Analysis of modular bioengineered antimicrobial lanthipeptides at nanoliter scale

Area of Science:

• Biochemistry
• Molecular Biology
• Medicinal Chemistry

Background:

• Antibiotic resistance is a growing global health threat, necessitating the development of new antimicrobial agents.
• Ribosomally synthesized and post-translationally modified antimicrobial peptides (RiPPs) represent a promising class of natural products with potent antibacterial activities.
• Lanthipeptides, a subgroup of RiPPs, are characterized by the presence of lanthionine rings, which are crucial for their structure and function.

Purpose of the Study:

• To engineer novel antimicrobial peptides with improved or altered activity profiles.
• To develop a high-throughput screening method for efficient characterization of engineered peptides.
• To establish design guidelines for future identification of peptide-based anti-infectives.

Main Methods:

• Large-scale engineering of RiPPs through combinatorial shuffling of peptide modules from 12 natural lanthipeptides.
• Utilizing a promiscuous post-translational modification machinery for processing engineered peptides.
• Development and application of nanoFleming, a miniaturized and parallelized high-throughput inhibition assay for experimental characterization.

Main Results:

• Generation of new-to-nature lanthipeptide variants with distinct biochemical properties.
• Identification of over 100 hit molecules exhibiting antimicrobial activity.
• Discovery of variants with enhanced activity against pathogenic bacteria and shifted activity profiles.

Conclusions:

• Combinatorial engineering of RiPPs, coupled with high-throughput screening, is an effective strategy for discovering novel antimicrobial agents.
• The developed nanoFleming assay facilitates rapid and efficient evaluation of antimicrobial peptide candidates.
• Extrapolated design guidelines will accelerate the identification and optimization of peptide-based anti-infectives to combat antibiotic resistance.