| Source: |
Lu, Z, Shen, Q, Bandari, N C, Evans, S, McDonnell, L, Liu, L, Jin, W, Luna-Flores, C H, Collier, T, Talbo, G, McCubbin, T, Esquirol, L, Myers, C, Trau, M, Dumsday, G, Speight, R, Howard, C B, Vickers, C E & Peng, B 2024, 'LowTempGAL : a highly responsive low temperature-inducible GAL system in Saccharomyces cerevisiae', Nucleic Acids Research, vol. 52, no. 12, pp. 7367-7383. https://doi.org/10.1093/nar/gkae460 |
| Description: |
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 Gal80psupressing 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. |