Recent advances in enzyme engineering for improved deconstruction of poly(ethylene terephthalate) (PET) plastics

In the last ~20 years, a multitude of natural enzymes have been discovered that can catalyze the breakdown of the common plastic poly(ethylene terephthalate) (PET).While enzymatic PET recycling is an attractive alternative end-of-life route for this waste plastic, the enzymes are not yet optimized for efficient and economical industrial use.Here, we discuss recent advances in engineering these PETdegrading enzymes, which include PET, bis(2-hydroxyethyl) terephthalate (BHET), and 2-hydroxyethyl terephthalic acid (MHET) hydrolases, toward industrially-relevant engineering goals.We place emphasis on trends from past efforts in rational and semi-rational design and emerging areas in directed evolution/high throughput screening and computational design for engineering these enzymes.Many of the same properties that make plastics attractive in use-e.g., durability, non-reactivity, chemical, light, and thermal resistance-allow them to persist and accumulate in the environment 1 .Coupled with low recycling rates 2 (i.e., high discard rates into landfills and the environment) and limited end-of-life pathways [3][4][5] , plastic pollution has become a cause for global concern, with mounting recognition of its environmental and health effects [6][7][8] .Chemical recycling offers an alternative end-of-life pathway for plastics, in contrast to conventional, mechanical recycling 9,10 .In mechanical recycling, old plastics can be reformed to new materials, but characteristically, with diminished properties, and with a limit on the number of times they can be mechanically recycled 11 .Mechanical recycling typically involves the steps of: sorting, washing, shredding/grinding, and melting, followed by re-extrusion of new materials 12 .In chemical recycling, plastics are broken down to constituent chemical species, either monomers or oligomers, which can then be used to create like-new plastics, or as feedstocks for plastics upcycling 13,14 .Chemical recycling methods include glycolysis, methanolysis, pyrolysis, gasification, and hydrolysis 12,13 .Enzymatic recycling (or biodegradation), via enzymatic hydrolysis, is a promising route for plastic recycling.Of particular promise is using enzymatic recycling for contaminated or colored plastic waste streams, as enzymatic recycling is more agnostic to feedstock quality vs. other methods, such as mechanical recycling 15,16 .Thus, enzymatic recycling could complement other types of recycling by creating and expanding value in waste feedstocks.Recent years have witnessed the discovery of naturally-occurring enzymes capable of breaking down manmade plastics to their building block, chemical constituents 17,18 .Hence, enzymatic recycling has gained increased attention, leading some to envision a biorefinery approach to dealing with post-consumer plastic waste streams 13,18 .Arguably the most successful and prolific efforts in enzymatic plastic recycling have been with poly(ethylene terephthalate) (PET)-common in single-use packaging and textiles, and one of the most-produced plastics-as the target 17,19 .The first observations of enzymes (in this case, the thermophilic soil bacterium Thermobifida fusca 20 , which can efficiently degrade plant cell walls 21 ) degrading PET (Fig. 1a) were reported almost 20 years ago; since then, PET depolymerization with enzymes has increased orders of magnitude in efficiency and speed, from reactions taking several weeks to several hours 19 .However, issues remain with enzymatic recycling.Natural enzymes are, generally, not yet optimized for industrial scale biodegradation [17][18][19] .Several properties lacked by these enzymes that will be essential to their industrial adoption include: high catalytic activity, high substrate and product tolerances, high thermostability, high expression and solubility, and acidic pH tolerance (Fig. 1b).Recent efforts have been made to relieve the shortcomings of these enzymes by protein engineering.In this Review, we first overview the state of industrial enzymatic PET recycling, to frame and motivate efforts in enzyme engineering.Next, we highlight the current state-of-the-art approaches used to engineer enzymes toward improved PET depolymerization efficiency, focusing on PET hydrolases (also called PETases), bis(2-hydroxyethyl) terephthalate (BHET) hydrolases (also called BHETases), and 2-hydroxyethyl terephthalic acid (MHET) hydrolases (also called MHETases) (Fig. 1a).Further, we discuss future opportunities for researchers, toward advancing protein engineering of these classes of enzymes past existing bottlenecks.We anticipate this summarization and guidance will facilitate continuing work in the field, toward improved, economically-and environmentallycompetitive industrial enzymatic PET deconstruction and recycling.