High-Throughput FACS Method for Directed Evolution of Substrate Specificity

Directed evolution is a powerful technology for the isolation of enzyme variants with enhanced properties such as organic solvent tolerance (1 ), thermostability (2 ), glutaraldehyde resistance (3 ), and altered substrate specificity (4 ,5 ). Typically, enzyme variants are isolated using low throughput screening techniques such as agar plate or microtiter well plate assays. Recently, Fluorescence Activated Cell Sorting (FACS) has emerged as a tool offering significant promise for the high-throughput screening of very large libraries of mutants with single cell resolution (6 ). Arising from the simultaneous collection of multiple parameters, FACS is inherently capable of rapidly evaluating libraries for several different catalytic activities simultaneously with no increase in time required for library screening. We have exclusively used a Becton Dickinson FACSCaliber instrument, perhaps the most popular low-end sorter commercially available.