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Updated: Jun 3, 2026
Antimicrobial Peptides Produced by Selective Pressure Incorporation of Non-canonical Amino Acids
Published on: May 4, 2018
Sequence diversity in the lasso peptide framework: discovery of functional microcin J25 variants with multiple amino
1Department of Chemical and Biological Engineering, Princeton University, Princeton, New Jersey 08544, USA.
Abstract:
Microcin J25 (MccJ25) is a ribosomally synthesized antimicrobial peptide that has an unusual threaded lasso structure in which the C-terminal "tail" of the peptide is fed through a macrocyclic "ring" formed by the N-terminal residues. Production of MccJ25 in Escherichia coli is dependent upon a four-gene cluster encoding the structural gene mcjA, two maturation enzymes mcjB and mcjC, and an immunity factor, mcjD, in the form of an MccJ25 export pump. Here we have developed a system for orthogonal control of the expression of mcjA and mcjD, thus permitting independent control of MccJ25 production and export/immunity in E. coli. We used this system to screen saturation mutagenesis libraries targeted to either the ring or tail portions of MccJ25 and discovered nearly 100 new MccJ25 variants that retain antimicrobial function. While multiple amino acid substitutions in the tail portion of the peptide are well-tolerated, mutagenesis of the ring portion of the peptide is detrimental to the antimicrobial function of MccJ25. We demonstrated that the decreased function of the ring variants is due to the inability of these variants to be transported to the cytoplasm of susceptible strains. Additionally, we found several MccJ25 variants from the tail library with improved efficacy toward the MccJ25-sensitive strains E. coli and Salmonella enterica serovar Newport with the best variants exhibiting a nearly 5-fold increase in potency. The results described here provide further evidence that diverse amino acid sequences can be tolerated by the rigid lasso peptide fold.
Insights
Researchers engineered Microcin J25 (MccJ25), an antimicrobial peptide, for enhanced function. Mutating the tail region yielded potent variants, while ring mutations impaired activity, revealing insights into lasso peptide structure-function relationships.
Area of Science:
- Microbiology
- Molecular Biology
- Biochemistry
Background:
- Microcin J25 (MccJ25) is a ribosomally synthesized antimicrobial peptide with a unique lasso structure.
- Its production in Escherichia coli involves a four-gene cluster (mcjA, mcjB, mcjC, mcjD) for peptide synthesis, maturation, and immunity.
- The mcjD gene encodes an export pump providing immunity to MccJ25.
Purpose of the Study:
- To develop a system for independent control of MccJ25 production and export/immunity.
- To screen mutagenesis libraries of MccJ25 to identify functional variants.
- To investigate the structure-function relationship of the MccJ25 lasso peptide.
Main Methods:
- Developed an orthogonal gene expression system for mcjA and mcjD in E. coli.
- Screened saturation mutagenesis libraries targeting the ring and tail regions of MccJ25.
- Assessed antimicrobial activity of MccJ25 variants against sensitive strains like E. coli and Salmonella enterica.
Main Results:
- Discovered nearly 100 new MccJ25 variants retaining antimicrobial function.
- Identified that tail region mutations are generally well-tolerated, while ring mutations significantly impair function.
- Demonstrated that impaired ring variants are not transported to the cytoplasm.
- Found MccJ25 variants with up to 5-fold increased potency against sensitive strains.
Conclusions:
- The study provides a system for dissecting MccJ25 production and immunity.
- Mutagenesis studies reveal the critical role of the MccJ25 ring structure for antimicrobial activity and cellular uptake.
- Tail region modifications can enhance MccJ25 potency, highlighting the tolerance of the lasso peptide fold to sequence diversity.
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