Efficient mild depolymerization of polyester plastics accomplished by engineered PETase via directed evolution of flexible loops

• Published in: Cell reports physical science 2024-11, Vol.5 (11), p.102295, Article 102295
• Main Authors: Liu, Yidi, Lin, Huanliu, Wei, Zhisheng, Bai, Shujin, Chen, Sheng, Wu, Jing, Liu, Zhanzhi
• Format: Article
• Language: English
• Subjects: directed evolution; flexible loops; Ideonella sakaiensis PETase; mild depolymerization; polyethylene terephthalate
• ISSN: 2666-3864; 2666-3864
• DOI: 10.1016/j.xcrp.2024.102295

Due to inadequate disposal and recycling of polyethylene terephthalate (PET) plastic products, there has been growing concern about the severe environmental consequences. Biocatalytic degradation of PET has emerged as a promising solution. The PET hydrolase IsPETase from Ideonella sakaiensis exhibits distinctive substrate activity at mild temperatures, but its practical application is limited by restricted catalytic efficiency. In this study, the highly flexible loops of IsPETase are targeted for engineering after a detailed structural analysis. A customized dual-fluorescence high-throughput assay is then developed to facilitate the directed evolution of these selected loops in order to enhance IsPETase’s catalytic performance, specifically at mild temperatures. Through implementation of this system, the evolved IsPETase mutant M4-Q demonstrates exceptional PET biodegradation activity at mild temperatures and advantages in degrading various aliphatic-aromatic copolyesters. These findings offer innovative insights into the enzymatic degradation of PET and other polyester plastic waste. [Display omitted] •Three highly flexible loops of IsPETase are chosen after detailed structural analysis•A high-throughput assay based on pH-sensitive fluorescent probes is established•The M4-Q mutant shows improved catalytic efficiency on PET and other polyesters•Molecular dynamics simulation reveals the structure-function relationship of M4-Q Liu et al. enhance the catalytic efficiency of IsPETase through the directed evolution of its flexible loops. The evolved M4-Q mutant exhibits superior biodegradation of PET and other polyester plastics at mild temperatures, offering a promising strategy for addressing plastic waste and advancing biocatalytic degradation technologies.