Fabrication, Evolution, and Mutual Conversion of d-Fucose-Activatable and -Repressible Acetyltransferase upon...

Area of Science:

• Synthetic Biology
• Molecular Biology
• Protein Engineering

Background:

• Protein fusions can lead to the emergence of novel molecular functions, including regulatory switches.
• The d-fucose-responsive transcription factor AraC is a known regulatory protein.

Purpose of the Study:

• To investigate how linker length in protein fusions influences the emergence and behavior of molecular switches.
• To engineer d-fucose-regulated chloramphenicol acetyltransferase (CAT) activity using protein fusion strategies.

Main Methods:

• Insertion of chloramphenicol acetyltransferase (CAT) into a d-fucose-responsive AraC mutant using diverse linker libraries.
• Analysis of switch emergence (on-switch vs. off-switch) based on linker length.
• Directed evolution and random mutagenesis to study the evolution of switching efficiency and regulatory relationships.

Main Results:

• Longer linkers favored the emergence of 'on-switches', while short or zero linkers resulted in 'off-switches'.
• Engineered switches rapidly evolved efficiency and maintained d-fucose-inducible regulation of CAT activity.
• Fusion proteins demonstrated interconvertible one-input/two-output split gate functions and evolved mutual regulatory relationships with partner proteins.

Conclusions:

• Linker length is a critical determinant in the type of molecular switch (on/off) that emerges from protein fusions.
• Protein fusion and mutagenesis offer a versatile platform for engineering novel regulatory systems and evolving complex protein interactions.
• Emergent regulatory relationships can be readily altered by mutations, leading to diverse functional outcomes and rapid adaptation.