Proof of concept

Accumulating plastic waste and the scarcity of protein-rich foods are an ongoing crisis. PET-2-Protein is a pipeline for turning Polyethylene terephthalate (PET), into single-cell proteins thus proposing one solution to both problems. The purpose of this page is to take a look at the process as a whole as shown in Figure 1, to discuss each of the steps, and lastly to discuss some of the pros and cons of the approach.

PET-2-Protein

The first step in the PET-2-Protein pipeline is the production of PETase and MHETase, the two enzymes capable of depolymerizing PET chains¹. In our experiments with enzyme expression, we chose E. coli as the expression host, as it is one of the most thoroughly studied organisms and one of the easiest to work with from the perspective of synthetic biology. Furthermore, there is previous evidence that extracellular production of both PETase² and MHETase³ is feasible with E. coli. We sought to investigate the E. coli-based extracellular production of FAST-PETase, which is a recently developed PETase variant with improved PET depolymerization capabilities⁴.

Apart from the enzymes, PET waste itself is obviously the primary material required for our process. Sourcing should not be an issue considering that more than 350 million tons of plastics are produced annually, of which about 10% is PET⁵. Moreover, it is unfortunately likely that PET production will continue to increase in the years to come due to it being ubiquitous in commercial packaging.

Next, the PET waste would be combined with purified enzymes in an incubator, where depolymerization would occur in temperatures ranging from 30 to 50°C. Considering the size differences between the constituent PET monomers compared to intact PET chains and the enzymes, a membrane-based filtration strategy could be viable to extract EG and TPA from the incubator.

Finally, the extracted PET monomers would be fed to various microorganisms to produce a protein-rich biomass. The general strategy would be to leverage certain natural strains from genera such as Comanonas or Rhodococcus, which are capable of growing with PET monomers as the primary carbon source⁶. In addition to basing this step of the concept on literature^7,8, we conducted directed lab evolution experiments showing that natural strains are very capable of growing on PET monomers. We furthermore leveraged genome-scale metabolic modeling to analyze the carbon flux of the PET monomers in various organisms, and designed parts for engineering assimilation pathways for EG and TPA into E. coli, and P. putida respectively.

In the final step, the resulting biomass would be dehydrated into a powder. As about 50-80% of the dry weight of bacteria is made up of protein, the resulting powder could potentially be used in a variety of downstream solutions as a cheap and sustainable protein source.

Outlook

In terms of PET depolymerization, the main selling point of the enzymatic approach is the relatively ambient temperature of around 30 to 50°C required for the degradation of PET chains⁴. To put this into perspective, chemical depolymerization techniques can require sustained temperatures of around 150 to 250°C⁹. From the perspective of single-cell proteins, the advantage is protein concentration coupled with the fact that only about 1% of water is required compared to livestock, with carbon emissions also being smaller⁶.

However, like any proof-of-concept, there are also drawbacks and unanswered questions that would need to be considered before any real-life implementation could take place. Firstly, while E. coli is a great strain for prototyping in the lab, it is not necessarily optimal for the extracellular mass production of enzymes. For this reason, more robust hosts such as Bacillus subtilis¹⁰, Pseudomonas putida¹¹, or even fungi should be considered for scale-up.

Secondly, while PET waste is inarguably an abundant resource, it is still one of the easier kinds of plastic to recycle. For example, as pointed out by Shafiullah Bhuiyan in our meeting with him, other kinds of single-use plastics such as polyethylene (PE) used in shopping bags can be more difficult to recycle, and could therefore be an even more compelling substrate for our project. In fact, there are already some results suggesting that enzymatic degradation of PE could likewise be viable¹². Another issue pointed out in the meeting was that as waste is not properly sorted, acquiring suitable PET waste might not be as trivial as one would think. Furthermore, the crystallinity of PET affects depolymerization efficiency¹³, which could prove to be an issue for real-life implementations.

Lastly, as the end product is envisioned to be used as food or feed, strict measures would be needed in place to ensure that the end product would not contain harmful material, such as traces of PET. As a European team, the local GMO and food regulations would also have to be taken into account. Especially if the microbes growing on PET monomers were engineered, this could prove to be a major issue for the concept.

Even with some of the drawbacks discussed, the presented concept is theoretically viable as discussed both in the literature and in part shown by our results. If the concept would prove infeasible due to safety or economic concerns, it could be adapted to produce other non-food-related high-value chemicals via further metabolic engineering, as the carbon flux from the PET monomers is fed through central metabolism.

References

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2. Shi L, Liu H, Gao S, Weng Y, Zhu L. Enhanced Extracellular Production of Is PETase in Escherichia coli via Engineering of the pelB Signal Peptide. Journal of Agricultural and Food Chemistry. 2021;69(7):2245–2252. doi:10.1021/acs.jafc.0c07469
3. Sagong H-Y, Seo H, Kim T, Son HF, Joo S, Lee SH, Kim S, Woo J-S, Hwang SY, Kim K-J. Decomposition of the PET Film by MHETase Using Exo-PETase Function. ACS Catalysis. 2020;10(8):4805–4812. doi:10.1021/acscatal.9b05604
4. Lu H, Diaz DJ, Czarnecki NJ, Zhu C, Kim W, Shroff R, Acosta DJ, Alexander BR, Cole HO, Zhang Y, et al. Machine learning-aided engineering of hydrolases for PET depolymerization. Nature. 2022;604(7907):662–667. doi:10.1038/s41586-022-04599-z
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