A Layered Genetic Design Enables the Yeast Galactose Regulon to Respond to Cyanamide

Abstract

The commonly used expression systems in Saccharomyces cerevisiae typically rely on either constitutive or galactose-regulated promoters. The lack of inducible systems in S. cerevisiae limits the precise temporal regulation of protein function and yeast metabolism. We herein repurposed the galactose-regulated system to make it respond to cyanamide. By using a cyanamide-inducible DDI2 promoter to control Gal4 expression in CEN.PK2−1C with Δgal80, a tight and graded cyanamide-inducible GAL system with an enhanced signal output was constructed. Subsequently, we demonstrated that the cyanamide-inducible GAL system was capable of tightly regulating the pentafunctional Aro1 protein to achieve conditional shikimate pathway activity. Taken together, the cyanamideinducible GAL system could be implemented for both fundamental research and applied biotechnology.

Copper-Induced In Vivo Gene Amplification in Budding Yeast

“…2 C), we constructed a 2μ plasmid containing the gene expression cassette of eGFP-2A-PEST-CUP1 and transformed it into JY-Cyan-∆cup1. Since the cyanamide-inducible GAL system is tightly regulated by cyanamide [ 26 ], we observed that the engineered strain exhibited viability only when the cyanamide inducer was added to trigger the expression of eGFP-2A-PEST-CUP1 (Fig. 2 D).…”

“…Upon the construction of copper-susceptible S. cerevisiae and the validation of functional expression of the 2A-PEST-CUP1 module, we subsequently aimed to investigate whether the gene expression cassette could be integrated at the δ sites by applying the copper selection [ 29 , 39 ]. Since the ability of yeast strains to resist Cu 2+ was affected not only by the number of integrated copies but also by the expression level of CUP1 gene, we adopted our recently constructed cyanamide-inducible GAL system [ 26 ], so that the gene expression cassette can be tightly regulated by cyanamide concentrations. This approach ensures that even a low amount of inducer could lead to much higher selection pressure for multi-integration events.…”

“…Escherichia coli strain TOP10 was used for general plasmid constructions, and the strain was maintained at 37 °C in Luria–Bertani medium [tryptone (10 g/l), yeast extract (5 g/l), and NaCl (10 g/l)]. Strain JY-Cyan with a cyanamide-inducible GAL system [ 26 ] was used as the starting yeast strain for further genetic modifications. The engineered yeast strains were cultured at 30 °C in the yeast–peptone–dextrose (YPD) medium [tryptone (20 g/l), yeast extract (10 g/l), and glucose (20 g/l)] or in the yeast–nitrogen–base–glucose (YNBD) medium with appropriate dropouts.…”

“…[ 23 ] reported the temperature-sensitive Gal4 mutant Gal4M9 for constructing a temperature-dependent regulatory system. By refactoring the galactose-regulated (GAL) system, it can respond to copper ions [ 24 , 25 ], cyanamide [ 26 ] and cell population [ 27 ].…”

Reshaping the yeast galactose regulon via GPCR signaling cascade

“…Strains with α-factor under glucose-repressible promoters were integrated with the P GAL1 -EGFP expression cassette at the gal7-10-1 locus. 59 14 mL sterile tubes containing 2 mL YPD medium were inoculated with fresh overnight cultures to an initial OD 600 of 0.1. The cell cultures were incubated at 30°C and 200 rpm, and 100 μL of cell culture in YPD were collected by centrifugation at 3000 g and resuspended with equal volume water before measuring OD 600 and green fluorescence intensities.…”