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Management of polyethylene terephthalate (PET) plastic waste remains a challenge. PET-hydrolyzing enzymes (PHEs) such as IsPETase and variants like FAST-PETase demonstrate promising PET depolymerization capabilities at ambient temperatures and can be utilized to recycle and upcycle plastic waste. Whole-cell biocatalysts displaying PHEs on their surface offer high efficiency, reusability, and stability for PET depolymerization. However, their efficacy in fully breaking down PET is hindered by the necessity of two enzymes: PETase and MHETase. Current whole-cell systems either display only one enzyme or struggle with performance when displaying larger enzymes such as the MHETase–PETase chimera. We developed a Saccharomyces cerevisiae-based whole-cell biocatalyst for complete depolymerization of PET into its constituent monomers with no accumulation of intermediate products. Leveraging a cellulosome-inspired trifunctional protein scaffoldin displayed on the yeast surface, we co-immobilized FAST-PETase and MHETase, forming a multi-enzyme cluster. This whole-cell biocatalyst achieved complete PET depolymerization at 30 °C, yielding 4.95 mM terephthalic acid (TPA) when tested on a PET film. Furthermore, we showed improved PET depolymerization ability by binding FAST-PETase at multiple sites on the scaffoldin. The whole cells had the added advantage of retained activity over multiple reusability cycles. This breakthrough in complete PET depolymerization marks a step toward a circular plastic economy.
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Characterization and engineering of a two-enzyme system for plastics depolymerization
Plastics pollution represents a global environmental crisis. In response, microbes are evolving the capacity to utilize synthetic polymers as carbon and energy sources. Recently, Ideonella sakaiensis was reported to secrete a two-enzyme system to deconstruct polyethylene terephthalate (PET) to its constituent monomers. Specifically, the I. sakaiensis PETase depolymerizes PET, liberating soluble products, including mono(2-hydroxyethyl) terephthalate (MHET), which is cleaved to terephthalic acid and ethylene glycol by MHETase. Here, we report a 1.6 Å resolution MHETase structure, illustrating that the MHETase core domain is similar to PETase, capped by a lid domain. Simulations of the catalytic itinerary predict that MHETase follows the canonical two-step serine hydrolase mechanism. Bioinformatics analysis suggests that MHETase evolved from ferulic acid esterases, and two homologous enzymes are shown to exhibit MHET turnover. Analysis of the two homologous enzymes and the MHETase S131G mutant demonstrates the importance of this residue for accommodation of MHET in the active site. We also demonstrate that the MHETase lid is crucial for hydrolysis of MHET and, furthermore, that MHETase does not turnover mono(2-hydroxyethyl)-furanoate or mono(2-hydroxyethyl)-isophthalate. A highly synergistic relationship between PETase and MHETase was observed for the conversion of amorphous PET film to monomers across all nonzero MHETase concentrations tested. Finally, we compare the performance of MHETase:PETase chimeric proteins of varying linker lengths, which all exhibit improved PET and MHET turnover relative to the free enzymes. Together, these results offer insights into the two-enzyme PET depolymerization system and will inform future efforts in the biological deconstruction and upcycling of mixed plastics.
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- Award ID(s):
- 1714556
- PAR ID:
- 10285033
- Date Published:
- Journal Name:
- Proceedings of the National Academy of Sciences
- Volume:
- 117
- Issue:
- 41
- ISSN:
- 0027-8424
- Page Range / eLocation ID:
- 25476 to 25485
- Format(s):
- Medium: X
- Sponsoring Org:
- National Science Foundation
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Abstract BackgroundDepolymerizing polyethylene terephthalate (PET) plastics using enzymes, such as PETase, offers a sustainable chemical recycling route. To enhance degradation, many groups have sought to engineer PETase for faster catalysis on PET and elevated stability. Considerably less effort has been focused toward expressing large quantities of the enzyme, which is necessary for large-scale application and widespread use. In this work, we evaluated severalE. colistrains for their potential to produce soluble, folded, and activeIsPETase, and moved the production to a benchtop bioreactor. As PETase is known to require disulfide bonds to be functional, we screened several disulfide-bond promoting strains ofE. colito produceIsPETase, FAST-PETase and Hot-PETase. ResultsWe found expression in SHuffle T7 Express results in higher active expression ofIsPETase compared to standardE. coliproduction strains such as BL21(DE3), reaching a purified titer of 20 mg enzyme per L of culture from shake flasks using 2xLB medium. We characterized purifiedIsPETase on 4-nitrophenyl acetate and PET microplastics, showing the enzyme produced in the disulfide-bond promoting host has high activity. Using a complex medium with glycerol and a controlled bioreactor,IsPETase titer reached 104 mg per L for a 46-h culture. FAST-PETase was found to be produced at similar levels in BL21(DE3) or SHuffle T7 Express, with purified production reaching 65 mg per L culture when made in BL21(DE3). Hot-PETase titers were greatest in BL21(DE3) reaching 77 mg per L culture. ConclusionsWe provide protein expression methods to produce three important PETase variants. Importantly, forIsPETase, changing expression host, medium optimization and movement to a bioreactor resulted in a 50-fold improvement in production amount with a per cell dry weight productivity of 0.45 mgPETasegCDW−1 h−1, which is tenfold greater than that forK. pastoris. We show that the benefit of using SHuffle T7 Express for expression only extends toIsPETase, with FAST-PETase and Hot-PETase better produced and purified from BL21(DE3), which is unexpected given the number of cysteines present. This work represents a systematic evaluation of protein expression and purification conditions for PETase variants to permit further study of these important enzymes.more » « less
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Katherine McMahon, University of (Ed.)Plastics, such as polyethylene terephthalate (PET) from water bottles, are polluting our oceans, cities, and soils. While a number of Pseudomonas species have been described that degrade aliphatic polyesters, such as polyethylene (PE) and polyurethane (PUR), few from this genus that degrade the semiaromatic poly- mer PET have been reported. In this study, plastic-degrading bacteria were isolated from petroleum-polluted soils and screened for lipase activity that has been associ- ated with PET degradation. Strains and consortia of bacteria were grown in a liquid carbon-free basal medium (LCFBM) with PET as the sole carbon source. We moni- tored several key physical and chemical properties, including bacterial growth and modi!cation of the plastic surface, using scanning electron microscopy (SEM) and attenuated total re"ectance-Fourier transform infrared spectroscopy (ATR-FTIR) spec- troscopy. We detected by-products of hydrolysis of PET using 1H-nuclear magnetic resonance (1H NMR) analysis, consistent with the ATR-FTIR data. The full consortium of !ve strains containing Pseudomonas and Bacillus species grew synergistically in the presence of PET and the cleavage product bis(2-hydroxyethyl) terephthalic acid (BHET) as sole sources of carbon. Secreted enzymes extracted from the full consor- tium were capable of fully converting BHET to the metabolically usable monomers terephthalic acid (TPA) and ethylene glycol. Draft genomes provided evidence for mixed enzymatic capabilities between the strains for metabolic degradation of TPA and ethylene glycol, the building blocks of PET polymers, indicating cooperation and ability to cross-feed in a limited nutrient environment with PET as the sole carbon source. The use of bacterial consortia for the biodegradation of PET may provide a partial solution to widespread planetary plastic accumulation.more » « less
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The widespread consumption of PET worldwide has necessitated the search for environment‐friendly methods for PET degradation and recycling. Among these methods, biodegradation stands out as a promising approach for recycling PET. The discovery of duo enzyme system PETase and MHETase in 2016, along with their engineered variants, has demonstrated significant potential in breaking down PET. Previous studies have also demonstrated that the activity of the enzyme PETase increases when it is immobilized on nanoparticles. To achieve highly efficient and complete PET depolymerization, we immobilized both FAST‐PETase and MHETase at a specific ratio on magnetic nanoparticles. This immobilization resulted in a 2.5‐fold increase in product release compared with free enzymes. Additionally, we achieved reusability and enhanced stability of the enzyme bioconjugates.more » « less
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Poly(ethylene terephthalate) (PET) is a highly recyclable plastic that has been extensively used and manufactured. Like other plastics, PET resists natural degradation, thus accumulating in the environment. Several recycling strategies have been applied to PET, but these tend to result in downcycled products that eventually end up in landfills. This accumulation of landfilled PET waste contributes to the formation of microplastics, which pose a serious threat to marine life and ecosystems, and potentially to human health. To address this issue, our project leveraged synthetic biology to develop a whole‐cell biocatalyst capable of depolymerizing PET in seawater environments by using the fast‐growing, nonpathogenic, moderate halophile Vibrio natriegens. By leveraging a two‐enzyme system—comprising a chimera of IsPETase and IsMHETase from< Ideonella sakaiensis —displayed on V. natriegens, we constructed whole‐cell catalysts that depolymerize PET and convert it into its monomers in salt‐containing media and at a temperature of 30°C.more » « less
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Accepted Manuscript
- Journal Article:
- https://doi.org/10.1073/pnas.2006753117
