Description

Part 1 Overview

At the beginning of the project, in order to consider the rights and interests of stakeholders, and let more experts in the field to join us, in the early stage we conducted a lot of literature review, collected more than 600 questionnaires, targeted to interview the stakeholders of the industry, in order to fully understand the ideas of the public and targeted action. At the same time, in the process of design experiments, we also communicated with many experts in related fields, such as Mr.Li Kai (research scientist) from Shanghai Jiao Tong University and Dr. Li Cheng from Polynovo Biotech, who provided us with valuable suggestions and expressed full recognition to our project. OVERVIEW

Figure 1 Flowchart of our projet

The team reviewed a large number of literatures and selected two highly efficient plastic-degrading enzymes that can degrade more than 99% of PET 1 2 3. However, both enzymes are required to exert maximal activity under 40–50℃ and more alkaline conditions.

Therefore, SUPERB predicts potential mutant sites through machine learning and combines enzymes with mutations, such as deglycosylation, to improve thermostability and acid-base tolerance, enabling efficient degradation of plastic under mild and neutral conditions. Through molecular docking to simulate the binding ability of mutant enzymes and substrates, the optimal mutant enzyme was selected to further improve degradation efficiency. This was combined with an optimized promoter, signal peptide, and copy number to construct a highly expressed strain, improving the expression amount of plastic-degrading enzyme in Pichia.

Finally, the degradation products TPA and EG were transformed into BC, including Pichia pastoris and Komagataeibacter xylinus, and the fermentation conditions were optimized to obtain high-yield BC, achieving value-added utilization of products.

Part 2 Implementation

Engineered strain

Pichia pastoris, a strain of methylnutritional yeast, is an important chassis cell in the enzyme production industry due to its ease of gene manipulation, secretion and expression, and high-density fermentation. Current reports point out that the PET hydrolase expressed by Pichia pastoris has higher heat resistance, which can obtain higher PET hydrolysis efficiency in a prolonged reaction duration. Therefore, Pichia pastoris was selected as the host of the PET enzyme, and the promoter and signal peptide were optimized at the same time. In the promoter GAP, AOX1, AOXm and the signal peptide MF, α signal peptide species, the optimal combination method was selected to further improve the expression mode of the PET enzyme and the catalytic efficiency of the enzyme, and the high expression strain of the plastic degradation enzyme was constructed.

SUPERB increase the copy number of IsPETasePA and FAST-PETase-212/277. By using the same caudal enzyme restriction sites Bgl II and BamH I at both ends of the pAO815 series carrier developed by Invitrogen Company and the expression box, the caudal enzyme restriction expression box was separated and inserted into the BamH I site to produce a tandem repeat expression box. Two copies of ISPETasePA-2C and FAST-PETase-212/277-2C are obtained.

Mutated Enzyme

SUPERB identified two highly efficient PET-degrading enzymes, IsPETasePA and FAST-PETase-212/277, through literature research. To further enhance their thermal stability, SUPERB used machine learning to predict mutation sites and combined it with deglycosylation engineering to improve the enzymes’ tolerance to pH extremes. This resulted in four mutants of IsPETasePA (mutation sites at 29, 59, 122, and 183) and five mutants of FAST-PETase-212/277 (mutation sites at 92, 169, 190, 212, and 223).

Subsequent validation through wet experiments and molecular docking identified the optimal mutant, THR-122-PRO, derived from IsPETasePA. Under conditions of 30°C and pH 6.0, the enzyme activity of THR-122-PRO increased by 475.1%, indicating a significant enhancement in its ability to degrade plastics under acidic conditions.

Dual-enzyme system

IsPETasePA which possesses a flexible substrate-binding pocket to accommodate PET,and is able to use PET as its major energy and carbon source. When grown on PET, this strain produces two enzymes capable of hydrolyzing PET and the reaction intermediate(MHET).

FAST-PETase-212/277 exhibits the highest overall PET depolymerization rate under a mild condition at 50 ℃ and enables complete decomposition of various commercial PET commodities in a week.

MHETase can further hydrolyze MHET and BHET into TPA and EG, resulting in the complete degradation of PET into TPA. We try to enhancing the biodegradation of PET by an IsPETasePA and MHETase dual-enzyme system and the MHETase and FAST-PETase-212/277 dual-enzyme system.

Fusion protein

Multienzyme agglomerates are membraneless organelles formed by liquid-liquid phase separation of biomolecules, which serve to isolate and concentrate substrates, intermediates and enzymes in the cell, thereby improving the efficiency of biochemical reactions Constructing multi-enzyme condensates using interacting peptides can simulate multi-enzyme complexes in nature, improve the local concentration and efficiency of chemical reactions, and reduce the occurrence of side reactions. Therefore, according to the above principle, we propose that two proteins can be linked by short peptides to construct a fusion protein. We selected two interacting polypeptides SZ1 and SZ2 and inserted them into our plasmid to construct PETase-mCherry-SZ2, RGG-SZ1, combining them to get better expression, degradation rate and some of the properties we want.

Biotransformation

It has been reported that terephthalic acid (TPA) and ethylene glycol (EG) can significantly increase the production of bacterial cellulose (BC) 4.

It is known that terephthalic acid and ethylene glycol can significantly increase the production of bacterial cellulose. Therefore, SUPERB utilizes the degradation products of PET to promote the production of bacterial cellulose (BC). SUPERB identifies strains with this capability through literature review and evaluates their BC production capacity through experiments. The best-performing strain is then mixed-cultured with Pichia pastoris, while the nutrient composition of the medium and fermentation conditions are optimized to achieve the goal of plastic degradation and reutilization. This method not only enables the environmentally friendly and efficient use of PET degradation products, promoting resource recycling and environmental protection, but also advances the low-cost and efficient industrial production of bacterial cellulose.

Part 3 Future Work

Multienzyme complex

In this competition, we found that PET degradation product MHET can greatly reduce the enzyme activity. We constructed a multi-enzyme condensate composed of MHETase, IsPETasePA and FAST-PETase-212/277, in order to efficiently degrade PET and minimize the influence of MHET on enzyme activity.

Due to time constraints, we only connect MHETase with the basic IsPETasePA and FAST-PETase-212/277.In the future, we also hope to combine the the higher enzyme activity after the mutation has been explored IsPETasePA with MHETase to improve the effect of PET degradation again, moreover, again, we can use machine learning to mutate the MHETase to improve the degradation efficiency of the dual-enzyme system.

Multiple copy plasmid

In our experiment, we have designed two two-copy plasmids, IS-2C and FAST-2C, which can greatly improve the expression level of Pichia Pastoris. However, among the plasmids we constructed, we failed to construct the FAST-2C plasmid. Therefore, in order to continue to improve our protein expression level, So we decided to continue with the multi-copy plasmid in the next experiment. In the future, we will also make multiple copies of our modified plasmids with machine learning and molecular docking and our multi-enzyme system in order to obtain higher protein expression

Fusion protein

In this competition, we constructed two plasmids, PETase-mcherry-SZ2 and RGG-SZ1, in which SZ1 and SZ2, as interacting peptides, successfully linked the two enzymes. In the future, we hope to interact our mutated enzymes better degrade PET, or try to express them in Pichia pastoris.

Crystallinity

In the whole experiment, we used many new methods to improve the efficiency and expression of plastic-degrading enzymes, such as through machine learning and molecular docking, and found many new parts. In addition, we constructed polypeptides interacting with multi-copy plasmids and multi-enzyme complexes, which linked multiple enzymes together by intermolecular forces.

We have successfully found plastic-degrading enzymes with high thermal stability, strong acid-base tolerance and improved enzymatic activity among many mutated enzymes. In addition, the primary exploration of multi-copy number, double enzyme system, fusion protein and so on was carried out, in order to pave the way for the follow-up work. It will be further explored in the future.

Current research mostly focuses on the degradation of shapeless PET plastic. There are still many limitations, such as the fact that PET plastic enzymes degrade low crystallinity PET plastic rapidly, but have poor effects on high crystallinity PET plastic. The most common PET life bottles have high crystallinity 5, making direct enzymatic degradation inappropriate.

Therefore, adequate pretreatment of high-crystallinity PET plastics is required. Existing pretreatment methods include electron irradiation, microbial pretreatment 6 7 8 , microwave pretreatment 9 10 , ultrasonic pretreatment 11 12 , grinding 13 14 , carrier swelling 15, and transesterification 16 , to improve the enzyme contact area or enhance the binding capacity of enzyme and substrate 17 18 . However, these methods are costly and cannot be applied on a large scale.

Some studies have shown that the enzymatic digestion efficiency of PET plastics can be improved by a cross-blending pretreatment strategy 19 . It was found that mixing PET with polycaprolactone (PCL) reduced crystallinity, enhanced hydrophilicity, and significantly improved enzymatic lysis efficiency.

In the future, SUPERB plans to continue focusing on enzyme modification through enzyme engineering and other means under mild conditions.

References

Footnotes

  1. GAO Y, ZHENG Y, QI Z, et al. Enhancing the biodegradation of bis(2‐hydroxyethyl) terephthalate by an IsPETasePA and MHETase dual‐enzyme system [J]. Journal of Chemical Technology & Biotechnology, 2024, 99(8): 1860-70. ↩

  2. CHEN C-C, LI X, MIN J, et al. Complete decomposition of poly(ethylene terephthalate) by crude PET hydrolytic enzyme produced in Pichia pastoris [J]. Chemical Engineering Journal, 2024, 481: 148418. ↩

  3. Shosuke Yoshida et al. A bacterium that degrades and assimilates poly(ethylene terephthalate). Science, 351, 1196-1199 (2016). ↩

  4. Stinson R M, Obendorf S K. Simultaneous diffusion of a disperse dye and a solvent in PET film analyzed by Rutherford backscattering spectrometry [J]. Journal of Applied Polymer Science, 1996, 62(12): 2121-34. ↩

  5. Li H, Fu J, Wang H, et al. Enhancing the hydrophilic modification effect of electron beam (eb) irradiation upon PET fabrics by introducing plasma pretreatment [J]. Materials Review, 2018, 32(2B): 626. ↩

  6. Huang Q S, Yan Z F, Chen X Q, et al. Accelerated biodegradation of polyethylene terephthalate by Thermobifida fusca cutinase mediated by Stenotrophomonas pavanii [J]. Science of the Total Environment, 2022, 808. ↩

  7. Zhang J F, Wang X C, Gong J X, et al. A study on the biodegradability of polyethylene terephthalate fiber and diethylene glycol terephthalate [J]. Journal of Applied Polymer Science, 2004, 93(3): 1089-96. ↩

  8. Hirota Y, Naya M, Tada M, et al. Analysis of soil fungal community structure on the surface of buried polyethylene terephthalate [J]. Journal of Polymers and the Environment, 2021, 29(4): 1227-39. ↩

  9. Zhou Ping, Xue Baoxia, Sun Bukun. Effect of microwave radiation on the structure and properties of PET fibers in aqueous medium [J]. Journal of Tianjin University of Technology, 2012, 31 (05): 5-8. ↩

  10. Choi H M, Cho J Y. Microwave-mediated rapid tailoring of PET fabric surface by using environmentally-benign, biodegradable urea-choline chloride deep eutectic solvent [J]. Fibers and Polymers, 2016, 17(6): 847-56. ↩

  11. Pellis A, Gamerith C, Ghazaryan G, et al. Ultrasound-enhanced enzymatic hydrolysis of poly(ethylene terephthalate) [J]. Bioresource Technology, 2016, 218: 1298-302. ↩

  12. Patidar R, Khanna S, Moholkar V S. Physical features of ultrasound-assisted enzymatic degradation of recalcitrant organic pollutants [J]. Ultrasonics Sonochemistry, 2012, 19(1): 104-18. ↩

  13. Gan L, Xiao Z, Pan H, et al. Efficient production of micron-sized polyethylene terephthalate (PET) powder from waste polyester fiber by physicochemical method [J]. Advanced Powder Technology, 2021, 32(2): 630-6. ↩

  14. Zhou L Y, Zhao G Y, Jiang W. Effects of catalytic transesterification and composition on the toughness of poly(lactic acid)/poly(propylene carbonate) blends [J]. Industrial and Engineering Chemistry Research, 2016, 55(19): 5565-73. ↩

  15. La Mantia F P, Morreale M, Botta L, et al. Degradation of polymer blends: a brief review [J]. Polymer Degradation and Stability, 2017, 145: 79-92. ↩

  16. Tsironi T N, Chatzidakis S M, Stoforos N G. The future of polyethylene terephthalate bottles: challenges and sustainability [J]. Packaging Technology and Science, 2022, 35(4): 317-25. ↩

  17. DING Z, XU G, MIAO R, et al. Rational redesign of thermophilic PET hydrolase LCCICCG to enhance hydrolysis of high crystallinity polyethylene terephthalates [J]. Journal of Hazardous Materials, 2023, 453: 131386. ↩

  18. CHEN Z, DUAN R, XIAO Y, et al. Biodegradation of highly crystallized poly(ethylene terephthalate) through cell surface co-display of bacterial PETase and hydrophobin [J]. Nature Communications, 2022, 13(1). ↩

  19. Cross-blending pretreatment strategy to improve the enzymatic hydrolysis efficiency and harmless treatment of PE _ [D], 2023. ↩