Quantitative characterization of the inorganic phosphate gene regulatory networks in S. cerevisiae and bottom up engineering an orthogonal gene network

Gene regulatory networks (GRNs) play a crucial role in an organism's response to changing environmental conditions. Cellular behaviors typically result from the integration of multiple gene outputs, and their regulation often demands precise control of numerous genes. A comprehensive understanding of GRNs is essential for replicating their native function and engineering cellular behavior. In this work, we systematically characterized the yeast inorganic phosphate gene regulatory network at the single-cell level under well-controlled environmental conditions. Our analysis identified a robust, perfectly adapted Pho regulon state under intermediate Pi conditions and revealed a previously unknown intermediate nuclear localization state of the transcriptional master regulator Pho4. We also examined the robustness of the yeast phosphate GRN by adjusting the concentration of master regulator Pho4. We developed a Z3EV-mediated inducible phosphate regulatory network to effectively tune Pho4 expression. By adding different concentrations of ß-estradiol, which yeast cells do not consume, we modulated Pho4 expression levels. We determined that Pho4 translocation robustness is limited to a specific range of Pho4 expression levels. At higher Pho4 levels, Pho4 can saturate promoter binding sites, resulting in saturated activation and demonstrating robustness to changes in Pho4 concentration. We constructed a synthetic orthogonal GRN using synthetic transcription factors and promoters on a yeast artificial chromosome. The synthetic transcription factor PhoZF was designed by replacing the basic domain of native Pho4 with a Zinc Finger binding domain, and the synthetic promoters were created by substituting consensus binding motifs in native Pho4-regulated promoters and a minimal CYC1 promoter with the Zinc Finger binding motifs. The synthetic transcription factor effectively activated the synthetic promoters, generating sufficient fold changes to fine-tune input-output levels. Our findings laid the groundwork for the bottom-up assembly of a complete synthetic gene regulatory network to replace native networks.