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

Area of Science:

• Synthetic Biology
• Biotechnology
• Metabolic Engineering

Background:

• Temperature is a critical parameter in precision fermentation for biomanufacturing.
• Existing genetic systems often lack rapid and precise temperature responsiveness.

Purpose of the Study:

• To develop a highly responsive, low-temperature-inducible genetic system (LowTempGAL) in Saccharomyces cerevisiae.
• To engineer rapid and stringent induction for biomanufacturing applications.

Main Methods:

• Utilized temperature biosensors (heat-inducible degron and aggregation domain) to regulate GAL activator Gal4p.
• Implemented a second-layer control using low-temperature repression of GAL80.
• Engineered 'turbo' mechanisms with basal Gal4p expression and a Gal3p mutant (Gal3Cp) for enhanced responsiveness.

Main Results:

• Developed leaky LowTempGAL systems with initial delayed and weak responses.
• Optimized systems achieved rapid, stringent, high-level induction upon temperature shift (37-33°C to ≤30°C).
• Proteomics identified factors limiting induction, guiding engineering efforts.

Conclusions:

• Presented a synthetic biology approach for creating responsive Boolean-type genetic circuits using leaky biosensors.
• Demonstrated the LowTempGAL system's potential for rapid, precise control in biomanufacturing.
• Highlighted the importance of intricate system design for optimizing genetic circuits.