Issue 8, 2023

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 library screening by fluorescence activated cell sorting (FACS). We screened a RgDAAOx variant library containing ∼106 clones while lowering the D-Ala substrate concentration over three sorting rounds in order to isolate variants with low Km. After three rounds of FACS sorting and regrowth, we isolated and fully characterized four variants displayed on the yeast surface. We identified variants with a more than 5-fold lower Km than the parent sequence, with an apparent increase in substrate binding affinity. The mutations we identified were scattered across the RgDAAOx structure, demonstrating the difficulty in rationally predicting allosteric sites and highlighting the advantages of scalable library screening technologies for evolving catalytic enzymes.

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

Supplementary files

Article information

Article type
Paper
Submitted
03 Jan 2023
Accepted
15 Apr 2023
First published
20 Apr 2023
This article is Open Access
Creative Commons BY-NC license

React. Chem. Eng., 2023,8, 1960-1968

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

C. Küng, R. Vanella and M. A. Nash, React. Chem. Eng., 2023, 8, 1960 DOI: 10.1039/D3RE00002H

This article is licensed under a Creative Commons Attribution-NonCommercial 3.0 Unported Licence. You can use material from this article in other publications, without requesting further permission from the RSC, provided that the correct acknowledgement is given and it is not used for commercial purposes.

To request permission to reproduce material from this article in a commercial publication, please go to the Copyright Clearance Center request page.

If you are an author contributing to an RSC publication, you do not need to request permission provided correct acknowledgement is given.

If you are the author of this article, you do not need to request permission to reproduce figures and diagrams provided correct acknowledgement is given. If you want to reproduce the whole article in a third-party commercial publication (excluding your thesis/dissertation for which permission is not required) please go to the Copyright Clearance Center request page.

Read more about how to correctly acknowledge RSC content.

Social activity

Spotlight

Advertisements