Development of a Chromosomally Integrated Metabolite-Inducible Leu3p-α-IPM “Off-On” Gene Switch

Abstract

BackgroundPresent technology uses mostly chimeric proteins as regulators and hormones or antibiotics as signals to induce spatial and temporal gene expression.Methodology/Principal FindingsHere, we show that a chromosomally integrated yeast ‘Leu3p-α-ΙΡΜ’ system constitutes a ligand-inducible regulatory “off-on” genetic switch with an extensively dynamic action area. We find that Leu3p acts as an active transcriptional repressor in the absence and as an activator in the presence of α-isopropylmalate (α-ΙΡΜ) in primary fibroblasts isolated from double transgenic mouse embryos bearing ubiquitously expressing Leu3p and a Leu3p regulated GFP reporter. In the absence of the branched amino acid biosynthetic pathway in animals, metabolically stable α-IPM presents an EC50 equal to 0.8837 mM and fast “OFF-ON” kinetics (t50ON = 43 min, t50OFF = 2.18 h), it enters the cells via passive diffusion, while it is non-toxic to mammalian cells and to fertilized mouse eggs cultured ex vivo.Conclusions/SignificanceOur results demonstrate that the ‘Leu3p-α-ΙΡΜ’ constitutes a simpler and safer system for inducible gene expression in biomedical applications.

The pathway intermediate 2‐keto‐3‐deoxy‐L‐galactonate mediates the induction of genes involved in D‐galacturonic acid utilization in Aspergillus niger

“…The middle domain of the Leu3p protein masks the C‐terminal activation domain by an intramolecular interaction in the absence of α‐isopropylmalate (α‐IPM), a metabolic intermediate of the leucine biosynthesis pathway. In the presence of α‐IPM, which accumulates during leucine starvation, this self‐masking is prevented, resulting in active Leu3p and activation of leucine biosynthesis genes . The Gal4p and Leu3p transcription factors localize to the nucleus regardless of the presence or absence of inducer molecules .…”

A “Hit and Run” Approach to Inducible Direct Reprogramming of Astrocytes to Neural Stem Cells

“…We have previously reported a novel heterologous ligand-inducible regulatory “OFF-ON” genetic switch, based on the yeast transcription factor Leu3p (Leu3p-α-IPM; Poulou et al, 2010 ). This system is based on a transcription factor (Leu3p) involved in the regulation of the leucine pathway in yeast, whose function is controlled by α-IPM, a metabolite involved in leucine biosynthesis itself (Kohlhaw, 2003 ).…”

“…This system is based on a transcription factor (Leu3p) involved in the regulation of the leucine pathway in yeast, whose function is controlled by α-IPM, a metabolite involved in leucine biosynthesis itself (Kohlhaw, 2003 ). Leu3p acts as an active repressor binding to its UAS LEU DNA element, turning into an activator of the transcription in the presence α-IPM (Sze et al, 1992 ), an ideal inducer since it exhibits lipid solubility, metabolic stability, rapid “OFF-ON” kinetics with no apparent toxicity to mammalian cells, to fertilized mouse eggs cultured ex vivo and to animals alike (Poulou et al, 2010 ). Although the leucine biosynthetic pathway is found only in prokaryotes, fungi and plants, Leu3p has been shown to be fully functional In mammalian cells in culture (Sze and Kohlhaw, 1993 ; Remboutsika, 1994 ; Guo and Kohlhaw, 1996 ) and in primary mouse embryonic fibroblasts isolated from double transgenic mouse embryos bearing ubiquitously expressing Leu3p and a Leu3p regulated GFP reporter (Poulou et al, 2010 ).…”

Development of a “turn off–on” whole‐cell biosensor for sulforaphane detection based on the ultrasensitive activator HrpRS

“…“Turn off” whole‐cell biosensors frequently lack sensitivity and have problems related to difficult visualization due to their working mechanism. In order overcome these issues, synthetic biologists have implemented a series of OFF–ON genetic switches, which are based on native switches that switch the output “turn off” to “turn on,” so as to reprogram the cell behavior 29,30 . The most characteristic switch is the toggle switch, which consists of any two repressible promoters arranged in a mutually inhibitory network 31–35 .…”

“…In order overcome these issues, synthetic biologists have implemented a series of OFF-ON genetic switches, which are based on native switches that switch the output "turn off" to "turn on," so as to reprogram the cell behavior. 29,30 The most characteristic switch is the toggle switch, which consists of any two repressible promoters arranged in a mutually inhibitory network. [31][32][33][34][35] Since this requires additional regulatory systems and ligand induction, it is not feasible to utilize such bistable logic switches for the precise monitoring of small molecules.…”