Abstract

The combination of diversity generation methods and ultrahigh-throughput screening (uHTS) technologies is key to efficiently explore nature's sequence space and elucidate structure-function relationships of enzymes. Beneficial substitutions often cluster in a few regions and simultaneous amino acid substitutions at multiple positions (e.g., by OmniChange) will likely lead to further improved enzyme variants. An extensive screening effort is required to identify such variants, as the simultaneous randomization of four codons can easily yield over 10(5) potential enzyme variants. The combination of flow cytometer-based uHTS with cell-free compartmentalization technology using (w/o/w) double emulsions (InVitroFlow), provides analysis capabilities of up to 10(7) events per hour, thus enabling efficient screening. InVitroFlow is an elegant solution since diversity loss through a transformation of host cells is omitted and emulsion compartments provide a genotype-phenotype linkage through a fluorescence readout. In this study, a multisite saturation mutagenesis and an OmniChange library with four simultaneously saturated positions in the active site of CelA2 cellulase were screened using InVitroFlow. Screening of over 36 million events, yielded a significantly improved cellulase variant CelA2-M3 (H288F/H524Q) with an 8-fold increase in specific activity compared to the parent CelA2-H288F (83.9 U/mg) and a 41-fold increased specific activity (674.5 U/mg) compared to wildtype CelA2 (16.6 U/mg) for the substrate 4-MUC (4-methylumbelliferyl-beta d-cellobioside).