Directed evolution of an α1,3-fucosyltransferase using a single-cell ultrahigh-throughput screening method

Sci Adv. 2019 Oct 9;5(10):eaaw8451. doi: 10.1126/sciadv.aaw8451. eCollection 2019 Oct.


Fucosylated glycoconjugates are involved in a variety of physiological and pathological processes. However, economical production of fucosylated drugs and prebiotic supplements has been hampered by the poor catalytic efficiency of fucosyltransferases. Here, we developed a fluorescence-activated cell sorting system that enables the ultrahigh-throughput screening (>107 mutants/hour) of such enzymes and designed a companion strategy to assess the screening performance of the system. After three rounds of directed evolution, a mutant M32 of the α1,3-FucT from Helicobacter pylori was identified with 6- and 14-fold increases in catalytic efficiency (k cat/K m) for the synthesis of Lewis x and 3'-fucosyllactose, respectively. The structure of the M32 mutant revealed that the S45F mutation generates a clamp-like structure that appears to improve binding of the galactopyranose ring of the acceptor substrate. Moreover, molecular dynamic simulations reveal that helix α5, is more mobile in the M32 mutant, possibly explaining its high fucosylation activity.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Bacterial Proteins / chemistry
  • Bacterial Proteins / genetics
  • Bacterial Proteins / metabolism*
  • Binding Sites
  • Biocatalysis
  • Crystallography, X-Ray
  • Directed Molecular Evolution*
  • Flow Cytometry
  • Fucosyltransferases / chemistry
  • Fucosyltransferases / genetics
  • Fucosyltransferases / metabolism*
  • Galactose / chemistry
  • Galactose / metabolism
  • Helicobacter pylori / enzymology
  • High-Throughput Screening Assays / methods*
  • Molecular Docking Simulation
  • Mutagenesis, Site-Directed
  • Protein Binding
  • Protein Structure, Tertiary
  • Single-Cell Analysis
  • Substrate Specificity


  • Bacterial Proteins
  • Fucosyltransferases
  • Galactose