Directed Evolution of Glycosyltransferases by a Single-Cell Ultrahigh-Throughput FACS-Based Screening Method

Yumeng Tan; Xue Zhang; Yan Feng; Guang-Yu Yang

doi:10.1007/978-1-0716-2152-3_14

Directed Evolution of Glycosyltransferases by a Single-Cell Ultrahigh-Throughput FACS-Based Screening Method

Methods Mol Biol. 2022:2461:211-224. doi: 10.1007/978-1-0716-2152-3_14.

Authors

Yumeng Tan¹, Xue Zhang¹, Yan Feng¹, Guang-Yu Yang²

Affiliations

¹ State Key Laboratory of Microbial Metabolism, School of Life Sciences and Biotechnology, Shanghai Jiao Tong University, Shanghai, China.
² State Key Laboratory of Microbial Metabolism, School of Life Sciences and Biotechnology, Shanghai Jiao Tong University, Shanghai, China. yanggy@sjtu.edu.cn.

PMID: 35727453
DOI: 10.1007/978-1-0716-2152-3_14

Abstract

Engineering of glycosyltransferases (GTs) with desired substrate specificity for the synthesis of complex oligosaccharides has been of great scientific and industrial interest. Here we describe an ultra-high-throughput fluorescence activated cell sorting (FACS) method for the directed evolution of GTs, at the single cell level. This assay relies on the exquisite substrate specificity of lactose permeases (LacY) that are located in the cell membrane, which distinguish selectively the fluorescent glycosylated products from the unreacted substrates. The method described here allows facile screening 10⁶-10⁷ mutants per hour. We demonstrate the application of this technique in the screening of libraries of α1,3-fucosyltransferase.

Keywords: Directed evolution; Fluorescence activated cell sorting; Glycosyltransferases; Ultra-high throughput screening.

Publication types

Research Support, Non-U.S. Gov't

MeSH terms

Biological Assay*
Directed Molecular Evolution / methods
Flow Cytometry / methods
Glycosyltransferases* / genetics
Glycosyltransferases* / metabolism
Substrate Specificity

Substances

Glycosyltransferases