A Timed Off-Switch for Dynamic Control of Gene Expression in Corynebacterium Glutamicum

Area of Science:

• Synthetic biology
• Metabolic engineering
• Microbial biotechnology

Background:

• Dynamic control of gene expression is crucial for metabolic engineering.
• Existing inducible systems require costly inducers.
• Timed gene expression shutdown systems are less common but highly desirable.

Purpose of the Study:

• To develop and characterize a synthetic regulatory circuit for timed gene expression shutdown in Corynebacterium glutamicum.
• To utilize the VanR/P* regulatory system with phenolic compounds (ferulic acid, vanillin, vanillic acid) for dynamic gene control.
• To engineer C. glutamicum for enhanced production of pyruvate, l-alanine, and l-valine.

Main Methods:

• Engineered C. glutamicum by replacing the native promoter of the aceE gene with the VanR/P* system.
• Investigated the effect of varying concentrations of phenolic compounds on biomass yield and metabolite production.
• Overexpressed l-valine biosynthesis genes (ilvBNCE) in the engineered strain.

Main Results:

• Biomass yield showed a linear increase with supplemented effector (phenolic compound) concentration.
• Gene expression ceased upon depletion of phenolic compounds, with residual glucose utilized for metabolite production.
• Increasing effector concentration shifted product spectrum from pyruvate to l-alanine to l-valine.
• Engineered strain achieved efficient l-valine production (0.36 mol/mol glucose) after ilvBNCE overexpression.

Conclusions:

• The VanR/P* system provides a robust and cost-effective method for timed gene expression control in C. glutamicum.
• Phenolic compounds serve as readily available and inexpensive effectors for dynamic metabolic pathway regulation.
• This system offers significant potential for optimizing microbial cell factories for the production of specific metabolites.