Directed evolution of Rhodotorula gracilisd-amino acid oxidase using single-cell hydrogel encapsulation and ultrahigh-throughput screening.

ABSTRACT: Engineering catalytic and biophysical properties of enzymes is an essential step en route to advanced biomedical and industrial applications. Here, we developed a high-throughput screening and directed evolution strategy relying on single-cell hydrogel encapsulation to enhance the performance of d-Amino acid oxidase from Rhodotorula gracilis (RgDAAOx), a candidate enzyme for cancer therapy. We used a cascade reaction between RgDAAOx variants surface displayed on yeast and horseradish peroxidase (HRP) in the bulk media to trigger enzyme-mediated crosslinking of phenol-bearing fluorescent alginate macromonomers, resulting in hydrogel formation around single yeast cells. The fluorescent hydrogel capsules served as an artificial phenotype and basis for pooled librar

SUBMITTER: Kung C 

PROVIDER: S-EPMC10366730 | biostudies-literature | 2023 Jul

REPOSITORIES: biostudies-literature