Design and application of a lactulose biosensor.

Area of Science:

• Molecular Biology
• Biotechnology
• Enzyme Engineering

Background:

• The Escherichia coli lac operon repressor (LacI) controls gene expression in response to lactose.
• Engineering LacI for altered effector specificity can create novel regulatory tools and biosensors.
• Developing specific biosensors is crucial for applications like directed evolution and metabolic engineering.

Purpose of the Study:

• To engineer the LacI repressor for specific induction by lactulose.
• To develop a whole-cell biosensor utilizing the engineered LacI for lactulose detection.
• To apply the biosensor in the directed evolution of cellobiose 2-epimerase (C2E) for enhanced lactulose production.

Main Methods:

• Generated a LacI saturation mutagenesis library.
• Employed Fluorescence Activated Cell Sorting (FACS) for dual screening of mutants.
• Constructed a whole-cell lactulose biosensor using the selected mutant LacI-L5.

Main Results:

• Isolated mutant LacI-L5, specifically induced by lactulose and not other tested disaccharides.
• Successfully developed a whole-cell lactulose biosensor with LacI-L5.
• Applied the biosensor to select a cellobiose 2-epimerase (C2E) mutant with a ~32-fold enhanced expression level for lactulose production.

Conclusions:

• Engineered LacI-L5 demonstrates high specificity for lactulose, serving as a novel regulatory tool.
• The developed whole-cell lactulose biosensor is highly efficient for directed evolution applications.
• This study highlights the potential of engineering LacI for creating customized molecular biosensors for practical use.