SignificanceSynthetic polymers are ubiquitous in the modern world but pose a global environmental problem. While plastics such as poly(ethylene terephthalate) (PET) are highly versatile, their resistance to natural degradation presents a serious, growing risk to fauna and flora, particularly in marine environments. Here, we have characterized the 3D structure of a newly discovered enzyme that can digest highly crystalline PET, the primary material used in the manufacture of single-use plastic beverage bottles, in some clothing, and in carpets. We engineer this enzyme for improved PET degradation capacity and further demonstrate that it can also degrade an important PET replacement, polyethylene-2,5-furandicarboxylate, providing new opportunities for biobased plastics recycling.

At the molecular level, polymers are long chains in which the emergent material properties are dictated by the movement, arrangement, and interactions of these chains. Key factors that contribute to how the polymer chains move and rearrange are the molecular identity and arrangement, crystallinity, and molecular weight. Generally, the monomer identity influences the final application of the polymer by dictating many properties, such as the glass transition temperature (Tg). 12 As the Tg represents a softening of the material, it is a prime factor in determining the final polymer application. Flexible molecules in the backbone, which can relax faster, may result in low Tg materials with applications such as PE bags or rubber (i.e. polybutadiene). 13 Meanwhile, rigid molecules or molecules that result in stronger interchain interactions (and relax on longer timescales) can result in high Tg materials, ideal for reinforced applications. In general, when materials are at temperatures below the Tg, the polymer chains are kinetically arrested, exhibiting higher strengths. Even though monomer identity is often the largest contribution to Tg, it is not the only factor, as molecular weight, 14 tacticity, 15 and crystallinity 16 also contribute. While nearly all polymers exhibit a Tg characteristic of their amorphous region, semi-crystalline polymers will also exhibit concomitant melting behaviour crystalline in their crystalline regions, making them semi-crystalline. Crystallinity has a direct impact on polymer properties, as increases in crystallinity augment the strength of the final product and reduce the permeability of liquids and gases. Co-monomers (e.g., isophthalic acid in poly(ethylene terephthalate) (PET)) are often used to lower or completely remove crystallinity to make polymers easier to process or more transparent. 17 Finally, molecular weight, and the distributions of molecular weights, have some effect on the thermomechanical polymer properties (e.g., increasing molecular weight leads to higher Tg, modulii, etc.). However, over a critical molecular weight, nearly all thermomechanical polymer properties are constant. The exception to this generalization is the viscosity of a polymer melt, which scales with the molecular weight to the 3-3.5 power (η ~ MW 3-3.5 ) and also encapsulates properties such as diffusivity. These factors together contribute to polymer recalcitrance by limiting polymer mobility and accessibility to chemical linkages, posing a challenge for catalytic plastics deconstruction.

Organisms use diverse mechanisms involving multiple complementary enzymes, particularly glycoside hydrolases (GHs), to deconstruct lignocellulose. Lytic polysaccharide monooxygenases (LPMOs) produced by bacteria and fungi facilitate deconstruction as does the Fenton chemistry of brown-rot fungi. Lignin depolymerisation is achieved by white-rot fungi and certain bacteria, using peroxidases and laccases. Meta-omics is now revealing the complexity of prokaryotic degradative activity in lignocellulose-rich environments. Protists from termite guts and some oomycetes produce multiple lignocellulolytic enzymes. Lignocellulose-consuming animals secrete some GHs, but most harbour a diverse enzyme-secreting gut microflora in a mutualism that is particularly complex in termites. Shipworms however, house GH-secreting and LPMO-secreting bacteria separate from the site of digestion and the isopod Limnoria relies on endogenous enzymes alone. The omics revolution is identifying many novel enzymes and paradigms for biomass deconstruction, but more emphasis on function is required, particularly for enzyme cocktails, in which LPMOs may play an important role.

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.

This study presents a comprehensive process, economic, environmental, and socioeconomic analysis of the enzymatic recycling of poly(ethylene terephthalate), which is the most widely used synthetic polyester. The analyses predict that PET deconstruction using enzymes can achieve cost parity with terephthalic acid manufacturing as well as substantial reductions in both supply chain energy use and greenhouse gas emissions relative to virgin polyester manufacturing. This study also highlights key research areas for further impactful development of biocatalysis-enabled plastics recycling.