Development of a temperature-responsive yeast cell factory using engineered Gal4 as a protein switch

Area of Science:

• Metabolic Engineering
• Synthetic Biology
• Biotechnology

Background:

• Biosynthesis of secondary metabolites in yeast often faces a conflict between optimizing cell growth and maximizing product accumulation.
• Existing fermentation strategies struggle to efficiently balance these competing cellular demands, limiting overall production yields.
• Dynamic control systems are needed to decouple growth and production phases for enhanced efficiency in yeast cell factories.

Purpose of the Study:

• To develop a temperature-dependent dynamic control strategy for two-stage fermentation in yeast.
• To engineer a novel protein switch for precise temporal regulation of metabolic pathways.
• To demonstrate the efficacy of this system in decoupling cell growth and product accumulation using lycopene biosynthesis as a model.

Main Methods:

• Directed evolution was employed to create a temperature-sensitive Gal4 mutant (Gal4M9) acting as a protein switch.
• The Gal4M9 switch was implemented in a ΔGAL80 yeast strain to control the expression of pathway genes under P_GAL promoters.
• EGFP reporter assays validated temperature-responsive induction, and lycopene production was quantified to assess system performance compared to a wild-type control.

Main Results:

• The engineered Gal4M9 system successfully decoupled yeast growth from lycopene production upon a temperature shift during fermentation.
• The two-stage fermentation strategy resulted in a 44% increase in biomass accumulation compared to the control strain.
• Lycopene production was significantly enhanced, with a 177% increase observed in the engineered strain relative to the control.

Conclusions:

• Temperature-based dynamic control is a viable strategy for decoupling cell growth and product biosynthesis in yeast.
• This novel system offers precise temporal regulation of metabolic pathways, overcoming limitations of traditional fermentation methods.
• This represents the first instance of using temperature as an input signal for metabolic pathway regulation in yeast cell factories, paving the way for improved biomanufacturing.