Feedforward growth rate control mitigates gene activation burden

Area of Science:

• Synthetic Biology
• Metabolic Engineering
• Molecular Biology

Background:

• Heterologous gene expression imposes a metabolic burden on cellular resources.
• This burden can lead to reduced growth rates and instability in engineered microbial systems.
• Existing methods lack dynamic control to mitigate these negative effects.

Purpose of the Study:

• To engineer a novel feedforward controller for dynamic gene activation in bacteria.
• To compensate for the non-physiological burden of gene expression on cellular resources.
• To enable sustained and stable heterologous gene expression without compromising cell growth.

Main Methods:

• Introduction of a feedforward controller activating a modified SpoT enzyme (SpoTH) upon gene of interest (GOI) activation.
• Utilizing an inducible RelA+ expression cassette to precisely control basal ppGpp levels and nominal growth rate.
• Co-culture experiments to assess population-level gene activation stability.

Main Results:

• The controller successfully compensated for the burden of GOI activation, preventing growth rate defects.
• Without the controller, GOI activation decreased growth rate by over 50%.
• The controller enabled persistent population-level activation of the GOI in co-culture, unlike control strains.

Conclusions:

• The developed feedforward controller is a tunable, modular, and portable tool for dynamic gene activation.
• This system allows for precise control over gene expression without impacting bacterial growth.
• It offers a significant advancement for synthetic biology applications requiring stable and inducible gene expression.