1. LowTempGAL: a highly responsive low temperature-inducible GAL system in Saccharomyces cerevisiae.
- Author
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Lu Z, Shen Q, Bandari NC, Evans S, McDonnell L, Liu L, Jin W, Luna-Flores CH, Collier T, Talbo G, McCubbin T, Esquirol L, Myers C, Trau M, Dumsday G, Speight R, Howard CB, Vickers CE, and Peng B
- Subjects
- Repressor Proteins metabolism, Repressor Proteins genetics, DNA-Binding Proteins metabolism, DNA-Binding Proteins genetics, Cold Temperature, Galactose metabolism, Biosensing Techniques, Saccharomyces cerevisiae genetics, Saccharomyces cerevisiae metabolism, Saccharomyces cerevisiae Proteins metabolism, Saccharomyces cerevisiae Proteins genetics, Gene Expression Regulation, Fungal, Transcription Factors metabolism, Transcription Factors genetics
- Abstract
Temperature is an important control factor for biologics biomanufacturing in precision fermentation. Here, we explored a highly responsive low temperature-inducible genetic system (LowTempGAL) in the model yeast Saccharomyces cerevisiae. Two temperature biosensors, a heat-inducible degron and a heat-inducible protein aggregation domain, were used to regulate the GAL activator Gal4p, rendering the leaky LowTempGAL systems. Boolean-type induction was achieved by implementing a second-layer control through low-temperature-mediated repression on GAL repressor gene GAL80, but suffered delayed response to low-temperature triggers and a weak response at 30°C. Application potentials were validated for protein and small molecule production. Proteomics analysis suggested that residual Gal80p and Gal4p insufficiency caused suboptimal induction. 'Turbo' mechanisms were engineered through incorporating a basal Gal4p expression and a galactose-independent Gal80p-supressing Gal3p mutant (Gal3Cp). Varying Gal3Cp configurations, we deployed the LowTempGAL systems capable for a rapid stringent high-level induction upon the shift from a high temperature (37-33°C) to a low temperature (≤30°C). Overall, we present a synthetic biology procedure that leverages 'leaky' biosensors to deploy highly responsive Boolean-type genetic circuits. The key lies in optimisation of the intricate layout of the multi-factor system. The LowTempGAL systems may be applicable in non-conventional yeast platforms for precision biomanufacturing., (© The Author(s) 2024. Published by Oxford University Press on behalf of Nucleic Acids Research.)
- Published
- 2024
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