Directed evolution of an efficient and thermostable PET depolymerase

The recent discovery of IsPETase, a hydrolytic enzyme that can deconstruct polyethylene terephthalate) (PET), has sparked great interest in biocatalytic approaches to recycle plastics. Realization of commercial use will require the development of robust engineered enzymes that meet the demands of industrial processes. Although rationally engineered PETases have been described, enzymes that have been experimentally optimized via directed evolution have not previously been reported. Here, we describe an automated, high-throughput directed evolution platform for engineering polymer degrading enzymes. Applying catalytic activity at elevated temperatures as a primary selection pressure, a thermostable IsPETase variant (HotPETase, T-m = 82.5 degrees C) was engineered that can operate at the glass transition temperature of PET. HotPETase can depolymerize semicrystalline PET more rapidly than previously reported PETases and can selectively deconstruct the PET component of a laminated multimaterial. Structural analysis of HotPETase reveals interesting features that have emerged to improve thermotolerance and catalytic performance. Our study establishes laboratory evolution as a platform for engineering useful plastic degrading enzymes.

Automated summary

The recent discovery of IsPETase, a hydrolytic enzyme that can degrade polyethylene terephthalate (PET), has sparked interest in using biocatalytic approaches to recycle plastics. In this study, we describe an automated, high-throughput directed evolution platform for engineering polymer degrading enzymes. By applying catalytic activity under elevated temperatures, a thermostable IsPETase variant (HotPETase) was successfully engineered to operate at the industrial processing temperature of PET. This HotPETase enzyme showed improved efficiency in degrading PET compared to previously reported enzymes and could selectively breakdown the PET component of multi-materials.