Peptide-Dependent Growth in Yeast via Fine-Tuned Peptide/GPCR-Activated Essential Gene Expression.

Building multicellular microbial consortia that communicate with each other and perform programmed functionalities is the next milestone for synthetic biology. Achieving cell-cell communication within these communities requires programming of the transduction of an extracellular signal into a customized intracellular response. G-protein-coupled receptors (GPCRs) are attractive candidates for engineering signal transduction as they can sense extracellular events with high sensitivity and specificity and transduce them into complex intracellular programs. We recently developed a scalable cell-cell communication language based on fungal mating GPCRs and their secreted peptide ligands. This language allows the assembly of engineered yeast strains into multicellular communication networks and allows them to be made interdependent by peptide signaling. In peptide signaling, one cell secretes a peptide that supports the growth of another cell at nanomolar concentrations, a scalable approach for engineering interdependence. Here we address the challenge of correlating the doubling time of cells with an increasing external peptide concentration by linking GPCR activation to the expression of an essential gene. The required fine-tuning of downstream signaling is achieved via the transcriptional titration of a set of orthogonal GPCR-activated transcription factors, a series of corresponding promoters with different output dynamics, and the use of chemically recoded peptide ligands with varying activation potentials. As such, our work establishes three control points that allow the tuning of the basal and maximal activation of the GPCR response, fold change activation, and response sensitivity. The presented results enable the implementation of peptide-dependent and peptide-tunable growth but could also facilitate the design and calibration of more complex GPCR-controlled synthetic functionality in the future.