Antimicrobial Peptides Produced by Selective Pressure Incorporation of Non-canonical Amino Acids

Abstract

Nature has a variety of possibilities to create new protein functions by modifying the sequence of the individual amino acid building blocks. However, all variations are based on the 20 canonical amino acids (cAAs). As a way to introduce additional physicochemical properties into polypeptides, the incorporation of non-canonical amino acids (ncAAs) is increasingly used in protein engineering. Due to their relatively short length, the modification of ribosomally synthesized and post-translationally modified peptides by ncAAs is particularly attractive. New functionalities and chemical handles can be generated by specific modifications of individual residues. The selective pressure incorporation (SPI) method utilizes auxotrophic host strains that are deprived of an essential amino acid in chemically defined growth media. Several structurally and chemically similar amino acid analogs can then be activated by the corresponding aminoacyl-tRNA synthetase and provide residue-specific cAA(s) → ncAA(s) substitutions in the target peptide or protein sequence. Although, in the context of the SPI method, ncAAs are also incorporated into the host proteome during the phase of recombinant gene expression, the majority of the cell's resources are assigned to the expression of the target gene. This enables efficient residue-specific incorporation of ncAAs often accompanied with high amounts of modified target. The presented work describes the in vivo incorporation of six proline analogs into the antimicrobial peptide nisin, a lantibiotic naturally produced by Lactococcus lactis. Antimicrobial properties of nisin can be changed and further expanded during its fermentation and expression in auxotrophic Escherichia coli strains in defined growth media. Thereby, the effects of residue-specific replacement of cAAs with ncAAs can deliver changes in antimicrobial activity and specificity. Antimicrobial activity assays and fluorescence microscopy are used to test the new nisin variants for growth inhibition of a Gram-positive Lactococcus lactis indicator strain. Mass spectroscopy is used to confirm ncAA incorporation in bioactive nisin variants.

Nisin Variants Generated by Protein Engineering and Their Properties

“…Both residue-specific and site-specific approaches have been taken to incorporate ncAAs into proteins [ 62 ]. A residue-specific approach involves the feeding of amino acid analogues to nutritionally deficient strains to replace canonical residues without gene manipulation [ 63 ]. Derivatives of tryptophan, phenylalanine, proline and methionine have all been incorporated into target positions of nisin by selective pressure incorporation (SPI) [ 63 , 64 , 65 ].…”

Incorporation of non-standard amino acids into proteins: challenges, recent achievements, and emerging applications

“…The Schultz group incorporated several NSAAs into nisin by expressing the nisA encoding structural peptide, nisB encoding dehydratase, and nisC encoding cyclase with the coexpression of OTS for amber suppression in E. coli and found that αchloroacetamide-containing NSAA incorporation resulted in recombinant nisin variants with novel macrocyclic topologies (Zambaldo et al 2017). The Budisa group employed the selection pressure incorporation method to enable the residue-specific incorporation of six proline analogs into the antimicrobial peptide nisin (Nickling et al 2018). These researchers also produced bioactive nisin variants containing Nε-Boc-L-lysine both in Lactobacillus lactis and E. coli.…”

Residue-Specific Exchange of Proline by Proline Analogs in Fluorescent Proteins: How "Molecular Surgery" of the Backbone Affects Folding and Stability

“…Our approach is based on multiple residue-specific incorporation of ncAAs in response to sense codons (sense codon reassignment), whereby within the target gene, the number of positions for Pro analog insertion can be manipulated via site-directed mutagenesis 44 . A similar approach was applied in our previous report on the preparation of recombinant peptides with antimicrobial properties 45 . In this work, we have applied the SPI method, which allows all proline residues to be replaced by related analogs, to generate proteins expected to possess distinct physicochemical properties not present in proteins synthesized with the canonical amino acid repertoire.…”