Engineered cell differentiation and sexual reproduction in probiotic and mating yeasts

Abstract

G protein-coupled receptors (GPCRs) enable cells to sense environmental cues and are indispensable for coordinating vital processes including quorum sensing, proliferation, and sexual reproduction. GPCRs comprise the largest class of cell surface receptors in eukaryotes, and for more than three decades the pheromone-induced mating pathway in baker's yeast Saccharomyces cerevisiae has served as a model for studying heterologous GPCRs (hGPCRs). Here we report transcriptome profiles following mating pathway activation in native and hGPCR-signaling yeast, and use a model-guided approach to correlate gene expression to morphological changes. From this we demonstrate mating between haploid cells armed with hGPCRs and endogenous biosynthesis of their cognate ligands. Furthermore, we devise a ligand-free screening strategy for hGPCR compatibility with the yeast mating pathway and enable hGPCR-signaling in the probiotic yeast Saccharomyces boulardii. Combined, our findings enable new means to study mating, hGPCR-signaling, and cell-cell communication in a model eukaryote and yeast probiotics.

Improving Therapeutic Protein Secretion in the Probiotic YeastSaccharomyces boulardiiusing a Multifactorial Engineering Approach

“…E. coli cells were grown in lysogeny broth (LB) (5g/L yeast extract, 10 g/L tryptone, 10g/L NaCl) at 37 °C supplemented with ampicillin (100 μg/mL), kanamycin (50 μg/mL) or chloramphenicol (34 μg/mL), as appropriate. Saccharomyces boulardii ATCC-MYA796Δ ura3 was used to construct NPA-secreting strains (Jensen et al, 2022). Yeast cultures for plasmid transformation and genome editing were grown in yeast extract-peptone-dextrose (YPD) medium (50 g/L YPD Broth (Sigma-Aldrich)).…”

“…E. coli cells were grown in lysogeny broth (LB) (5g/L yeast extract, 10 g/L tryptone, 10g/L NaCl) at 37 °C supplemented with ampicillin (100 µg/mL), kanamycin (50 µg/mL) or chloramphenicol (34 µg/mL), as appropriate. Saccharomyces boulardii ATCC-MYA796Δura3 was used to construct NPA-secreting strains (Jensen et al, 2022). Yeast cultures for plasmid transformation and genome editing were grown in yeast extract-peptonedextrose (YPD) medium (50 g/L YPD Broth (Sigma-Aldrich) The copyright holder for this preprint this version posted December 31, 2022. ; https://doi.org/10.1101/2022.12.30.522352 doi: bioRxiv preprint cultures were grown in synthetic complete media (pH 7.04) containing 0.67% (w/v) Yeast Nitrogen Base Without Amino Acids (Sunrise Science Products), 1.54 g/L Yeast Synthetic Media Dropout Mix (uracil, i.e.…”