Computational Redesign of a PETase for Plastic Biodegradation under Ambient Condition by the GRAPE Strategy

Abstract

Nature has provided a fantastic array of enzymes that are responsible for essential biochemical functions but not usually suitable for technological applications. Not content with the natural repertoire, protein engineering holds promise to extend the applications of improved enzymes with tailored properties. However, engineering of robust proteins remains a difficult task since the positive mutation library may not cooperate to reach the target function in most cases owing to the ubiquity of epistatic effects. The main demand lies in identifying an efficient path of accumulated mutations. Herein, we devised a computational strategy (greedy accumulated strategy for protein engineering, GRAPE) to improve the robustness of a PETase from Ideonella sakaiensis. A systematic clustering analysis combined with greedy accumulation of beneficial mutations in a computationally derived library enabled the redesign of a variant, DuraPETase, which exhibits an apparent melting temperature that is drastically elevated by 31 °C and a strikingly enhanced degradation toward semicrystalline poly(ethylene terephthalate) (PET) films (30%) at mild temperatures (over 300-fold). Complete biodegradation of 2 g/L microplastics to water-soluble products under mild conditions is also achieved, opening up opportunities to steer the biological degradation of uncollectable PET waste and further conversion of the resulting monomers to high-value molecules. The crystal structure revealed the individual mutation match with the design model. Concurrently, synergistic effects are captured, while epistatic interactions are alleviated during the accumulation process. We anticipate that our design strategy will provide a broadly applicable strategy for global optimization of enzyme performance.

Assessment of thePETaseconformational changes induced by poly(ethylene terephthalate) binding

“…Indeed, point mutations have been used for stabilizing protein structure and some substitutions were proposed in flexible regions of the protein 31 . Recently, Cui et al, have successfully redesign Is PETase to improve its robustness using a systematic clustering analysis combined with the greedy accumulation of beneficial mutations in a computationally derived library 32 . Therefore, the thermal stability of PETase may be crucial for effective PET degradation and may provide biocatalysts as candidates for industrial PET recycling processes.…”

“…31 Recently, Cui et al, have successfully redesign IsPETase to improve its robustness using a systematic clustering analysis combined with the greedy accumulation of beneficial mutations in a computationally derived library. 32 Therefore, the thermal stability of PETase may be crucial for effective PET degradation and may provide biocatalysts as candidates for industrial PET recycling processes. In this work, we have examined in detail the protein conformational changes and residue fluctuations using essential dynamics to suggest potential target regions of IsPETase for mutagenesis.…”

Structural Insights into Carboxylic Polyester-Degrading Enzymes and Their Functional Depolymerizing Neighbors

“…Moreover, Cui and coworkers developed a computational strategy for the mutational analysis of PETase and the design of a multiple mutant with enhanced thermostability when compared with the wild-type protein. This mutant designated as "duraPETase" contains the substitutions S214H-I168R-W159HS188Q-R280A-A180I-G165A-Q119Y-L117F-T140D, and it was able to degrade amorphous PET, but also longer polymers such as PBT with an optimum reaction temperature of 40 • C [60]. However, both engineered PETase mutants showed low efficiency in the degradation of crystalline high-density polymers, suggesting that the compactness of the material limits the protein access to the free chemical groups.…”

Engineering Microbes to Bio-Upcycle Polyethylene Terephthalate

“…Indeed, they achieved a 90% conversion of pre-treated post-consumer PET in less than 10 h, with a mean productivity of 16.7 g TPA L −1 h −1 with a yield of 27.9 g TPA g enzyme −1 , and demonstrate the green route of the circular economy. In concert with computation studies, protein engineering shows the potential to develop efficient PET-hydrolyzing enzymes with improved crystalline PET activity, expanded substrate specificity, alleviated product inhibitions, and thermostability (Cui et al, 2021).…”