Overview
Problem
We are in the midst of a plastic pollution crisis. Plastic has become an essential component of our daily lives, but our dependence on it is not without consequences. Plastic is an incredible product for countless industries, as it is very durable and versatile; however, it’s also not easily degradable. Moreover, the ease and affordability of plastic production make it difficult to rationalize recycling plastic. These are just a couple of the factors as to why only 9% of plastic is recycled in Canada (Canada, 2023). Plastic constantly escapes into the environment, such that each week, we consume an average of 5g of plastic – about a credit card’s worth (Francis, 2022)! To improve recycling methods, we were inspired to create project Recircuit.
Solution
Using Recircuit, our aim will be to bridge the gap created by plastic's resistance to decomposition and the cost associated with recycling. This will be accomplished by developing a bacterium capable of utilizing PET plastic, commonly found in transparent water bottles, as feedstock for PHB synthesis, a form of bioplastic. Recycling with a biological system not only has the potential to be cheaper than chemical methods, but such a system can repurpose PET into a more environmentally friendly alternative, taking a few weeks to degrade instead of hundreds of years. As a result, we can reduce PET waste by transforming it into a bioplastic, which, after use, will be easier to degrade.
PHB, while not as strong as PET, could be better suited for single-use plastics, that often aren’t recycled properly, so by replacing PET with PHB, there won’t be as adverse an impact when escaping into the landfill or environment.
Our solution is to incorporate the PET depolymerization, TPA degradation, and PHB synthesis pathways into one organism. We are building upon previous work, such as the Calgary 2017 iGEM team’s work on PHB synthesis in Escherichia coli. We are also working with E. coli as our chassis, but the PET and TPA degradation pathway could also be engineered into Pseudomonas putida, which natively has the PHB synthesis pathway, and is also a promising bacterium for biotechnology purposes.
What Inspired Us?
Plastics are one of the most used materials and are difficult to recycle; not only are they difficult to collect but they are also environmentally harmful to reprocess, made of toxic materials, and not economical to recycle. As a result, plastic has become a large contributor to microplastic water contamination and has made significant contributions to greenhouse gas emissions that lower air quality. This has inspired us to tackle this issue by focusing on the three Rs that our team has been taught since primary school “Reduce, Reuse, Recycle”. We hope to directly tackle the recycling aspect with our project by introducing a new “R”: recircuit. Much of the available research has focused on specific reactions related to plastics recycling, especially PET polymerization or the breakdown of TPA, a monomer of PET. However, Recircuit will extend this by building a gene circuit that links the input of PET plastic to the output of PHB bioplastics.
Repurposing System
Metabolic Pathway Overview
The metabolic pathway of terephthalate (TPA) is dependent on the availability of NADPH + H+ and subsequent enzymes for the transformation to 1,2-Dihydroxy-3,5-cyclohexadiene-1,4-dicarboxylic acid (DCD) through a hydration reaction. NADPH + H+ interacts with TphA1 acting as a reductase component utilizing a plant-type [2Fe-2S] iron-sulfur cluster, FAD binding domain, and a NAD binding domain, allowing the donation of two H + atoms resulting in the release of NADP+ (Sasoh et al., 2006). The product then interacts with the oxygenase component, TphA2A3 which consists of two subunits TphA2 and TphA3 with one unit being the large and small subunit, respectively (Sasoh et al., 2006). The combination of these two reactions with TPA in the presence of oxygen (O2) results in the formation of DCD which contains an additional two hydroxyl groups with respect to TPA. DCD is further processed to protocatechuate (PCA) through the reaction of TphB which is a primary process of re-forming the reactants used for the TphA reactions (Sasoh et al., 2006). DCD and TphB in the presence of NADP+ causes a decarboxylation reaction to occur forming PCA and the byproducts of NADPH + H+ and CO2 (Sasoh et al., 2006). Through the PCA 4,5-clevage pathway forms the product of pyruvate and oxaloacetate which are key components for the tricarboxylic acid cycle and subsequent ATP synthesis (Kamimura et al., 2010).
PET Degradation
Following PET degradation, TPA is further degraded into protocatechuic acid (PCA) by a gene cluster known as tphRICIA2IA3IBIA1I isolated from Comanomas sp. strain E6 (Sasoh et al., 2006). This gene cluster contains three subunits that make up TPA 1,2 dioxygenase (TPADO), which converts TPA into 1,2-dihydroxy-3,5-cyclohexadiene-1,4-dicarboxylate (DCD). These subunits include tphA2 and tphA3, which make up the large and small subunit of TPADO, as well as tphA1, which functions as the reductase component of TPADO (Sasoh et al., 2006). DCD is then converted into PCA by a DCD dehydrogenase expressed by tphB. The remaining two genes in this gene cluster are tphR and tphC, which are believed to function as an IcIR-type transcriptional regulator and a periplasmic TPA binding receptor, respectively (Sasoh et al., 2006). Due to the large size of the genes, they were ordered as two separate level 0 parts designed to be constructed using golden gate assembly. The first part included the genes tphC and tphA2, and the second part had the genes tphA3, tphB, and tphA1. Ribosome binding sites were included between each of the genes. These two parts, a tph promotor, and a tph terminator were used to construct a plasmid capable of breaking down TPA resulting in the production of PCA.
TPA Degradation
PHB Production
Polyhydroxybutyrate (PHB) is a short chain polyhydroxyalkanoate (PHA) (Pettinari & Egoburo, 2021). PHB is a bioaccumulant that is produced in micro-organisms during periods of nutrient depletion (Pettinari & Egoburo, 2021). PHB production is greatest in conditions of low nitrogen and a surplus of carbon (Ushani, 2020). The key genes in PHB are phbA, phbB, and phbC (Du & Webb, 2011). These are found in numerous PHB producers. These genes have also been successfully cloned into organisms such as E. coli (Du & Webb, 2011). phbA catalyzes a condensation reaction of two acetyl coA molecules. Acetyl CoAd reductase catalyses the reduction of acetoacetyl-CoA to R-3-hydroxybutyryl CoA. Third, the enzyme PHB synthase catalyzes the polymerization of R-3-hydroxybutyryl-CoA to form PHB (Du & Webb, 2011). E. coli can naturally convert glucose into acetyl CoA, which acts as a precursor for PHB production via the transformationally added phaCAB operon from pseudomonas putida. PHAs have thermomechanical properties similar to manmade polymers like polypropylene but are decomposable in the environment (Ushani et al., 2020). These are carbon storing compounds can be used in place of fossil-derived polymers, making them a greener option. PHB is biodegradable, biocompatible, and versatile (Ushani et al., 2020). Its many merits are attributable to its low permeability and less leaky nature compared to polyethylene and polypropylene, as well as its superiority in food packaging due to its lack of need for antioxidation practices (Ushani et al., 2020). Furthermore, PHB has pharmacological applications as a drug delivery technology. It is also used in tissue engineering as a scaffold for implants (dos Santos et al., 2017). Since PHB production is low in optimal conditions (Pettinari & Egoburo, 2021), our team sought to optimize PHB secretion to increase the yield of eco-friendly plastic that is produced in the process of PET conversion.
TPA Detection
To show that the TPA regulation method in Comamonas can be adapted to other chasses in a modular manner, we developed a TPA biosensor. There are three steps we must engineer our E. coli to do: 1) Transport TPA into the cell 2) Express a TPA-binding activator 3) Express cyan fluorescent protein under a TPA-induced promoter
Under a high activity promoter, will be the tphC, tpiA, and tpiB genes- they are crucial to the transport of TPA. TphC is a periplasmic TPA-binding protein that participates in the transport of TPA with the transporter membrane proteins TpiA and TpiB (Hosaka et al., 2013). The tpiBA system is also involved in the uptake of IPA, OPA, PCA and citrate, having the ability to interact with other substrate-binding proteins like TphC (Hosaka et al., 2013). Terephthalate Regulator (TphR) is an IclR-type transcriptional regulator (ITTR) which activates the tph promoter when bound to TPA (Kasai et al., 2010), which in this case, induces CFP expression.
Our TPA detection system can help us monitor the degradation of TPA via changes in fluorescence. Initially, when TPA is added to the system we would expect high levels of fluorescence since the TPA-inducible promoter will be continually activated. Once TPA starts to be degraded, there will be less TPA present and available to bind to the P_thpc promoter, thus reducing CFP expression and subsequent fluorescence. Using fluorescence as an indicator of degradation allows us to monitor reaction progress easily in real-time and the efficiency of the reaction.
An alternative design would be to switch tphC, tpiB, and tpiA with tpaK, which encodes a terephthalate transporter that comes from Rhodococcus jostii. Having both constructs would allow us to compare which is the most efficient transport system and combine the same pathway from different organisms (C. Testosteroni and R. Jostii) it in our Recircuit bioconversion system.
Phasin Mutant
Phasins are the major poly(3-hydroxybutyrate) (PHB) granule-associated proteins that play a key role in the intracellular localization and equal distribution of PHB granules. Phasins affect the size, number, and distribution of PHB granules due to their amphiphilic nature through the non-covalent interactions between the hydrophobic PHB and hydrophilic cytoplasm (Mezzina, M. P. et al., 2016). Production levels of PHB are increased in the presence of phasin largely due to the increased surface-to-volume ratio (York GM et al., 2001). Studies have shown that the phasin PhaPRe (Ralstonia eutropha) increases the activity of class II PHA synthases activity, PhaC1 and PhaC2, from Pseudomonas aeruginosa in vitro (Mezzina, M. P. et al., 2016), so having a more efficient phasin will likely also improve phasin synthesis. We will be working with PhaF, the phasin protein of Pseudomonas putida. A general characterization of phasins is an α-helix conformation. This conformation is observed to change according to the environment. Phasins will form oligomers, such as dimers and dodecamers (Mezzina, M. P. et al., 2016). Oligomerization is a common organization mechanism for phasins, supported by a leucine‐zipper motif in their amino acid sequences (Maestro, B. et al., 2017).
PhaF’s PHB-binding domain, BioF, is composed of four segments, B1-B4, where B1 has a distincly higher hydrophobicity, such that it has the greatest impact on binding affinity for PHB. We will improve upon PhaF by changing all four segments to the sequence of B1, with the intention to improve PHB-binding, thus improving PHB production and accumulation (Maestro, B. et al., 2017).
Increasing the production and transportation of PHB presents various possible medical applications, including medical devices, pharmaceutical carriers, and tissue engineering. A specific example of this includes the delivery of hydrophobic drugs directly to target cells by fusing PhaP with various ligands, attached to PHA/PHB nanoparticles which carry the drug. This will allow drug delivery to cells that have receptors recognized by the ligands (Mezzina, M. P. et al., 2016). Furthermore, PHB can be further industrialized on a larger scale for food packaging and coatings, decreasing the plastic waste typically used in these fields (Mohan et al., 2016). PHB has also been seen to show possible antibacterial properties when modified, leading to a strongly hydrophilic surface, largely effective against Escherichia coli (Slepička et al., 2015).
Plastic Biodegradation Research at Queen's University
Our institution, Queen’s University, has several professors who have research involved in plastic biodegradation, including our two PIs. They’re also part of a larger research program known as Open Plastic, led by Canadian researchers who’re dedicated to reducing plastic pollution through biological methods.
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