p.T284Q/P285S/L286I/S287H/V288I Thr284Gln/Pro285Ser/Leu286Ile/Ser287His/Val288Ile (p.T312Q/P313S/L314I/S315H/V316I Thr312Gln/Pro313Ser/Leu314Ile/Ser315His/Val316Ile in primary sequence with 28 amino-acids signal peptide) Multiple amino acid substitutions in the acyl-binding loop 284-TPLSV-288 284-ThrProLeuSerVal-288 close to the active site. replaced by ProProLeuArgSer QSIHI clone 8. Slow self-reactivation after inhibition by paraoxon
Ultrahigh-throughput screening (uHTS) techniques can identify unique functionality from millions of variants. To mimic the natural selection mechanisms that occur by compartmentalization in vivo, we developed a technique based on single-cell encapsulation in droplets of a monodisperse microfluidic double water-in-oil-in-water emulsion (MDE). Biocompatible MDE enables in-droplet cultivation of different living species. The combination of droplet-generating machinery with FACS followed by next-generation sequencing and liquid chromatography-mass spectrometry analysis of the secretomes of encapsulated organisms yielded detailed genotype/phenotype descriptions. This platform was probed with uHTS for biocatalysts anchored to yeast with enrichment close to the theoretically calculated limit and cell-to-cell interactions. MDE-FACS allowed the identification of human butyrylcholinesterase mutants that undergo self-reactivation after inhibition by the organophosphorus agent paraoxon. The versatility of the platform allowed the identification of bacteria, including slow-growing oral microbiota species that suppress the growth of a common pathogen, Staphylococcus aureus, and predicted which genera were associated with inhibitory activity.
