Directed evolution of an efficient and thermostable PET depolymerase

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.