Fig. 5: Sorting and recovery of C. rosea mutants based on the activity of cell-wall-degrading enzymes.

a Peak intensity histogram and enzymatic assays using FL-GlcNAc (right) and FL-GalNAc (left) as the substrate for single-spore libraries. For the histogram, the fluorescence intensity of each droplet was measured in a mutant population after incubation at 27 °C. The intensity of the peaks between the wavelengths 510 nm and 520 nm were used for gating. To sort droplets, the intensity gate was set at the 0.9 quantile of the mutant population fluorescence (shown by the dotted line). The total number of peaks (N) and integration time (int_t) are indicated on the plot. The top 10% of the droplets were recovered on plates and cultured to obtain distinct mutant colonies (MG for the FL-GalNAc-sorted spores, MC for the FL-GlcNAc-sorted spores). b Three cell-wall-degrading enzymes were assayed to determine the top active mutant strains using 4-MU-GlcNAc (indicating chitinase activity), 4-MU-GalNAc (indicating N-acetylgalactosaminidase activity), or 4-MU-Glc (indicating β-1,3-glucanase activity). Fold change values were obtained with an endpoint enzymatic assay (pH 5.1, 30 min) and normalized to the activity of the wild-type (N = 3).