Abstract
In response to the global challenge of plastic waste, our iGEM project aims to develop innovative biological pathways for the upcycling of polyethylene terephthalate (PET) plastic into high-value products, such as BHB (Beta Hydroxybutyrate).
Our approach designs synthetic pathways in E. coli and P. putida, enabling them to convert PET waste into Polyhydroxybutyrate (PHB) and later BHB. Utilizing computational tools and Flux Balance Analysis (FBA), we will optimize the engineered pathways to enhance the flux towards PHB production. This will include the use of kinetic models to predict and refine enzyme activity based on varying substrate concentrations.
By designing and constructing novel biological parts, we aim to establish an innovative production pathway that aligns with circular economy principles. This will not only facilitate the conversion of PET plastic waste into valuable products but also provide a sustainable solution to a pressing environmental issue.
Introduction
The global plastic waste crisis is currently one of the most prevalent and detrimental environmental issues. Conventional methods, such as chemical and mechanical recycling, are costly, energy-intensive, and often result in material devaluation and pollutant emissions. To address this, we engineered a synthetic biology solution that upcycles polyethylene terephthalate (PET) into high-value products, such as polyhydroxybutyrate (PHB), beta-hydroxybutyrate (BHB) and protocatechuic acid (PCA), through novel biological pathways in Escherichia coli and Pseudomonas putida. Experimental validation using HPLC and Bradford assays confirmed the functionality of key constructs, including the PETase enzyme, TPA transporter, and PHB-to-BHB conversion pathway. Our results demonstrate the feasibility of transforming PET waste into valuable, biologically produced materials, advancing circular economy principles and paving the way for scalable, eco-friendly biorecycling solutions.
Our project integrated a comprehensive strategy to ensure both technical, environmental, and commercial viability. Technically, we employed metabolic engineering, utilizing flux-balance analysis to optimize biochemical yield. The project's environmental footprint was evaluated via a life cycle assessment (LCA) to anticipate industrial-scale complications. Commercially, a SWOT analysis was conducted to critically determine our strategic position, formulating plans to capitalize on inherent advantages, address weaknesses, leverage opportunities, and counter potential threats. Furthermore, we initiated a community outreach program, developing an original curriculum to broaden access to synthetic biology.
Problem
Plastic waste is a growing global crisis, with 460 million metric tons of waste being produced annually (Ritchie et al., 2023). Current mainstream methods of plastic recycling, such as chemical recycling or mechanical recycling, are efficient in plastic degradation, but fall short in their ability to deal with plastic waste, and often emit more pollutants than is prevented (Ciriminna & Pagliaro, 2023). These methods are often unsustainable and costly, requiring labor-intensive procedures and further additives (Lee et al., 2024). Thus, plastic is often wasted, and even if it is recycled, these plastics often remain in circulation for decades, contaminating soil, air, and water. This damages ecosystems and harms the wildlife within them, eventually also threatening human wellbeing (Dokl et al., 2024). Specifically, microplastics have been shown to be especially harmful; their presence has been detected in our food supplies, drinking water, and even human tissues, raising concerns about their effects on health, such as oxidative stress, metabolic disorder, organ dysfunction, etc. (Li et al., 2023). Chemicals associated with plastic production and degradation are associated with endocrine disruption, inflammatory responses, and chronic illnesses, further emphasizing the detrimental effect of plastic on human health (Pilapitiya & Ratnayake, 2024).
Moreover, while effective, the current processes used in chemical synthesis are labor-intensive, costly, and limit scalability, restricting accessibility to niche markets (Stephens et al., 2024). Given the issue of plastic, both on earth’s natural ecosystems and on public health, we sought to create a synthetic biology solution that harnesses plastic into useful high value products (HVPs). Furthermore, a synthetic biology pathway would create a more optimized, environmentally friendly solution that could be linked to a circular economy. Thus, we call ourselves PURPLE: Plastic Upcycling to Reduce Pollution and Loop the Economy, because when it comes to protecting the planet, we’re not just green — we’re PURPLE.
Background
Our project specifically targets polyethylene terephthalate (PET), a semi-crystalline polyester renowned for its durability and widespread use in packaging, textiles, and engineering materials (Joseph et al., 2024). This very durability, however, makes its environmental persistence a significant challenge, motivating our focus on biological degradation as a sustainable solution. The project was inspired by recent discoveries of microbial PETases, a class of esterase enzymes found in organisms such as bacteria from the genus Nocardia; these enzymes catalyze the cleavage of the ester linkage between two oxygens that links subunits of PET. Building upon foundational research, including the PET hydrolases characterized by Arnal et al. (2023), our approach leverages this natural mechanism. Rather than a simple breakdown, we aim to transform PET waste into a resource by converting terephthalic acid (TPA) and ethylene glycol (EG) monomers into high-value products (HVP), a promising pathway supported by recent metabolic engineering studies (Qiu et al., 2024). This creates a circular economy model, turning environmental pollutants into valuable industrial feedstocks.
In exploring and researching potential high-value products derived from our pathway, we identified compounds that have industrial applications. There is growing demand for high-value and sustainable materials as industries shift towards eco-friendly alternatives from traditional plastics and chemicals. Polyhydroxybutyrate (PHB) is a biodegradable plastic replacement for conventional petroleum-based plastics with a market predicted to reach 679.1 million USD by 2034 (Polyhydroxybutyrate (PHB) Market, 2025). Its 16.6% annual market growth rate owes to its ability to break down completely into carbon dioxide and water under composing conditions, making it an excellent plastic substitute. Protocatechuic acid (PCA) is a valuable aromatic compound used in pharmaceuticals, cosmetics, and as an industrial precursor such as making vanillin (Li et al., 2024). Beta-hydroxybutyrate (BHB) targets the fitness and supplements industry. Our project was built upon the work done by our team last year, Concordia Shanghai 2024, which focused on BHB synthesis for athletes. However, we noted a possible application of our project that can be implemented in a circular economy in the context of bioremediation; specifically, plastic waste could be a feedstock for synthesizing high value products, such as BHB. BHB is a ketone body naturally produced by the liver that when taken orally achieves ketosis without the need for dietary carbohydrate restriction. Current BHB supplements are mostly chemically synthesized ketone salts, which are prone to heavy metal contamination and are likely to cause an overconsumption in salt. By producing synthetically made BHB via hydrolyzing PHB, we provide a cleaner and biologically sustainable alternative to current supplements.
Solution
Through the engineering of microorganisms capable of breaking down PET waste in controlled, aqueous environments, our team provides an alternative to existing recycling methods that helps avoid any toxic byproducts and greenhouse gas emissions. By converting PET into high-value products, we open up broader opportunities within the bioremediation field. This will not only enable the conversion of PET plastic waste into valuable products but also provide a more sustainable solution to one of the largest environmental problems of our time.
Our iGEM project addresses the global plastic crisis by engineering a circular bio-economy where waste PET plastic is upcycled into high-value products, closing the loop on pollution. We achieve this through a series of engineered biological pathways in E.coli and Pseudomonas putida. First, in E. coli, pET3a+ plasmid is being used with T7 promoter and BBa_B0034 RBS. PET is broken down into TPA by a mutated PETase enzyme coded by LCC-ICCG gene with H218y mutation. The TPA is then imported into the cell via a TPA transporter and converted into PCA by a multi-enzyme system including terephthalate dioxygenase (TphA1, A2, A3) and a dehydrogenase (DCDDH). This PCA can be funneled into central metabolism through the pca gene cluster (PcaGH, B, C, D, IJ, F), which cleaves the aromatic ring and ultimately produces Acetyl-CoA. This Acetyl-CoA serves as the building block for PHB, a biodegradable plastic, through the action of the phaCAB operon (PhaA, PhaB, PhaC). Since E.coli can’t metabolize TPA naturally, we also engineered a parallel, more direct route in P. putida, where TPA can be converted directly into PHB using the same phaCAB operon since TPA can be broken down into acetyl-CoA naturally in P.putida. The plasmid being used for this construct is SEVA plasmid with a Pm promoter. Finally, to create a valuable supplement, we engineered a pathway where PHB is hydrolyzed into beta-hydroxybutyrate (BHB) by PHB depolymerase enzyme (PHAZ_TALFU). Our experimental data has successfully validated key parts of this pipeline, confirming the functionality of our PETase, TPA transporter, and PHB depolymerase, proving the feasibility of transforming plastic waste into sustainable materials and products.
One of the high-value products, BHB, also addresses the problem with current BHB supplements. The biological synthesis of BHB, as opposed to current chemical methods, offers superior enantiomeric control, producing only the d-isomer of BHB and minimizing the risk of contamination. It is identical to the endogenous D-3HB, and has significantly reduced heavy metal content (Yao et al. 2021). Below are our genetic constructs.
PET to TPA Pathway in E. Coli
The construct shown in Figure 5 is the PET to TPA pathway. Part BBa_E0040 is the GFP protein that allows for visual confirmation of circuit activity. Part BBa_K3478888 is the LCC-ICCG gene with a H218Y mutation. The introduction of a histidine to tyrosine substitution at position 218 in Petase shows an increase in Petase activity (Orr et al., 2024). LCC- ICCG codes for the protein Petase that degrades PET into TPA and EG. EG will not go on to further processes and will be discarded.
TPA to PCA pathway in E.coli
This construct in E.coli encodes a transporter system allowing TPA to enter E.coli and enzymes allowing for the breakdown of TPA into PCA. To ensure that the TPA transporter is on the cell before the production of TPA, two promoters were engineered into the construct: the T7 promoter (BBa_I719005) and a constitutive promoter (BBa_J23100). The T7 promoter is induced after the TPA transporters are synthesized. BBa_K808011 (tphA1) is the gene that codes for the enzyme terephthalate dioxygenase reductase. In this construct, BBa_K808012 (tphA2) and BBa_K808013 (tphA3) work together, along with tphA1, to form the complex terephthalic acid 1,2-dioxygenase system (TERDOS). TERDOS catalyzes the reaction that degrades TPA. BBa_K2013010 codes for the enzyme 1,2-dihydroxy-3,5-cyclohexadiene-1,4-dicarboxylate dehydrogenase (DCDDH) which decarboxylates the product of TERDOS to form PCA. This pathway of tphA1, tphA2, tphA3 and DCDDH are used due to its high efficiency in BL21 DE3 E.coli cell. The enzymes TPADO (tphA1, tphA2, tphB2) and DCDDH form a natural, sequential metabolic pathway in native TPA-degrading bacteria Comamonas sp. This feature allows those genes to have optimal heterologous expression, high catalytic efficiency, and proper protein assembly in E. Coli. Compared with other constructs also derived from native TPA- degrading bacteria (such as Ideonella sakaiensis and Rhodococcus jostii RHA1), the TPA degrading genes derived from Comamonas sp showed the highest efficiency (Li et al., 2024). By comparing the area under the peak from HPLC for cells transformed with this construct with WT E.coli cells, we showed that the TPA transporter successfully took in TPA into the cell.
Theoretical pathway of PCA to Acetyl-Coa in E.coli
This theoretical pathway in E.coli converts PCA into Acetyl-CoA and succinyl-CoA. The gene PCAK codes for the PCA transporter on E.coli that allows PCA to enter the E.coli cell. Figure 4 shows the function of each PCA gene. The gene PCAGH codes for the enzyme protocatechuate 3,4-dioxygenase, which is responsible for the ring-cleavage of PCA, forming Beta- carboxyl-cis-cis-muconate. PCAB codes for the enzyme 3-carboxy-cis,cis-muconate cycloisomerase which catalyzes anti cycloisomerization, leading to the formation of γ-carboxymuconolactone. BBa_K2091002 (PCAC) codes for the enzyme γ-carboxymuconolactone decarboxylase which then, through decarboxylation, converts γ-carboxymuconolactone into Beta- ketoadipate enol-lactone. PCAD codes for the enzyme β-ketoadipate enol-lactone hydrolase which would catalyze a hydrolysis reaction that adds a water molecule on β-ketoadipate enol-lactone's lactone ring, forming β-ketoadipate. PCAIJ codes for the enzyme β-ketoadipate:succinyl-CoAtransferase, which forms Beta- ketoadipyl- CoA by adding a coenzyme A group on Beta- ketoadipate. PCAF codes for the enzyme β-ketoadipyl-CoA thiolase, which breaks the carbon-carbon bond in the middle of the β-ketoadipyl-CoA molecule to form succinyl- CoA and acetyl CoA. Acetyl-Coa would then go on to the next construct for further reactions. This pathway is efficient for acetyl-CoA production because they form a specialized, coordinated, and energetically favorable catabolic system for breaking down aromatic compounds directly into central metabolites. The enzymes in this pathway have high specificity for their substrates, minimizing side reactions and ensuring a smooth, efficient flow of carbon through the pathway (Buchan et al., 2000).
Acetyl-Coa to PHB pathway
This construct in E.coli converts Acetyl-CoA into PHB then uses natural E.coli metabolic pathways to secrete the product out of the cell. phaC, phaA, and phaB come together to form the phaCAB operon. The phaCAB operon codes for an enzyme that converts acetyl-coa to PHB. Specifically, phaA codes for the enzyme Acetyl-CoA acetyltransferase that converts Acetyl-Coa into Acetoacetyl-Coa. phaB codes for the enzyme Acetoacetyl-Coa reductase which consumes NADPH as an energy source to convert Acetoacetyl-Coa into (R)-3-hydroxybutyryl-CoA. This is the reason why BBa_K1674004 is added. BBa_K1674004 is zwf gene which codes for the enzyme NADPH reductase that reduces NADP+ into NADPH. By adding the zwf gene into the construct, there will be a higher yield of NADPH which would result in a more efficient process of synthesizing PHB from Acetyl-Coa. phaC codes for the enzyme PHA synthase, which converts (R)-3-hydroxybutyryl-CoA into PHB. BBa_K2560091 codes for Phasin-HIyA, a protein combining phasin—which binds electrostatically to intracellular PHB—with the C-terminal secretion signal from the HlyA toxin. The HlyA tag hijacks E. coli's type one secretion system, causing the bound phasin-PHB complex to be transported out of the cell, allowing PHB to be excreted out of the E.coli cell which removes the need for cell lysis.
TPA to PHB synthesis pathway in P.putida cells
In P.putida KT2440 cells, we selected the Pm promoter which is coded by BBa_K4757009 (Kernel does not show the proper symbol for this promoter). phaC, phaA and phaB form the same phaCAB operon used in E.coli cells, which converts Acetyl-Coa to PHB. Since P.putida can naturally metabolize TPA and break down TPA into Acetyl-Coa naturally, only the PhaCAB operon is needed for the final conversion to PHB from acetyl-CoA.BBa_K1674004 encodes for NADPH reductase, which reduces NADP+ to NADPH, allowing for a higher concentration of NADPH in the system. This increased concentration of NADPH makes PHB synthesis more efficient, since phaB encodes Acetoacetyl-Coa reductase, which uses NADPH as an energy source. BBa_K4728007 codes for phaF and has the same function as the Phasin- HIyA protein in the Acetyl-CoA to PHB pathway in E.coli, which is to secrete PHB out of the cell. Phasin-HIyA is not used in this construct because the gene would not be translated into the final protein in Pseudomonas cells.
PHB to BHB pathway in E.coli cells
This construct in E.coli depolymerizes PHB into BHB. The system Includes an inducible promoter, RBS, coding sequence for PHAZ_TALFU, linker, coding sequence for GFP, and a terminator. Once we add IPTG, the system produces PHAZ_TALFU, which is a PHB depolymerase that catalyzes the hydrolysis of PHB into monomers of BHB, as well as GFP, which acts as a reporter. The linker will link the GFP molecule and BHB together, which can allow us to determine the levels of PHAZ_TALFU, and relatively the concentrations of BHB, by detecting GFP. By measuring fluorescence using FLUOstar Omega microplate reader we confirmed that this construct successfully expresses PHAZ_TALFU and synthesizes Polyhydroxybutyrate depolymerase. Using a fluorescein standard curve, it was also calculated that the highest concentration of Polyhydroxybutyrate depolymerase is shown to be 0.31 uM when 0.5 mM IPTG is induced for 16 hours.
A life cycle analysis (LCA) was also performed to measure the environmental footprint of recycling PET into BHB, focusing on carbon dioxide emissions from plastic recycling, transportation, processing, and purification. The LCA showed that the carbon emissions, under the functional unit of 1 kg of PET, ranged from 164.42 to 211.81 kg of CO₂, revealing key emission hotspots that can be further improved upon to achieve the goal of net-zero carbon output.
Since our project focuses on an issue that impacts the broader community, we recognized the importance of raising awareness in plastic reduction, collaborating with Ecolve to aid in launching projects and campaigns that align with our project goal of upcycling plastic waste into practical products. Additionally, we noted a gap in educational resources regarding synthetic biology. Understanding that providing knowledge and skills in synthetic biology allows students to think critically and develop solutions to global issues such as plastic waste, we sought to mend this gap by developing an opportunity for synthetic biology education. We achieved this by developing an accessible, hands-on synthetic biology curriculum with integrated workshops and labs, which we tested with pre- and post-lab surveys within our community. Results indicated that prior to participation, 50% of students were unfamiliar with aseptic technique, DBTL thinking, applications of reporter proteins, and gene regulation. However, after the workshop, 75% of the students reported having an increased understanding of these aspects. Furthermore, there was a 25% increase in agreement that project-based activities were more engaging and effective than traditional teaching methods. Overall, the synthetic biology curriculum and integrated workshops were successful.
Future Visions
We envision a future where our engineered construct contributes to reducing plastic waste and enabling the upcycling of PET, fostering a circular economy and protecting the planet. While our current production process carries a significant carbon footprint, we recognize these challenges as an opportunity for further improvement; switching to electric vehicles for transportation, using renewable energy sources, and optimising the biological pathway to use one bioreactor are some of many possible ways to alleviate the negative environmental impacts. Recognising the commercial potential of our project, with a clear path to becoming a viable company, we seek to refine our business plan to strengthen its feasibility. Additionally, plasmid restrictions in China prevented us from testing our Pseudomonas construct experimentally. Moving forward, we aim to optimize our processes, expand our experimental research, and continue developing our system toward a more efficient and sustainable future. In developing our accessible Agar Art Curriculum we seek to foster a wider, more inclusive awareness and understanding of synthetic biology amongst students of any background.