LowTempGAL: a highly responsive low temperature-inducible GAL system in Saccharomyces cerevisiae

• Published in: Nucleic acids research Vol. 52; no. 12; pp. 7367 - 7383
• Main Authors: Lu, Zeyu, Shen, Qianyi, Bandari, Naga Chandra, Evans, Samuel, McDonnell, Liam, Liu, Lian, Jin, Wanli, Luna-Flores, Carlos Horacio, Collier, Thomas, Talbo, Gert, McCubbin, Tim, Esquirol, Lygie, Myers, Chris, Trau, Matt, Dumsday, Geoff, Speight, Robert, Howard, Christopher B, Vickers, Claudia E, Peng, Bingyin
• Format: Journal Article
• Language: English
• Published: England Oxford University Press 08.07.2024
• Subjects: Biosensing Techniques; Cold Temperature; DNA-Binding Proteins - genetics; DNA-Binding Proteins - metabolism; Galactose - metabolism; Gene Expression Regulation, Fungal; Repressor Proteins - genetics; Repressor Proteins - metabolism; Saccharomyces cerevisiae - genetics; Saccharomyces cerevisiae - metabolism; Saccharomyces cerevisiae Proteins - genetics; Saccharomyces cerevisiae Proteins - metabolism; Synthetic Biology and Bioengineering; Transcription Factors - genetics; Transcription Factors - metabolism
• ISSN: 0305-1048; 1362-4962; 1362-4962
• DOI: 10.1093/nar/gkae460

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.