(518a) Flow Cytometry-Based High-Throughput Screening Systems for Directed Evolution of Proteases

Proteases are industrially important enzymes and advances in screening technologies such as flow cytometry (FC) based in vitro compartmentalization (IVC) offer novel opportunities to tailor protease properties by directed evolution. Here we present two flow cytometry-based screening systems for proteases, cell-free based ProCF-FCIVC and whole-cell based Pro-FCIVC, with a high-throughput (>7500 per second). The systems are based on the linkage between genotype and phenotypic activity by encapsulation of single cell together corresponding fluorogenic substrate in in vitro emulsion compartments, and subsequently analyzing and sorting those emulsions by flow cytometry machine. In the study, system related technologies such as generation of water-in-oil-in-water double emulsions, selection of suitable substrate and calibration of the sorting performance of flow cytometry were investigated and optimized. For the cell-free based ProCF-FCIVC, the system was developed using a model protease M57 isolated from a metagenomic library. Different parameters like in vitro expression of protease, enzymatic reaction in emulsions, and recovery of sorted DNA variants were investigated. Sorting and subsequent activity analysis of the M57 protease reference library demonstrated a proof of concept of the developed ProCF-FCIVC system. For the whole-cell based Pro-FCIVC system, an extracellular proteases-deficient Bacillus strain together with a subtilisin Carlsberg (SC) secretion system was used. Several main challenges such as avoiding the high background of natural occurring proteases, generating mutagenesis library in high diversity using Bacillus expression system, and optimizing the sorting strategies were studied. The developed whole-cell Pro-FCIVC system was validated by screening an epPCR library of SC for increased inhibitor resistance. In these two cases mutants with improved activities and interesting changes were found. These approaches exemplify the application of flow cytometry based HTS for reengineering of proteases for additional useful properties in future.