Ultrahigh-Throughput Directed Evolution of Polymer-Degrading Enzymes Using Yeast Display

Area of Science:

• Biotechnology
• Biochemistry
• Polymer Science

Background:

• Enzymatic degradation of synthetic polymers offers a sustainable approach to plastic recycling.
• Directed evolution is crucial for enhancing enzyme activity on non-natural substrates like plastics.
• Existing screening methods for enzyme evolution are limited in throughput, hindering rapid discovery.

Purpose of the Study:

• To develop a high-throughput platform for directed evolution of plastic-degrading enzymes.
• To significantly increase the screening capacity for identifying enhanced enzyme variants.
• To improve the efficiency of polyethylene terephthalate (PET) depolymerization.

Main Methods:

• Utilized yeast surface display to present individual enzyme mutants on yeast cells.
• Employed a fluorescence-based assay to detect enzyme activity upon cleavage of a synthetic polymer probe.
• Used fluorescence-activated cell sorting (FACS) for high-throughput isolation of active mutants.
• Performed DNA sequencing to identify beneficial mutations in directed evolution of leaf and branch compost cutinase (LCC).

Main Results:

• Developed a platform capable of screening >10^7 enzyme mutants, a 3-order-of-magnitude increase over previous methods.
• Identified mutations that substantially enhance the degradation kinetics of solid PET films by the LCC enzyme.
• Biochemical assays and molecular dynamics simulations indicated that the H218Y mutation improves enzyme-substrate binding to PET.

Conclusions:

• The yeast surface display platform dramatically accelerates the discovery and optimization of polymer-degrading enzymes.
• This approach enables the identification of enzyme variants with significantly improved activity on solid plastic substrates.
• The developed platform holds great promise for advancing eco-friendly plastic recycling technologies.