GSE110818
RNA-seq of strains grown under the control of  native galactose regulon or synthetic xylose regulon vs constitutive expression of galactose and xylose metabolic genes
In this study, we first assess the role of the GAL regulon in enabling efficient galactose utilization for cell growth by decoupling its regulatory responses from sugar catabolism. We provide evidence that regulon-controlled galactose assimilation is more efficient than constitutive expression of the catabolic genes in supporting fast growth rates to higher cell densities. Next, we assessed whether a regulon could enable more complete and efficient utilization of a nutrient that is non-native to this yeast – xylose. We first adapt the GAL regulon to respond to xylose through directed evolution of Gal3p, enabling coupling of nutrient stimulus with sensing, computation, and regulatory actuation. Next, by using a rational, model-guided approach, we test two different positive feedback signal transduction loop designs for the regulon and demonstrate their individual merits and weaknesses. We also show that implementation of a GAL-type xylose-responsive regulon can regulate multiple genes across the yeast genome and enable more homogeneous population-wide gene expression. By integrating a minimal set of heterologous catabolic genes into the synthetic regulon we demonstrate high cellular growth rates and high final cell densities on xylose as well as better growth in non-inducing carbon sources. Finally, we compare the genome-wide expression profiles of strains grown with regulon assistance and conventionally engineered strains to identify mechanistic reasons that account for the different phenotypes observed. We posit that this study strongly supports the need to re-evaluate how nutrient assimilation systems are currently implemented and introduces a new and unexplored paradigm of adapting a native regulon for efficient non-native sugar assimilation. Overall design: Four different samples with biological triplicates were analyzed in the study. The strains include, A) Wildtype yeast strain with intact GAL regulon grown in galactose B) Yeast strain with GAL4 knock out, but GAL1, GAL10 and GAL7 genes expressed under constitutive TEF1p, TPI1p and GPM1p promoters, grown in galactose, C) A GAL-adapted xylose regulon with XYLA*3, XKS1, TAL1 and GAL2-2.1, necessary for growth in xylose and grown in xylose and D) Strain without xylose regulon but constitutively expressing XYL*3, XKS1, TAL1 and GAL2-2.1, and grown in xylose.