Machine learning-aided engineering of hydrolases for PET depolymerization

Abstract

Plastic waste poses an ecological challenge^1-3 and enzymatic degradation offers one, potentially green and scalable, route for polyesters waste recycling⁴. Poly(ethylene terephthalate) (PET) accounts for 12% of global solid waste⁵, and a circular carbon economy for PET is theoretically attainable through rapid enzymatic depolymerization followed by repolymerization or conversion/valorization into other products^6-10. Application of PET hydrolases, however, has been hampered by their lack of robustness to pH and temperature ranges, slow reaction rates and inability to directly use untreated postconsumer plastics¹¹. Here, we use a structure-based, machine learning algorithm to engineer a robust and active PET hydrolase. Our mutant and scaffold combination (FAST-PETase: functional, active, stable and tolerant PETase) contains five mutations compared to wild-type PETase (N233K/R224Q/S121E from prediction and D186H/R280A from scaffold) and shows superior PET-hydrolytic activity relative to both wild-type and engineered alternatives¹² between 30 and 50 °C and a range of pH levels. We demonstrate that untreated, postconsumer-PET from 51 different thermoformed products can all be almost completely degraded by FAST-PETase in 1 week. FAST-PETase can also depolymerize untreated, amorphous portions of a commercial water bottle and an entire thermally pretreated water bottle at 50 ºC. Finally, we demonstrate a closed-loop PET recycling process by using FAST-PETase and resynthesizing PET from the recovered monomers. Collectively, our results demonstrate a viable route for enzymatic plastic recycling at the industrial scale.