Engineering of a Thermostable PETase by Directed Evolution

Abstract

Plastics are exceptionally useful materials. With a wide range of beneficial characteristics and capabilities, plastics have been instrumental in the progression of modern society by underpinning the development of new technologies across a spectrum of industries from food packaging to medicine. Yet many of the attributes that make these materials such a valuable commodity lends them to becoming a serious environmental threat. It has become impossible to ignore the duplicitous nature of anthropogenic polymers and the failure of current recycling methodologies to curb the ever-increasing plastic pollution problem. Biocatalytic depolymerisation has the potential to overcome some of the challenges faced by conventional recycling strategies, such as mechanical or chemical methods, but the absence of industry-ready enzymes has hindered wide-scale adoption of this technology. Indeed, for biological recycling to become a viable methodology, biocatalysts must be able to withstand tough plastic processing conditions and function competitively, both practically and economically, as compared to alternative methods. The research presented in this thesis describes a method to rapidly optimise plastic degrading enzymes to enhance their commercial applicability, detailing the design and deployment of a high-throughput directed evolution platform to engineer the poly(ethylene terephthalate) (PET) deconstructing enzyme from Ideonella sakaiensis (IsPETase). The resulting biocatalyst, HotPETase, has superior characteristics to its parent protein: it has the ability to function at elevated temperatures close to the glass transition temperature of PET (60-70oC) and to depolymerise a range of PET-containing materials, including highly crystalline PET, PET composites and PET copolymers, that would be present in realistic post-consumer waste streams. Structural characterisation of HotPETase sheds light on the features installed during directed evolution that augment stability and catalytic performance. To further understand the structural basis and catalytic mechanism of polymer deconstruction by IsPETase and its engineered variants, a method to trap and characterise a close analogue of a key catalytic intermediate is also presented, taking advantage of genetic code expansion technologies. It is anticipated that the engineering strategies described within, along with the enhanced characteristics and broad substrate scope of HotPETase, in conjunction with an elevated understanding of the mechanism of enzymatic PET deconstruction, will contribute to the future development of a suite of complementary, industrially-viable, plastic-deconstructing enzymes.

Date of Award	1 Aug 2022
Original language	English
Awarding Institution	The University of Manchester
Supervisor	Philip Day (Supervisor), Anthony Green (Supervisor), Nigel Scrutton (Supervisor) & Andrew Currin (Supervisor)