Precursor gene engineering expands the loop region of the lasso peptide microcin J25

Area of Science:

• Biochemistry
• Molecular Biology
• Synthetic Biology

Background:

• Microcin J25 (MccJ25) is a lasso peptide known for its potent RNA polymerase inhibition and unique mechanically interlocked structure.
• Previous attempts to create MccJ25 variants via precursor engineering focused on amino acid substitutions, often leading to low production due to poor tolerance by biosynthetic machinery.

Purpose of the Study:

• To explore the permissiveness of MccJ25 biosynthetic enzymes towards insertions within the peptide's loop region.
• To establish a new strategy for generating mechanically interlocked molecules and functionalizing the MccJ25 scaffold.

Main Methods:

• Investigated the MccJ25 biosynthetic pathway's tolerance to amino acid insertions in the loop region.
• Systematically introduced insertions of up to 15 amino acids at various loop positions.
• Assessed the production and structural integrity of generated MccJ25 variants.

Main Results:

• The MccJ25 biosynthetic enzymes demonstrated significant tolerance to insertions in the loop region.
• Expanded loops accommodated up to 15 additional amino acids, including diverse types.
• The lasso synthetase (McjC) possesses a cavity capable of tolerating these loop expansions.

Conclusions:

• The MccJ25 biosynthetic machinery is surprisingly permissive to loop region insertions, offering a new avenue for MccJ25 engineering.
• This work provides a novel strategy for constructing mechanically interlocked molecules.
• The MccJ25 scaffold can be modified to potentially graft diverse bioactive ligands.