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.

Engineered yeast cells simulating CD19+ cancers to control CAR T cell activation

“…CD19), candidate promoters for controlling regulation of effector module expression cassettes were selected based on our recent transcriptome analysis of differentially expressed genes linked to GPCR-mediated PRP activation ( Fig. 2A ) 52 . From this, we selected 11 native promoters with different expression characteristics, ranging from undetectable basal mRNA levels to the most highly expressed gene in S. cerevisiae , and spanning 6 orders of magnitude in differential mRNA abundance across GPCR stimulation levels, including genes regulated by the commonly used PRP-regulated promoters P FUS1 and P FIG1 ( Suppl.…”

“…We discovered that tuning of the output intensity was not only dependent on the choice of processing module promoter, but also PRP-controlled phenotypical changes of the engineered yeast. Shmooing, a morphological key indicator of PRP activation measured as cellular complexity 52 , was most intense at ≥0.01 µM, and generally peaking at 1 µM across all strains ( Fig. 2C ).…”

“…1 , Suppl. Table 1 ) and being commonly regarded as constitutive 52,61 . We determined that this PRP-induced boost amplifies gene expression multiplicatively from any promoter in yeast post-transcriptionally with an intensity that is PRP-stimulation-dependent (p<0.0001)( Fig.…”

“…Lastly, customizability in terms of choice of input and output signal allows for modularity and adaptation to user-defined cell assays. To fulfill these criteria, we sought to devise engineered G protein-coupled receptor (GPCR) biosensors, converting extracellular input in the form of GPCR ligands into MAP kinase-amplified intracellular signals through the well-characterized and highly modular signaling cascade of the S. cerevisiae pheromone response pathway (PRP), known to elicit dynamically regulated expression outputs [52][53][54][55][56] . From this, we were interested in combining such a biosensing technology with yeast surface display (YSD) to provide a controlled CD19 presentation output [57][58][59] .…”

“…Primers, DNA fragments, amplicons, plasmids, and gDNA were always dissolved in Milli-Q H2O and stored at -20°C. An overview of parts, backbones, constructed plasmids, and plasmids originating from other studies [52][53][54]57,58,67,70,71,84,86 can be found in Suppl. Table 11-12, and primers in Suppl.…”