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

Glycosaminoglycan synthases haveimmense potential inapplicationsinvolving synthesis of oligosaccharides, using enzymatic approachesand construction of cell factories that produce polysaccharides ascritical metabolic components. However, the use of high-throughputactivity assays to screen for the evolution of these enzymes can bechallenging because there are no significant changes in fluorescenceor absorbance associated with glycosidic bond formation. Here, usingincorporation of azido-labeled N-acetylhexosamineanalogs into bacterial capsule polysaccharides via bacterial metabolismand bioorthogonal chemistry, fluorophores were specifically introducedonto cell surfaces. Furthermore, correlations between detectable fluorescencesignals and the polysaccharide-synthesizing capacity of individualbacteria were established. Among 10 candidate genes, 6 members ofthe chondroitin synthase family were quickly identified in a recombinant Bacillus subtilis host strain. Additionally, directedevolution of heparosan synthase was successfully performed using fluorescence-activatedcell sorting of recombinant Escherichia coli O10:K5(L):H4, yielding several mutants with increased activity.Cell-based approaches that selectively detect the presence or absenceof synthases within an individual colony of bacterial cells, as wellas their level of activity, have broad potential in the explorationand engineering of glycosaminoglycan synthases. These approaches alsosupport the creation of novel strategies for high-throughput screeningof enzyme activity based on cell systems.