Cell-Based Assay Approaches for Glycosaminoglycan Synthase High-Throughput Screening: Development and Applications

Glycosaminoglycan synthases have immense potential in applications involving synthesis of oligosaccharides, using enzymatic approaches and construction of cell factories that produce polysaccharides as critical metabolic components. However, the use of high-throughput activity assays to screen for the evolution of these enzymes can be challenging because there are no significant changes in fluorescence or absorbance associated with glycosidic bond formation. Here, using incorporation of azido-labeled N-acetylhexosamine analogs into bacterial capsule polysaccharides via bacterial metabolism and bioorthogonal chemistry, fluorophores were specifically introduced onto cell surfaces. Furthermore, correlations between detectable fluorescence signals and the polysaccharide-synthesizing capacity of individual bacteria were established. Among 10 candidate genes, 6 members of the chondroitin synthase family were quickly identified in a recombinant Bacillus subtilis host strain. Additionally, directed evolution of heparosan synthase was successfully performed using fluorescence-activated cell sorting of recombinant Escherichia coli O10:K5(L):H4, yielding several mutants with increased activity. Cell-based approaches that selectively detect the presence or absence of synthases within an individual colony of bacterial cells, as well as their level of activity, have broad potential in the exploration and engineering of glycosaminoglycan synthases. These approaches also support the creation of novel strategies for high-throughput screening of enzyme activity based on cell systems.