PROJECT DESCRIPTION

Our journey

Our team was born in February 2023, and soon after, we started our ideation process. Finding a common problem proved to be more challenging than expected. After intensive brainstorming sessions, we came up with three different proposals, which we later presented to several experts. We are very grateful for the advice and assistance we received, as it played an essential role in directing us towards a viable project. Consequently, the team reached a consensus to address two pressing global challenges through the implementation of PET-2-Protein: plastic waste, and food scarcity. Our goal is to develop an optimized enzymatic plastic depolymerization system and demonstrate the feasibility of protein production utilizing polyethylene terephthalate (PET) as the primary source.

The challenges we wish to tackle

In today's world, plastic waste and food scarcity are two major global challenges that demand urgent attention. The Aalto-Helsinki 2023 iGEM team has taken the mission to address these challenges by integrating plastic recycling and protein production through synthetic biology.

The scale of plastic waste pollution is alarming, with over 350 million tons of plastics manufactured annually, and over 70% of plastic waste accumulating in landfills and oceans¹. This poses a significant environmental threat to ecosystems, marine life, and human health. Without any further changes to current policies, global plastic waste generation is projected to triple to one billion metric tons by 2060. In 2021, global plastic recycling was estimated at roughly 40 billion U.S. dollars, and the market has been estimated to grow. However, the global plastic waste industry has faced some changes in recent years due to China’s plastic import ban, which has resulted in a decline in plastic imports from rich nations². Currently, the goal of plastic recycling is to reduce the need for primary plastic production. The variety of many plastic types makes recycling difficult, and due to the low or inconsistent quality, recycled plastics can trade at discounts of up to 50% lower than the price of corresponding primary plastic categories². The competition between the virgin plastics market and recycled plastics make recycling less attractive since newly produced plastic has a higher relative material efficiency due to the ongoing availability of lower-cost feedstock².

Simultaneously, global food security remains a pressing concern, with over 820 million people undernourished and the need to increase food production by 70% before 2050³. Conventional methods are insufficient to meet this growing demand. Focusing on the Nordic countries, most of the agricultural activity focuses on meat production, even though it is a well-known fact that it has a huge carbon footprint, and takes up double the resources than plant-based food production. A recent study by Xu, X. et al⁴, estimated that almost 60% of food production greenhouse gas emissions are due to the production of animal-based foods. As the demand for food protein continues to rise, the development of novel and sustainable protein sources becomes environmentally and economically significant. There is great potential for producing protein-rich feed or food additives in the form of algae, yeasts, fungi, and plain bacterial cellular biomass. They have a lower environmental footprint compared with other plant or animal-based alternatives, since they can use low-value organic and inorganic side streams of our current non-cyclic economy to produce valuable matter.

Our project has a focus on circular economy, aiming to reduce waste and utilize resources more efficiently by converting waste into valuable proteins.

PET-2-Protein: Our solution to plastic pollution and food production

Our project, PET-2-Protein, aims to develop a proof-of-concept approach for converting PET into proteins. Naturally occurring PETase enzymes can break down PET plastics into monomers such as terephthalic acid (TPA) and ethylene glycol (EG). Enzymatic degradation of plastic waste is an eco-friendly alternative to chemical plastic recycling. However, our research aims to address the fundamental issue of plastic waste by focusing on plastic reduction rather than merely extending the life cycle of single-use plastics through recycling methodologies. Consequently, we not only designed an optimized system for producing plastic-degrading enzymes but also optimized the enzymatic depolymerization of PET into TPA and EG, and finally, the microbial conversion of those monomers into protein-rich biomass.

The project has three parts, which include recombinant enzyme production, plastic depolymerization, and finally bioconverting the produced monomers into single-cell proteins. Although naturally produced PETase and MHETase degrade PET fairly well, they lack robustness⁵. Therefore we used an engineered strain in an attempt to speed up the depolymerization of the PET plastic^5,6. We used two parallel plasmid cloning protocols in Escherichia coli BL21 (DE3) to express PETase (polyethylene terephthalate hydrolase) and MHETase (monohydroxyethyl terephthalate hydrolase) enzymes. The enzymes are secreted and purified, after which we proceed to test their efficacy in depolymerizing PET.

In the initial wet lab phase, we pretreated the PET plastic to facilitate the enzymatic depolymerization. We employed both chemical and thermal pretreatment methods to understand the impact of different pretreatment approaches on the subsequent depolymerization steps. The pretreated plastics were to be submerged into a liquid media with the enzymes, which break down the plastic into monomers.

Schaerer, L. G. et al ⁷ have explored the feasibility of using microorganisms for producing edible microbial protein powder using PET plastic as a carbon source. To identify potential strains for the bioconversion of TPA and EG into proteins, we reached out to Professor Techtmann. After careful consideration, we selected Pseudomonas putida, Escherichia coli, Comamonas testosteroni, and Rhodococcus opacus as the strains. The resulting protein-rich biomass would undergo analysis using High Performance Liquid Chromatography (HPLC), Protein Bradford assay, and Mass Spectrometry to ensure the absence of plastic remnants and toxins, while also achieving a high protein concentration⁸.

Looking ahead, we envision the application of this biomass as an artificial medium for biological pesticides in agriculture, contributing to sustainable food production. Figure 1 provides an overview of the project, offering a visual representation of our research journey.

Figure 1: Project overview

Project inspiration

During the ideation phase of the project, our team found two topics that captured our interest: biological solutions for plastic pollution and sustainable food production. Recognizing their importance in highly developed countries like Finland, we embarked on a series of meetings and discussions to shape our project's direction.

Our journey began with a meeting with our PIs, who provided valuable guidance and insights on potential topics. Further, Rahul Mangayil, a researcher at Aalto University, guided us to explore the possibilities of developing or refining a system for plastic degradation. His advice steered us toward the exciting prospect of developing an optimized PETase enzyme for PET depolymerization.

Continuing our quest for knowledge, we had the privilege of meeting Christopher Landowski, Co-Founder, and CTO at Onego Bio. The visit to their laboratory, where they produce animal-free egg protein through fermentation, proved to be an enlightening experience. During our discussion, Christopher introduced us to Nesli Sozer, a research professor at VTT, who suggested combining our two topics, plastic degradation, and protein production, to address both challenges simultaneously.

The expertise of Shameer Kodambiyakamenna, a postdoctoral researcher at the University of Helsinki, became invaluable as we explored the potential capabilities of our end product. Considering that our biomass would be obtained from a process involving genetically modified organisms, Shameer proposed the idea of utilizing it as nourishment for insects, which could serve as a natural and sustainable biological pesticide.

To further enrich our project, we reached out to Stephen Techtmann, an associate professor at Michigan Technological University, whose work shared similarities with our PET-2-Protein project. Our discussions with Stephen focused on identifying the most suitable microorganisms that could naturally generate the biomass we desired. As a result, our team decided to incorporate several bacterial strains to enhance the efficiency of transforming depolymerization products into profitable biomass.

Additionally, we contacted Ting Lu, a professor from the University of Illinois at Urbana-Champaign with an interest in synthetic bioengineering. His expertise in synthetic bioengineering helped us identify new possibilities for engineering strains to utilize PET monomers resulting from the depolymerization step.

Conclusion

Through the PET-2-Protein project, we aim to combat plastic waste pollution and contribute to global food security by harnessing the power of synthetic biology. By converting PET into valuable proteins, we can make a significant impact on the environment while promoting a sustainable future. We believe that by working together on this project, we can make an important contribution to creating a world that we can be proud to leave behind for future generations.

References