Catabolite repression and induction time effects for a temperature-sensitive GAL-regulated yeast expression system

Area of Science:

• Biotechnology
• Molecular Biology
• Biochemical Engineering

Background:

• Investigating temperature-sensitive (ts) GAL-regulated yeast expression systems.
• Addressing residual catabolite repression and induction timing impacts.
• Utilizing Saccharomyces cerevisiae with reg1 and ts mutations for controlled gene expression.

Purpose of the Study:

• To determine the effects of residual catabolite repression on a stable yeast expression system.
• To evaluate the importance of induction timing for optimizing productivity.
• To balance growth and protein yield in a regulated GAL expression system.

Main Methods:

• Employing a Saccharomyces cerevisiae strain with a reg1 mutation (inhibiting catabolite repression) and a ts mutation for temperature-inducible GAL promoters.
• Conducting batch cultures in SDC (semi-defined) and YPDa (complex) media with varying initial glucose concentrations (1-10 g l-1).
• Assessing beta-galactosidase specific activity and biomass yield at different glucose levels and induction time points (e.g., 14 h delay).

Main Results:

• Residual catabolite repression by glucose lowered lacZ expression (18-36% decrease with increasing glucose).
• Higher glucose concentrations significantly improved biomass yield, more than compensating for repression.
• Increasing initial glucose from 1 to 10 g l-1 in YPDa led to a 2.6-fold increase in beta-galactosidase volumetric activity.
• Delayed induction (14 h) improved cell yield but reduced specific activity, yielding modest (9-15%) overall productivity gains.

Conclusions:

• Optimizing glucose concentration is crucial for maximizing productivity in this yeast expression system.
• Despite residual repression, higher glucose levels enhance overall protein production through increased biomass.
• Strategic delay of induction can improve cell yield, offering a trade-off for optimizing productivity.