Application of the yeast pheromone system for controlled cell-cell communication and signal amplification

Abstract

Aims: The aim of the work is to exploit the yeast pheromone system for controlled cell–cell communication and as an amplification circuit in technical applications, e.g. biosensors or sensor‐actor systems. Methods and Results: As a proof of principle, we developed recombinant Saccharomyces cerevisiae cells that express enhanced green fluorescent protein (EGFP) in response to different concentrations of the alpha (α)‐factor mating pheromone. A respective reporter construct allowing the pheromone‐driven expression of EGFP was transformed into the S. cerevisiae strains BY4741 and BY4741 bar1Δ. Upon addition of synthetic α‐factor, the fluorescence strongly increases after 4 h. Furthermore, cells with constitutive α‐factor expression were able to induce the expression of EGFP in co‐cultivation with sensor cells only if both cell types were deleted for the gene BAR1, encoding α‐factor protease. For technical applications, the immobilization of functionalized cells may be beneficial. We show that pheromone‐induced expression of EGFP is effective in alginate‐immobilized cells. Conclusions: Based on S. cerevisiaeα‐factor, we developed a controlled cell–cell communication system and amplification circuit for pheromone‐driven expression of a target protein. The system is effective both in suspension and after cell immobilization. Significance and Impact of the Study: The developed set of recombinant yeast strains is the basis to apply the yeast pheromone system for signal production and amplification in biosensors or sensor‐actor systems.

Temporal and Spatial Properties of a Yeast Multi-Cellular Amplification System Based on Signal Molecule Diffusion

“…A bimodular biosensor like the formerly introduced multi-cellular communication and signal amplification system utilizes functionally separated cell types that are applied in mixtures [8]. Co-immobilization of different cell types minimizes diffusion distances of signaling molecules, such as α–factor, and may allow rapid activation of reporter cells.…”

“…A set of plasmids for controlled yeast pheromone signaling was described previously [8]. Briefly, constructs contain a 1.5 kb P ADH1 or 1.0 kb P FIG1 promoter element for constitutive and α–factor-inducible expression, respectively, of EGFP (fluorescence output) or MFα1 (α–factor production).…”

“…All of these systems are based on a single yeast strain that senses the analyte, which drives expression of a marker gene. Recently, we reported an amplification system based on at least two different S. cerevisiae cell types [8]. In detail, recombinant sensor cells (cell type I) respond to the presence of an analyte by expression and secretion of α–factor.…”

“…The recently described bimodular amplifier system [8] can be regarded as an extension of a unicellular sensor approach, in which the two functions—sensing of an analyte as the input signal and production of fluorescence as the output—are separated and connected by α–factor signaling. Particular advantages of this multi-step system are signal amplification and modularization.…”

Engineering a model cell for rational tuning of GPCR signaling

“…Narrowing the operational range of the melatonin-responsive MTNR1A sensor strains required more complex engineering as the Hill slope of a response can only be increased via mechanisms such as cooperativity 41 , sequesteration 49 or positive feedback 50 . As before, we utilized a community-based approach, but here using cell-to-cell communication to enable feedback at the population level 51,52 . A two-cell system was designed where the first cell acts as an amplifier, sensing via MTNR1A and responding by secreting ɑ-factor from the reintroduced gene.…”