanced in vitro activity by a novel multi-copy bead display approach for in vitro evolution. Retrieved September 13, 2020, from https://academic.oup.com/nar/article-lookup/doi/10.1093/nar/gks940
-Phua, S., Castillo, E., Anderson, J., & Hiltner, A. (2004, September 13). Biodegradation of a polyurethane in vitro. Retrieved September 13, 2020, from https://onlinelibrary.wiley.com/doi/abs/10.1002/jbm.820210207
-Yoshida, S., Hiraga, K., Takehana, T., Taniguchi, I., Yamaji, H., Maeda, Y., . . . Oda, K. (2016, March 11). A bacterium that degrades and assimilates poly(ethylene terephthalate). Retrieved October 02, 2020, from https://science.sciencemag.org/content/351/6278/1196.long

Educations

Education

We conducted a series of educational events for the general public from June to August 2020.
The educational events can be classified into four categories: social media, partnership, human practice and science communication.
The aims of the events were to raise public awareness on the global plastic pollution problem and to promote the potential applications of synthetic biology. We have also organized public talks covering the (1) Basic principles of synthetic biology, and (2) Applications of synthetic technology.

Human Practices

Online survey on plastic waste problem, and
- Encourage environmental-friendly practices in youth, adults and elderly
- Raised public awareness on plastic waste recycling and the role of synthetic biology in tackling a variety of environmental problems
Sharing Session by a guest speaker from the World Wide Fund for Nature.
- Mr So shared with us the practice of circular economy. This economic model is a regenerative system in which waste products are recycled to maximize economic efficiency and with minimal waste production. Circular economy not only benefits businesses, it also benefits our environment.
- Our team was inspired to explore the possibility of collaborating with certain recycling industries to transform wastes into useful resources.

Science Commmunication

Mini-talk:

Survey:

Symposium :

Youtube Platform : Collaboration on Video Making

Partnership and Collaborations

Partnership

Symposium:
Mini talk:
YouTube channel
Public talk
We started our first meeting with HKUST team on 21 July 2020. In the first meeting we had exchanged a lot of ideas on our experiment. Both of our team and HKUST had a similar ideas on developing a project to help alleviate plastic pollution. Therefore, we decided to collaborate with each other and agreed on collaborating on organizing several online activities on promoting the application of synthetic biology approaches to solve plastic pollution problems.

Collaboration

-Collaborating with HKU iGEM Team:

Identified possible challenges that our team may encounter regarding the interaction between engineered bacteria and plastics.
Our team held an internal meetup to discuss these issues.

-Collaborating with HKUST Team:

Both teams decided to co-organize community events to raise awareness about the plastic pollution crisis in the form of public talks, YouTube clips and seminars.
Both teams shared a common goal – designing activities to raise public awareness about the importance of reducing plastic wastes – we hosted several talks on this topic in real-time interactive format
Both teams collaborated in production of video clips on synthetic biology to engage the audience interest on the different revolutionary achievements of synthetic biology.

Contribution and Implementation

Contribution:

The modification of the T7 promoter

The C62-T7 mutant promoter is reported to facilitate enhanced in vitro transcriptional activity by more than 10-fold relative to the wild-type T7 promoter (Paul et al., 2013). An additional choice is provided for future iGEM teams needing a strong promoter to enhance gene expression.

New add-in items and modification of the plastic degradation biobricks in the iGEM part registry

The mutant papain-G23W (BBa_K3511000) and mutant PueA-R392F (BBa_K3511001) enzyme biobricks were created by us. These biobricks will serve as an additional resource for future iGEM teams working on a similar project theme, and could possibly help in their project design work plan.
The composite biobrick, BBa_K3511002 designed in this project can serve as useful tools for future iGEM teams aiming to develop novel solutions to tackle the plastic pollution problem.
Our team has introduced a single-base change to the genes encoding the papain and PueA enzymes which had been reported to have the ability to degrade polyurethane plastic. In silico analysis indicated an increase in binding affinity of both mutant enzymes for PU plastic.

Implementation:

Realizing that the environmental pollution issue is becoming more serious, our team intends to construct multi-plastic-degrading bacteria to reduce plastic wastes in a non-polluting manner.
It is our belief that our project can benefit plastic waste treatment industries and farming fields. Our powerful plastic-degrading bug can turn plastic trash into useful materials, without generating toxicants as incineration and landfills do.
We have predicted the models of two PU-degrading enzymes, papain and PueA, using the MODELLER software, which can serve as groundwork for further modification work.
The enzymatic rate of plastic degradation remains to be determined, and we plan to collect experimental data on this using SEM to investigate the plastic degradation efficiency of our engineered bacteria.
Biosecurity risk was taken into consideration. As our project involves the use of bacteria and genetic engineering technologies, the E. coli strain to be used is an attenuated laboratory strain that carries numerous mutations in its genome which would preclude its ability to survive in the natural environment.

-Paul, S., Stang, A., Lennartz, K., Tenbusch, M., & Überla, K. (2012, October 15). Selection of a T7 promoter mutant with enhanced in vitro activity by a novel multi-copy bead display approach for in vitro evolution. Retrieved September 13, 2020, from https://academic.oup.com/nar/article-lookup/doi/10.1093/nar/gks940

Team and Attribution

Acknowledgement:

-Dr. KONG Yuen Chong, Richard
-Dr. LAI Keng Po, Ball
-Dr. TSE Man Kit
-Dr. LAU Kai Chung
-Mr. HO Cheuk Hin
-Dr. CHEUNG Kwok Ming