Design of a tunable bacterial gene expression system using engineered σ factors

Area of Science:

• Bacterial transcription regulation
• Synthetic microbiology
• Protein engineering

Background:

• Extracytoplasmic function (ECF) σ factors regulate specific gene expression in bacteria.
• Promoter specificity and transcription initiation efficiency are governed by dynamic polypeptide segments (L3 loop and linker) within σ factors.
• Limited conservation of these segments hinders correlation between protein-DNA interactions and transcription efficiency.

Purpose of the Study:

• To characterize features governing gene expression dynamic range using chimeric Escherichia coli σ^E.
• To engineer σ factors for bespoke gene expression levels.
• To improve efficiency of microbial biosynthetic pathways.

Main Methods:

• Created chimeric σ^E by replacing L3 loop and linker with segments from 10 Mycobacterium tuberculosis ECF σ factors.
• Performed in vitro and in vivo measurements to assess effects of polypeptide replacements on gene expression.
• Utilized the chimeric σ^E library to modulate enzyme levels in a two-enzyme biosynthetic pathway in E. coli.

Main Results:

• Generated an experimentally validated dataset of gene expression levels for σ^E chimeras.
• Demonstrated increased desired product levels in a biosynthetic pathway by altering intracellular enzyme concentrations.
• Showed that modifying flexible loops of σ^E modulates target gene expression without altering the genomic makeup.

Conclusions:

• Engineering σ factors via modification of flexible loops offers a route to control gene expression.
• Chimeric σ^E library enables pre-determined conditional gene expression for synthetic microbiology applications.
• This approach facilitates pathway optimization without significant genetic engineering, improving microbial cell fitness.