Directed evolution of bacterial polysialyltransferases

Polysialyltransferases (polySTs) are glycosyltransferases that synthesize polymers of sialic acid found in vertebrates and some bacterial pathogens. Bacterial polySTs have utility in the modification of therapeutic proteins to improve serum half-life, and the potential for tissue engineering. PolySTs are membrane-associated proteins and as recombinant proteins suffer from inherently low solubility, low expression levels and poor thermal stability. To improve their physicochemical and biochemical properties, we applied a directed evolution approach using a FACS-based ultrahigh-throughput assay as a simple, robust and reliable screening method. We were able to enrich a large mutant library and, in combination with plate-based high-throughput secondary screening, we discovered mutants with increased enzymatic activity and improved stability compared to the wildtype enzyme. This work presents a powerful strategy for the screening of directed evolution libraries of bacterial polySTs to identify better catalysts for in vitro polysialylation of therapeutics.