Overcoming chromoprotein limitations by engineering a red fluorescent protein

Area of Science:

• Molecular Biology
• Microbiology
• Biotechnology

Background:

• Chromoproteins (CPs) are essential visual reporters for gene expression.
• Overexpression of darkly colored CPs in E. coli leads to significant fitness costs and genetic instability.
• Previous CP variants often resulted in loss of color due to mutations during cultivation.

Purpose of the Study:

• To identify a suitable alternative to chromoproteins for gene expression in E. coli.
• To investigate the causes of chromoprotein toxicity.
• To develop new color variants with minimal fitness costs.

Main Methods:

• Subcloning of a codon-optimized mRFP1 gene (mRFP1E) into E. coli.
• Analysis of E. coli fitness and color expression.
• Gel electrophoresis and size-exclusion chromatography to study CP aggregation.
• Semi-rational mutagenesis of mRFP1.

Main Results:

• mRFP1E produced dark coloration with minimal impact on E. coli fitness.
• CP toxicity correlated with expression levels, oligomerization, and inclusion body formation, not isoelectric point.
• Mutagenesis yielded new mRFP1 variants with diverse colors and maintained low fitness costs.

Conclusions:

• The mRFP1E series provides a superior alternative to traditional CPs for gene expression studies in E. coli.
• Understanding CP aggregation is key to mitigating toxicity.
• New mRFP1 variants facilitate comparative fitness studies across E. coli strains.