p.T284N/P285K/L286S/S287N Thr284Asn/Pro285Lys/Leu286Ser/Ser287Asn (p.T312N/P313K/L314S/S315N Thr312Asn/Pro313Lys/Leu314Ser/Ser315Asn 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 13. 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.
