Fig. 4: High-throughput screen for hyperactive Leu4p variants using the isopentanol configuration of the biosensor.

a Flow cytometry analysis of yeast libraries containing LEU4 variants generated with error-prone PCR-based mutagenesis of the full-length LEU4 (ep_Leu4, blue) or only the LEU4 regulatory domain (ep_Leu4_Reg, red), compared to control strains containing an empty vector (Vector, orange) or wild-type LEU4 (WT, cyan). Cell populations were normalized to the mode for comparison. b Flow cytometry analysis of the same libraries depicted in (a) after three rounds of FACS. c Isopentanol titers after 48 h fermentations of sorted strains with unique LEU4 sequences (orange), plasmid-cured derivatives (black), and strains obtained from retransforming YZy148 with each unique plasmid isolated from the corresponding sorted strain (cyan). Titers obtained from the basal strain YZy148 (leu4Δ leu9Δ bat1Δ LEU2) with (red) or without (blue) an empty vector, or transformed with plasmids containing wild-type LEU4 (LEU4^WT, green), LEU4^1–410 (brown), or LEU4^ΔS547 (magenta), are shown as controls. The numbers on the upper x-axis identify each strain harboring a unique LEU4 sequence. LEU4 mutants 1–13 are derived from mutagenizing the full-length LEU4; and mutants 14–24 from mutagenizing the LEU4 leucine regulatory domain. All data are shown as mean values. Open circles represent individual data points. Error bars represent the standard deviation of at least three biological replicates. A two-sided t-test was used to determine the statistical significance of the difference between isopentanol titers achieved by YZy148 transformed with a plasmid containing LEU4^ΔS547 (magenta), or LEU4 mutant #6 (LEU4^{E86D_K191N_K374R_A445T_S481R_N515I_A568V_S601A}). P = 0.0021; **P ≤ 0.01. Source data are provided as a Source Data file.