Directed Evolution of an Efficient and Thermostable PET Depolymerase

Abstract

The recent discovery of a hydrolytic enzyme, IsPETase, that can deconstruct poly(ethylene) terephthalate (PET), has sparked great interest in biocatalytic approaches to recycle plastics. Realisation of commercial utility will require the development of robust engineered enzymes that meet the demands of industrial processes. Although rationally engineered variants of PETases have been reported, enzymes that have been experimentally optimised through iterative rounds of directed evolution - the go-to method for engineering industrially useful biocatalysts – have not yet been described. Here, we report the development and implementation of an automated, high-throughput directed evolution platform for engineering polymer degrading enzymes. Evaluation of >13,000 IsPETase variants, applying catalytic activity at elevated temperatures as a primary selection pressure, afforded a HotPETase variant with 21 mutations that has a melting temperature of 82.5°C and can therefore operate near or above the glass transition temperature of PET (60-70°C). HotPETase can depolymerise semi-crystalline PET more rapidly than previously reported PETases and can selectively deconstruct the PET component of a laminated packaging multi-material. Structural characterisation of HotPETase reveals several interesting features that have emerged during evolution to improve thermotolerance and catalytic performance. Our study establishes laboratory evolution as a platform to engineer useful plastic degrading enzymes to underpin biocatalytic plastic recycling processes.

Understanding Consequences and Tradeoffs of Melt Processing as a Pretreatment for Enzymatic Depolymerization of Poly(ethylene terephthalate)

“…[ 46 ] This behavior is unlike those seen here and in both Yoshida et al.’s and Bell et al.’s work, potentially due to a number of factors including substrate form and thickness, crystallinity, or even enzyme‐specific properties. [ 26,47 ] Together, the observations from this work and the literature suggest that opportunities to improve depolymerization might be found in the determination of how enzymes bind to and migrate across or through the substrate. For instance, determining how polymer configuration (e.g., in‐plane, out‐of‐plane, loops, and folds) impacts enzyme binding could help guide preferences in substrate modification and improve depolymerization rates.…”

“…While enzyme modeling is outside the scope of this investigation, insights into the depolymerization behaviors of ex‐RPET strands can be seen via scanning electron microscope (SEM) images of degraded substrates ( Figure ) and compared to other findings in the literature. [ 26,46,47 ] Figure 2a,b shows the undegraded strand with a relatively pristine, smooth surface. Small amounts of surface disfigurement are attributed to handling and mounting of the samples with tweezers.…”

“…showed similar pitting behaviors where a PET substrate was riddled with pits of various depths that seem to impinge on each other instead of uniformly removing material in all directions. [ 47 ]…”

Balance-directed protein engineering ofIsPETase enhances both PET hydrolysis activity and thermostability

“…It means that the mesophilic PET depolymerase has accessibility to overcome this limitation. In the last 6 years, several monumental thermostable and durable variants of IsPETase have been engineered [18][19][20][21] ; however, these have not been reported to reach the stability and efficacy of thermophilic cutinases at high temperatures.…”