Source: The Regents of University of California submitted to NRP
RECYCLE PLASTIC WASTE USING ENGINEERED YEASTS DISPLAYING PLASTIC-DEGRADING ENZYMES AS A WHOLE-CELL BIOCATALYST
Sponsoring Institution
National Institute of Food and Agriculture
Project Status
ACTIVE
Funding Source
AFRI COMPETITIVE GRANT
Reporting Frequency
Annual
Accession No.
1032441
Grant No.
2024-67021-42551
Cumulative Award Amt.
$650,000.00
Proposal No.
2023-10822
Multistate No.
(N/A)
Project Start Date
Aug 1, 2024
Project End Date
Jul 31, 2027
Grant Year
2024
Program Code
[A1531]- Biorefining and Biomanufacturing
Recipient Organization
The Regents of University of California
200 University Office Building
Riverside,CA 92521
Performing Department
(N/A)
Non Technical Summary
Because of the low costs to manufacture, plastics have been widely used in agricultural and food systems, especially for food packaging.Polyethylene terephthalate (PET), a polyester plastic, is the most widely used plastic material for food packaging. The production and use of PET for food packaging are constantly increasing at a rate of 5.2% worldwide.Because PET is non-biodegradable, it can take years to degrade when discarded into the environment. Plastic pollution has received much attention and become a major environmental issue. Although some strategies (e.g., prevention measures, clean-up activities, and awareness instruments) have been raised, it is still not feasible to remove discarded plastics from the environment and fix the existing widespread plastic pollution. In recent years, chemical recycling of PET waste has been actively studied.For chemical recycling, PET polymers are broken down to their monomers of terephthalic acid (TPA) and ethylene glycol (EG) that can be refined or re-manufactured into high-value products, but these methods have several disadvantages, including high cost, complicated processing steps, and poor compatibility with down-stream refined processes.To perform an environmental-friendly and green depolymerization of PET waste, the discovery of plastic-degrading enzymes has opened a new avenue for PET degradation and recycling.?
Animal Health Component
40%
Research Effort Categories
Basic
20%
Applied
40%
Developmental
40%
Classification

Knowledge Area (KA)Subject of Investigation (SOI)Field of Science (FOS)Percent
40240201040100%
Knowledge Area
402 - Engineering Systems and Equipment;

Subject Of Investigation
4020 - Fungi;

Field Of Science
1040 - Molecular biology;
Goals / Objectives
In this project, we aim to engineer yeast surfaces to display plastic-degrading enzymes to degrade waste PET bottles. The expressed enzymes on yeast surfaces can be programmed to achieve a high biocatalytic activity to degrade PET waste. In this study, we will engineer yeasts (Saccharomyces cerevisiae, EBY100) to display two PET-degrading enzymes (PETase and MHETase) that can break down PET waste into its original monomers (TPA and EG). Under optimal conditions, we will then test the engineered yeasts to degrade commercial PET bottles in bioreactors, such as Coca-Cola, Pepsi-Cola, and Dasani bottles. The products (TPA and EG) will be recycled using downstream processes and quantified using high-performance liquid chromatography (HPLC). In addition, techno-economic analysis (TEA) will be applied to evaluate the economic feasibility and identify the 'hot spot' to improve the economic performance of the proposed technology.
Project Methods
TheSpecific Aimsof this proposal are:Aim 1: Engineer yeast strains displaying various PETasemutand MHETasemuton their cell surfaces and evaluate their ability to degrade PET.Various PETasemutand MHETasemutwill be expressed on yeast surfaces (EY-PETasemutor EY-MHETasemut), respectively. The mutant enzymes expressed on the yeast surface will be characterized using flow cytometry and confocal microscopy. Afterward, their performances to degrade PET film will be determined based on the enzymatic products (TPA and EG). Engineered yeasts (EY-PETaseoptimalor EY-MHETaseoptimal) with the highest enzymatic activities for PET degradation will be used in the following aims.Aim 2: Engineer yeast strains displaying both PETaseoptimaland MHETaseoptimalthat can degrade PET with a high efficiency.To completely degrade PET waste to high-value monomers, both PETaseoptimaland MHETaseoptimalwill be co-expressed on the same yeast surface at different ratios. The expression of both optimal PET-degrading enzymes on the same yeast surface will be characterizedusing both flow cytometry and confocal microscopy. The capacity for degrading PET waste using these yeasts will be evaluated based on the enzymatic products (TPA and EG).Aim 3: Degrade waste PET bottles in bioreactors using engineered yeasts displaying optimal PET-degrading enzymes and then recover high-value products.An up-scaled test in bioreactors will be carried out to optimize the bioprocessing conditions, map out the technological pitfalls, and provide key parameters for performing the subsequent techno-economic analysis. To this end, the PET bioconversion process ranging from pretreatment to enzymatic degradation and final product recovery and purification will be demonstrated in a 10 L bioreactor.Aim 4: Apply techno-economic analysis (TEA) to evaluate engineered yeasts displaying plastic-degrading enzymes to degrade plastic waste.System-level TEA will be conducted to evaluate the economic feasibility of applying the proposed technology to convert PET waste into TPA and EG. The resulting TEA model will be continuously updated based on the experimental results and provide key information forprocess improvement from economic perspectives.

Progress 08/01/24 to 07/31/25

Outputs
Target Audience:Primary audiences include graduate students, biological engineers, and industry. It is important to share our findings and apply this technique to solve environmental problems. Thus, primary audiences will be reached through interactions at local and national conferences, as well as open access to scientific publications. Collaborations with other biological engineers and industry will be another way to approach the primary audiences. In addition, we will train our undergraduate and graduate students who will rise to the challenges of environmental issues in the coming years. Additional means of dissemination of our findings include a training-the-trainer program, extension publications, and presentations at the state/regional/national venues. Changes/Problems:In our original plan, PI Chen's group will degrade PET-based food bottles using engineering yeasts, and co-PI Wang's group will apply the developed system to degrade PET plastics in a bioreactor. To maxmize the degration effiency, co-PI Wang needs tooptimize the degradtion parameters. To accomplish this project, the leading university (UC Riverside) plans to transfer $50,000 to co-PI Wang at Virignia Tech. Since the budget change is less than 10%, NIFA approval is not needed. What opportunities for training and professional development has the project provided?1) Two undergraduate students (George Maida and Merna Amine, senior year at the University of California, Riverside) learned to clone PETase-expressing plasmids and yeast transformation. 2) One graduate student (Kairui Zhai, a second-year Ph.D. student in the Department of Bioengineering at the University of California, Riverside) gave a poster presentation at the ACS National Conference in San Diego. The student was provided feedback by the communities of biological and environmental engineers. How have the results been disseminated to communities of interest?1) One poster presentation (Kairui Zhai) orally presented research findings for this project to an audience of biological and environmental engineers at the ACS National Conference in San Diego. 2) One peer-reviewed journal article has been accepted for publication in ACS Synthetic Biology. What do you plan to do during the next reporting period to accomplish the goals?1) Publish 1-2 peer-reviewed papers. 2) Present findings at meetings (including ACS, ASABE, and AIChE) to share the results with biological and environmental engineers who are working on plastic degradation and recycling. 3) Work with undergraduate students, graduate students, and stakeholders in California to develop a better understanding of the impacts of this study.

Impacts
What was accomplished under these goals? In this period,we developed engineered yeasts with the surface display of PET-degrading enzymes as whole-cell biocatalysts to degrade PET plastics. Using molecular engineering tools, we displayed PETase and MHETase on yeast surfaces (Saccharomyces cerevisiae EBY100) that were confirmed using confocal microscopy and a flow cytometer. In addition, we identified optimal reaction conditions (pH and temperature) of the yeast-displayed PETase and MHETase to improve the PET degradation efficiency. Furthermore, our developed engineered yeasts displaying PET-degrading enzymes were applied to commercial post-consumer drinking water bottles. The engineered whole-cell biocatalysts demonstrated a significant breakdown rate and hold promise for resolving the current crisis of PET pollution, potentially providing a financially feasible and environmentally friendly alternative to chemical recycling.

Publications

  • Type: Peer Reviewed Journal Articles Status: Accepted Year Published: 2025 Citation: Jiang, C.; Zhai, K.; Wright, R. C.; Chen, J., Engineered Yeasts Displaying PETase and MHETase as Whole-Cell Biocatalysts for the Degradation of Polyethylene Terephthalate (PET). ACS Synthetic Biology 2025. DOI: 10.1021/acssynbio.5c00209
  • Type: Conference Papers and Presentations Status: Accepted Year Published: 2025 Citation: This is an abstract presented at the ACS San Diego 2025.