An Improved Single Cell Ultrahigh Throughput Screening Method Based on In Vitro Compartmentalization

Abstract

High-throughput screening is a key technique in discovery and engineering of enzymes. In vitro compartmentalization based fluorescence-activated cell sorting (IVC-FACS) has recently emerged as a powerful tool for ultrahigh-throughput screening of biocatalysts. However, the accuracy of current IVC-FACS assays is severely limited by the wide polydispersity of micro-reactors generated by homogenizing. Here, an improved protocol based on membrane-extrusion technique was reported to generate the micro-reactors in a more uniform manner. This crucial improvement enables ultrahigh-throughput screening of enzymatic activity at a speed of >108 clones/day with an accuracy that could discriminate as low as two-fold differences in enzymatic activity inside the micro-reactors, which is higher than similar IVC-FACS systems ever have reported. The enzymatic reaction in the micro-reactors has very similar kinetic behavior compared to the bulk reaction system and shows wide dynamic range. By using the modified IVC-FACS, E. coli cells with esterase activity could be enriched 330-fold from large excesses of background cells through a single round of sorting. The utility of this new IVC-FACS system was further illustrated by the directed evolution of thermophilic esterase AFEST. The catalytic activity of the very efficient esterase was further improved by ∼2-fold, resulting in several improved mutants with k cat/K M values approaching the diffusion-limited efficiency of ∼108 M−1s−1.

Dissecting enzyme function with microfluidic-based deep mutational scanning

“…In addition to glycosidases, emulsion-based methods have been used to screen DNA/RNA polymerases, oxidoreductases, sulfatases, peroxidases, esterases, proteases, and even ribozymes (10,11,(33)(34)(35)(36)(37). The greatest challenge with emulsion-based screening is finding a fluorescent assay for one's particular enzyme of interest.…”

Double Emulsion Picoreactors for High-Throughput Single-Cell Encapsulation and Phenotyping via FACS

“…High data quality in single-cell analyses depends on the ability to discern which droplets contain single cells [31][32][33] . Previously, it has been difficult to attain predictable single-cell loading in DEs due to droplet polydispersity 39,47 . To achieve single-cell droplet FACS, picoliter DEs needed for FACS analysis must be highly uniform in size to yield accurate cell occupancy distributions 49 .…”

“…S4A). Prior techniques using picoliter DEs which encapsulate yeast or bacteria at high concentration estimate up to 50% of droplets contain single cells (λ > 0.5) 39,47 . However, these techniques suffer extremely high multiplet rates (>10%), preventing true single-cell resolution in downstream sequencing.…”

“…Unlike W/O droplets, DE droplets can be suspended in aqueous solutions, making them compatible with standard FACS instruments 45 . DEs have previously been combined with FACS for screening of bacterial or yeast mutant libraries 39,46,47 . However, these techniques suffer unpredictable cell occupancy and high multiple-cell loading ('multiplet') rates, confounding the downstream phenotype to genotype linkage.…”

High‐Throughput Approaches in Carbohydrate‐Active Enzymology: Glycosidase and Glycosyl Transferase Inhibitors, Evolution, and Discovery

“…Fragmentation on enzymatic cleavage separates the donor and acceptor fluorophores. [53,54] In vitro compartmentalization based fluorescence-activated cell sorting (IVC-FACS), [55] encapsulating ac ell within such an emulsion droplet, allows the evolution of surface-displayed [56] and secreted enzymes, [57] otherwise not possible using FACS. [49,50] Aside from the direct assays mentioned, the change in fluorescence signal can be achieved by afluorescent reporter protein such as GFP,w ith expression under the control of ap roduct-sugar inducible promoter.…”