Fig. 4: High-speed sorting for rapid directed evolution.
a MOMS efficiently screened a mixed population containing 0.5% VAN-3 and 99.5% wild-type (WT) yeast strains at a throughput of ~3.0 × 103 cells/s, processing 1.2 × 106 cells in under 7 minutes. Secretory yeast strains were enriched to >90% purity of VAN-3 cells. Vanillin production from individual clones was randomly sampled from pre- and post-sorting populations (n = 3 biological replicates, mean ± s.d., individual replicates plotted as dots). b For ECH enzyme evolution, MOMS was used to screen a mutant library of ~2.2 × 106 variants at ~3.0 × 103 cells/s, isolating the top 0.05% of strains with superior vanillin secretion within approximately 12 minutes. The enriched pool exhibited a ~1.93-fold increase in vanillin production relative to the parental population (n = 3 biological replicates, mean ± s.d., individual replicates plotted as dots). c Time-course high-performance liquid chromatography (HPLC) analysis of fermentation supernatants from VAN-3 and selected mutant strains showed a ~2.7-fold increase in vanillin titer after 48 hours (n = 3 biological replicates, mean ± s.d., individual replicates plotted as dots). d The schematic illustrates the sorted ECH mutant from the S-10 strain, with amino acid substitutions highlighted in red: I90N, Y169C, N212Q, and P213L. e For directed evolution of the vanillin transporter PP_0179, a library of 2 × 107 yeast variants was screened using MOMS to isolate top-performing strains with enhanced extracellular vanillin secretion. f Four high-performing transporter variants were validated through batch fermentation, each exhibiting increased vanillin titers (n = 3 biological replicates, mean ± s.d., individual replicates plotted as dots). g Two key mutations, K40R and V79D, identified in the top-performing strain, were found to enhance transporter efficiency and vanillin export. Source data are provided as a Source Data file.
