A Dual Fluorescence Assay Enables High-Throughput Screening for Poly(ethylene terephthalate) Hydrolases.
Kun LiuZiping XuZhiyi ZhaoYuexing ChenYating ChaiLi MaShengying LiPublished in: ChemSusChem (2023)
The drastically increasing consumption of petroleum-derived plastics hasserious environmental impacts and raises public concerns. Poly(ethylene terephthalate) (PET) is amongst the most extensively produced synthetic polymers. Enzymatic hydrolysis of PET recently emerged as an enticing path for plastic degradation and recycling. In-lab directed evolution has revealed the great potential of PET hydrolases (PETases). However, the time-consuming and laborious PETase assays hinder the identification of effective variants in large mutant libraries. Herein, we devise and validate a dual fluorescence-based high-throughput screening (HTS) assay for a representative IsPETase. The two-round HTS of a pilot library consisting of 2850 IsPETase variants yields six mutant IsPETases with 1.3-4.9 folds improved activities. Compared to the currently used structure- or computational redesign-based PETase engineering, this HTS approach provides a new strategy for discovery of new beneficial mutation patterns of PETases.
Keyphrases
- high throughput
- pet ct
- positron emission tomography
- computed tomography
- copy number
- pet imaging
- single molecule
- healthcare
- single cell
- small molecule
- human health
- wild type
- energy transfer
- clinical trial
- cross sectional
- nitric oxide
- dna methylation
- risk assessment
- randomized controlled trial
- climate change
- life cycle
- genome wide
- drug induced
- adverse drug