Fig. 2: One single genetic edit enables GPCR-signaling in probiotic yeast.
From: Engineered cell differentiation and sexual reproduction in probiotic and mating yeasts
A Homozygous mating-type switch in MATa/α diploid cells with CRISPR/Cas9 (left). Cas9 is pre-expressed in S. boulardii prior to transformation with a single gRNA plasmid that targets MATα in the genome for double-strand break (DSB). The endogenous MATa copy templates Cas9-induced double-strand break repair (not shown) and converts the cell into a MATa/a diploid. Gα expression is engineered from the PGK1 promoter, and heterologous GPCRs (hGPCRs) are expressed from 2 µ plasmids (right). B–E Median fluorescence intensities (MFI) of PFUS1-GFP reporter expression from plasmid pDAM194 following incubation with cognate ligands in dosages 0–100 µM. B. SB14 (MATa/α, left) and SB17 (MATa/a, right) were incubated with yeast pheromone (α-factor). C–E Cognate ligand sensing in hGPCR-signaling MATa/a cells for C A2bR sensing adenosine (SB48), D MT1 sensing melatonin (SB49), and E Mam2 sensing P-factor (SB50). Means and standard deviations represent three biological replicates. Statistical significance was determined using two-way analysis of variance (ANOVA) with Tukey’s multiple comparison test (B) or one-way ANOVA with Dunnett’s multiple comparison test in GraphPad Prism (**p ≤ 0.01) (C–E). Source data are provided as a Source Data file.
