Extending dynamic and operational range of the biosensor responding to l-carnitine by directed evolution

Area of Science:

• Biochemistry
• Metabolic Engineering
• Biosensor Technology

Background:

• L-carnitine is crucial for fatty acid metabolism and energy production in eukaryotes.
• It finds widespread use in healthcare and as a food additive.
• The transcriptional activator CaiF, key to l-carnitine metabolism, is activated by crotonobetainyl-CoA.

Purpose of the Study:

• To engineer a novel biosensor for detecting l-carnitine.
• To overcome the limitations of restricted detection range in existing biosensors.
• To enhance the sensitivity and utility of CaiF-based biosensors for practical applications.

Main Methods:

• Computer-aided design and simulation of CaiF protein structure.
• Alanine scanning to verify DNA binding sites.
• Functional Diversity-Oriented Volume-Conservative Substitution Strategy to modify key CaiF sites.
• Construction and characterization of a modified CaiF biosensor (CaiF^Y47W/R89A).

Main Results:

• A modified biosensor, CaiF^Y47W/R89A, was successfully constructed.
• The engineered biosensor demonstrated a significantly expanded concentration response range from 10^-4 mM to 10 mM.
• This represents a 1000-fold increase in response range and a 3.3-fold higher output signal intensity compared to the control.

Conclusions:

• The modified CaiF biosensor offers a substantially improved detection range for l-carnitine.
• These engineered variants hold significant potential for optimizing l-carnitine production processes.
• The study highlights the effectiveness of protein engineering in enhancing biosensor performance.