The incorporation of non-canonical amino acids (ncAA) is an elegant way for the chemical diversification of recombinantly produced antimicrobial peptides (AMPs). Residue- and site-specific installation methods in several bacterial production hosts hold great promise for the generation of new-to-nature AMPs, and can contribute to tackle the ongoing emergence of antibiotic resistance in pathogens. Especially from a pharmacological point of view, desirable improvements span pH and protease resistance, solubility, oral availability and circulation half-life. Although the primary focus of this report is on ribosomally synthesized and post-translationally modified peptides (RiPPs), we have included selected cases of peptides produced by solid phase peptide synthesis to comparatively show the potential and impact of ncAA introduction. Generally speaking, the introduction of ncAAs in recombinant AMPs delivers novel levels of chemical diversification. Cotranslationally incorporated, they can take part in AMP biogenesis either through direction interaction with elements of the post-translational modification (PTM) machinery or as untargeted sites with unique physicochemical properties and chemical handles for further modification. Together with genetic libraries, genome mining and processing by PTM machineries, ncAAs present not a mere addition to this process, but a highly diverse pool of building blocks to significantly broaden the chemical space of this valuable class of molecules. This perspective summarizes new developments of ncAA containing peptides. Challenges to be resolved in order to reach large-scale pharmaceutical production of these promising compounds and prospects for future developments are discussed.

The incorporation of non-canonical amino acids (ncAAs) into ribosomally synthesized and post-translationally modified peptides, e.g., nisin from the Gram-positive bacterium Lactococcus lactis, bears great potential to expand the chemical space of various antimicrobials. The ncAA Nε-Boc-L-lysine (BocK) was chosen for incorporation into nisin using the archaeal pyrrolysyl-tRNA synthetase–tRNAPyl pair to establish orthogonal translation in L. lactis for read-through of in-frame amber stop codons. In parallel, recombinant nisin production and orthogonal translation were combined in Escherichia coli cells. Both organisms synthesized bioactive nisin(BocK) variants. Screening of a nisin amber codon library revealed suitable sites for ncAA incorporation and two variants displayed high antimicrobial activity. Orthogonal translation in E. coli and L. lactis presents a promising tool to create new-to-nature nisin derivatives.

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.

Across scales, many biological phenomena, such as protein folding or bioadhesion and cohesion, rely on synergistic effects of different amino acid side chains at multiple positions in the protein sequence. These are often fine-tuned by post-translational modifications that introduce additional chemical properties. Several PTMs can now be genetically encoded and precisely installed at single and multiple sites by genetic code expansion. Protein nitration is a PTM of particular interest because it has been associated with several diseases. However, even when these nitro groups are directly incorporated into proteins, they are often physiologically reduced during or shortly after protein production. We have solved this problem by using an engineered Escherichia coli host strain. Six genes that are associated with nitroreductase activity were removed from the genome in a simple and robust manner. The result is a bacterial expression host that can stably produce proteins and peptides containing nitro groups, especially when these are amenable to modification. To demonstrate the applicability of this strain, we used this host for several applications. One of these was the multisite incorporation of a photocaged 3,4-dihydroxyphenylalanine derivative into Elastin-Like Polypeptides. For this non-canonical amino acid and several other photocaged ncAAs, the nitro group is critical for photocleavability. Accordingly, our approach also enhances the production of biomolecules containing photocaged tyrosine in the form of ortho-nitrobenzyl-tyrosine. We envision our engineered host as an efficient tool for the production of custom designed proteins, peptides or biomaterials for various applications ranging from research in cell biology to large-scale production in biotechnology.

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