19 18 D I A M O N D L I G H T S O U R C E A N N U A L R E V I E W 2 0 2 0 / 2 1 D I A M O N D L I G H T S O U R C E A N N U A L R E V I E W 2 0 2 0 / 2 1 Macromolecular Crystallography Group Beamline I03 Engineering enzyme cocktails to break down single-use plastics Related publication: Knott B. C., Erickson E., AllenM. D., Gado J. E., GrahamR., Kearns F. L., Pardo I.,Topuzlu E., Anderson J. J., Austin H. P., Dominick G., Johnson C.W., Rorrer N. A., Szostkiewicz C. J., CopiéV., Payne C. M.,Woodcock H. L., Donohoe B. S., BeckhamG.T. &McGeehan J. E. Characterization and engineering of a two-enzyme system for plastics depolymerization. Proc. Natl. Acad. Sci. 117 , 25476–25485 (2020). DOI: 10.1073/pnas.2006753117 Publication keywords: Polyethylene terephthalate; Recycling; Upcycling; Biodegradation; Serine hydrolase A n estimated 11 million tons of plastic waste enter our oceans annually, impacting wildlife, our food chain and our health. The UK government has set ambitious 2025 targets for plastics recycling, but effective means of achieving these are currently lacking. Therefore, an international teamof researchers is focusing on the discovery and engineering of enzymes that can help break down plastics for recycling. In previous work, they characterised the structure and function of PETase, a bacterial enzyme with the remarkable ability to deconstruct one of themost commonly polluting thermoplastics, polyethylene terephthalate, PET. This study looked in-depth at a partner enzyme called MHETase, secreted from the same bacterium, that can significantly speed up the breakdown process. The structures they collected on Diamond Light Source’s I03 Macromolecular Crystallography (MX) beamline are the highest resolution available and provide a detailed insight into the MHETase enzyme. Combined with detailed bioinformatics, biochemistry and molecular simulations, they show a highly synergistic relationship between the PETase and MHETase enzymes. The team investigated if tethering the proteins together could improve the breakdown and demonstrated that this was significantly faster than PETase alone or a PETase-MHETase cocktail. Enzymes offer a low-energy solution and the potential to allow infinite recycling, reducing our growing requirements for fossil resources. There is a lot of excitement around the potential for naturally-evolved enzymes to tackle our plastic waste. This publication reached the Altmetric Top 100 (#39) from the 3.4 million papers published in 2020. Plastic pollution represents a global environmental crisis, with growing levels of production from fossil resources contributing to greenhouse gas emissions and poor disposal resulting in the contamination of our land, rivers and oceans. Innovative solutions for dealing with our increasing appetite for plastics are urgently required. One of our most polluting single-use plastics is the polymer polyethylene terephthalate, commonly known as PET and recognisable with the triangle recycling label with the number one. This thermoplastic is most visible in the environment as discarded drinks bottles but is vastly more abundant in the form of polyester textiles. Both forms of this polymer have residence times in the natural environment of hundreds of years and while processes such as waves and sunlight break down these materials into smaller microplastics, their chemical structure remains largely intact. While technology exists to turn plastic bottles back into plastic bottles, even in countries with excellent infrastructure, typically this only represents around 7% of the total. Most are mechanically recovered and downcycled to lower value materials such as fibres for clothing and carpet. This material stream inevitably ends up being incinerated or dumped in landfill as there is little economic incentive to reclaim it with current processes and in less well- developed systems, large quantities escape to the environment. In 2018 we reported the characterisation of the enzyme PETase, secreted by the bacterium, Ideonella sakaiensis , originally discovered in a Japanese recycling centre 1,2 . This naturally occurring enzyme is capable of breaking the ester bonds that hold the two monomers, ethylene glycol and terephthalic acid, together to form the long chains that provide PET with its high resistance and lightweight properties. This biological activity opens the door to use enzymes to regenerate these monomers for the infinite recycling of this type of plastic (Fig. 1), a central goal for our team at the Centre for Enzyme Innovation at the University of Portsmouth. I. sakaiensis is known to secrete two key enzymes, PETase, which liberates soluble products, includingmono(2-hydroxyethyl) terephthalate (MHET) and a partner enzyme called MHETase, which cleaves this MHET to form terephthalic acid and ethylene glycol. In order to investigate how these enzymes work in tandem, we cloned and expressed MHETase to study its structural and functional adaptations. We collected multiple datasets from several crystal forms of MHETase on beamline I03, utilising a selenomethionine derivative to solve the phases by SAD. The highest resolution data, with diffraction extending to 1.6 Å, provided detailed insights into the active site that included key residues captured in multiple conformations. MHETase has a core domain with a fold resembling PETase but has an additional extensive lid domain that covers the active site (Fig. 2). The overall fold is similar to some ferulic acid esterases from the tannase family. Bioinformatic searches revealed other potential MHETases and two of these identified homologues were shown to be active. While they had lower efficiency, they provided useful insights into the key residues required for MHET turnover. We were able to rationalise the structural basis of substrate specificity through mutational analysis and furthermore, these structural models provided an excellent platform for QM/ MMmolecular simulations which allowed us to determine that the deacylation is the rate limiting in the MHETase two-step reaction. We were particularly interested in how the MHETase enzyme interacts with PETase to enhance the deconstruction of PET, so we took a matrix approach, analysing 144 reactions at varying concentrations and ratios. This revealed significant synergy when employing these enzyme cocktails, with terephthalate acid production increased. This inspired the design of several chimeric constructs for a multi-enzyme approach, where the two enzymes were directly connected via a short flexible linker (Fig. 3). Hydrolysis rates were increased further, up to six-fold higher than PETase alone, suggesting that further improvements may be possible through this type of protein engineering. Multi-enzyme systems have been previously described in the deconstruction of natural polymers including lignocellulose and chitin, with a range of enzyme mixtures produced by one species of bacteria, or sometimes large consortia working in parallel. A deeper understanding of these naturally evolved processes will provide further insights into how we may better engineer systems that can tackle polymers such as PET. In collaboration with our US colleagues at the National Renewable Energy Laboratory, our team is focused on the discovery and engineering of faster and more efficient enzyme systems. We aim to develop enhanced cocktails for polyesters, but also expand into enzymatic deconstruction of some of our other most polluting plastics. The generation of monomers from waste plastic allows the regeneration of food-grade quality plastics and with the addition of other bio-based monomers, creates the potential for upcycling into higher-value materials. Structure-function relationships will remain a central part of this work and we look forward to utilising a wider range of beamlines at Diamond as new advanced facilities continue to come onstream. Given the range and scale of plastic pollution, we are certain that an interdisciplinary approach will be essential, combining advances in mechanical techniques, chemical catalysts and biochemical approaches. This will require working in large consortia such as BOTTLE.org to which we share membership, allowing us to productively share resources and expertise. While we recognise that we must reduce our reliance on plastic, particularly single-use materials, we believe that the low energy footprint and carbon capturing advantages of closed-loop circular enzyme systems can have a significant positive impact on our environment. References: 1. Austin H. P. et al. Characterization and engineering of a plastic-degrading aromatic polyesterase. Proc. Natl. Acad. Sci. 115 , 201718804 (2018). DOI: 10.1073/pnas.1718804115 2. Yoshida S. et al. A bacterium that degrades and assimilates poly(ethylene terephthalate). Science (80-. ). 351 , 1196–1199 (2016). DOI: 10.1126/ science.aad6359 Funding acknowledgement: We thank Research England (E3) and the BBSRC (grant BB/P011918/1) for funding and the National Renewable Energy Laboratory (NREL) and BOTTLE. org Consortium funded through the US Department of Energy contract DEAC3608GO28308. Corresponding authors: Prof. John McGeehan, Centre for Enzyme Innovation, University of Portsmouth,
[email protected]; Miss Rosie Graham, Centre for Enzyme Innovation, University of Portsmouth,
[email protected] Figure 1: Enzymes can break down waste plastic polymers into building blocks that can be purified and re-polymerised, allowing infinite recycling of the materials as part of a circular plastics economy (Credit Prof. Andrew Pickford, CEI). Figure 2: Structural model of MHETase highlighting the PETase-like core domain in purple with the lid domain in pink. (Inset) Key residues lining the active site with bound benzoate providing insights into ligand interactions. Figure 3: Cartoon representation of a PETase-MHETase chimera, with PETase in green, MHETase in purple and the 8 amino acid linker in blue.