Incorporation of Non-Canonical Amino Acids into Antimicrobial Peptides: Advances, Challenges, and Perspectives

ABSTRACT

The emergence of antimicrobial resistance is a global health concern and calls for the development of novel antibiotic agents. Antimicrobial peptides seem to be promising candidates due to their diverse sources, mechanisms of action, and physicochemical characteristics, as well as the relatively low emergence of resistance. The incorporation of noncanonical amino acids into antimicrobial peptides could effectively improve their physicochemical and pharmacological diversity. Recently, various antimicrobial peptides variants with improved or novel properties have been produced by the incorporation of single and multiple distinct noncanonical amino acids. In this review, we summarize strategies for the incorporation of noncanonical amino acids into antimicrobial peptides, as well as their features and suitabilities. Recent applications of noncanonical amino acid incorporation into antimicrobial peptides are also presented. Finally, we discuss the related challenges and prospects.

INTRODUCTION

The misuse of traditional antibiotics and slow development of new antibiotics has resulted in antimicrobial resistance (AMR). It has been predicted that, without urgent intervention in controlling resistance, the estimated global deaths resulting from AMR would reach 10 million per year by 2050 and cost the world up to 100 trillion USD (1). It is a global health and economic issue which requires novel antimicrobial drugs with different killing mechanisms (2). Antimicrobial peptides (AMPs) are linear or cyclic peptides consisting of 10 to 100 amino acid residues (3–5). AMPs have been discovered in most life forms, making their classification a non-trivial task (6). AMPs can be grouped according to their source organisms, such as bacteriocins, fungal peptide antibiotics, plant thionins and defensins, insect defensins and cecropins, amphibian magainins and temporins, and defensins from higher vertebrates (7–9). They can also be classified based on their biosynthetic mechanisms, e.g., ribosomal synthesized or nonribosomal synthesized AMPs. Nevertheless, AMPs are most commonly categorized into four classes based on their secondary structures, i.e., linear α-helical peptides, β-sheet-containing peptides, peptides involving α- and β-elements, and linear extended structures (10). As of 13 August 2022, 6,032 natural and synthetic AMPs have been reported by the DRAMP (Data Repository of Antimicrobial Peptides, http://dramp.cpu-bioinfor.org/) database, and 77 AMPs have been developed as drug candidates (preclinical or clinical stage) (11).

Their mechanisms of action are variable, including disruption of cell membranes, inhibition of metabolic and translational elements, formation of nanonets, and stimulation of antimicrobial immune activity (12–16). The stereotypical mechanism of AMPs is interacting with and disrupting bacterial cell membranes. Bacterial cytoplasmic membranes are rich in anionic groups, such as the phospholipids phosphatidylglycerol, cardiolipin, and phosphatidylserine. Lipopolysaccharides (LPS) in the outer membranes of Gram-negative bacteria and teichoic acids in the cell walls of Gram-positive bacteria also provide electronegative charges to the bacterial surface. Most AMPs exhibit either a cationic, hydrophobic, or amphipathic nature, which facilitates interaction with the negatively charged bacterial membrane (17–20).This interaction results in perturbation and disintegration of the cell membrane, leading to the loss of nutrients and cell death. The development of resistance against AMPs was initially considered unlikely due to their rapid bactericidal effects, multiple potential targets, and avoidance of SOS or rpoS bacterial stress pathways (21–25). This is especially the case for AMPs such as teixobactin, which was recently isolated from the undescribed soil microorganism Eleftheria terrae and specifically targets conserved substrates (e.g., lipids II and III [26]), because it is relatively difficult to alter the membrane without harming its function and integrity (27, 28). Although some studies have reported resistance mechanisms against AMPs, including charge modification of the membrane surface, secretion of proteases, and enhanced activity of efflux pumps (12), AMPs remain promising candidates to overcome the increasing AMR crisis due to their broad antimicrobial activities and the decline in discoveries of new classes of antibiotics (29, 30).

Noncanonical amino acids (ncAAs) are amino acids beyond the 20 canonical amino acids (cAAs), also named unnatural amino acids (uAAs), nonstandard amino acids (nsAAs), and non-natural amino acids (nAAs) (31). The majority of ncAAs are precursors, analogues, or metabolic intermediates of cAAs, and they are widely found in nature. ncAAs can contain special side chains which are chemically and structurally distinctive from those of cAAs. The past decades have witnessed rapid growth in the diversity and scope of ncAA application (32–34). The incorporation of ncAAs can introduce novel characteristics into proteins, including alteration of enzyme catalytic activity and specificity (35, 36), functional assays of enzymes (37), and the development of novel nanomaterials (38). Moreover, some ncAAs can be utilized in cosmetics, such as l-homoserine, N-hydroxyglycine, and N-hydroxyserine, which exhibit higher water uptake compared to conventional moisturizing additives (38). ncAAs have also been introduced into AMPs. The ncAA-containing AMPs have several advantages over natural AMPs, such as bioavailability, organism selectivity, metabolic stability, overall toxicity, and control of charge distribution (39–41).

Here, we describe the basis of common approaches to ncAA incorporation and review the progress toward ncAA-containing modification of AMPs, with a primary focus on exciting advances within the past 5 years. For earlier studies, readers are referred to Baumann et al. (42) and Budisa (43). Furthermore, the choice of incorporation strategies is discussed. The challenges of ncAA incorporation into AMPs and the emerging applications of ncAA-containing AMPs are also presented.

METHODS FOR NCAAS INCORPORATION INTO AMPS

Solid-phase peptide synthesis.

To date, solid-phase peptide synthesis (SPPS) is the major chemical strategy for peptide synthesis (44–46). It is a stepwise assembly of a peptide chain attached to an insoluble resin support (Fig. 1A). Peptide chain synthesis starts from the carboxyl end of the N terminus of the peptide. The incoming amino acid is protected, commonly by 9-fluorenylmethoxycarbonyl (Fmoc) or tert-butyloxycarbonyl (tBoc), at the N terminus, and is coupled with the preceding chain after activation of the C terminus (47). Then, the N terminus is deblocked and the synthesis process goes to the next cycle. After full assembly, the peptide is cleaved from the resin by treatment with trifluoroacetic acid (TFA), which also removes the side chain-protecting groups. The Fmoc and tBoc groups can be removed by piperidine and diluted TFA, respectively. The crude linear peptide can be purified by one-step high-pressure liquid chromatography (47, 48). A series of AMPs containing ncAAs has been synthesized using SPPS, generating broad structural and functional diversities (48–51).

FIG 1.

Schemes of four noncanonical amino acid (ncAA) incorporation approaches. (A) Solid-phase peptide synthesis (SPPS). Fmoc, 9-fluorenylmethoxycarbonyl; tBoc, tert-butyloxycarbonyl; TFA, trifluoroacetic acid. (B) Cell-free protein synthesis (CFPS). aaRS, aminoacyl-tRNA synthetase. (C) Selective pressure incorporation (SPI). Blue and gray circles represent 20 canonical amino acids (cAAs). Blue circle with star represents specific cAA analog. (D) Genetic code expansion (GCE).

Chemical synthesis of peptides is limited by synthesis length and speed. SPPS of longer peptides (>50 amino acids) is a challenge due to inefficient coupling and side reactions. Furthermore, synthesis speeds of minutes to hours per amide bond make SPPS a time-consuming strategy. The development of automated flow-based approaches highly improved the efficiency of SPPS, allowing for the chemical synthesis of long peptides (up to 164 amino acids) with a cycle time of 2.5 min per amino acid (52, 53). However, SPPS could not cover the chemical complexity of some AMPs, such as lanthipeptides (also called lantibiotics for those which display antimicrobial activity), a major group of ribosomally synthesized and post-translationally modified peptides (RiPPs) produced by microorganisms, because of the difficulty of chemically mimicking post-translational modification (PTM) (32) (Table 1).

TABLE 1.

Comparison of ncAA incorporation approaches^a

Approach	Advantages	Disadvantages
SPPS	Well-established and standardized	Time-consuming
	Complete control of peptide assembly	Limited PTM systems
	Multi-type and multi-site incorporation of ncAAs	High cost of resins, ncAAs, and coupling reagents
	High-throughput synthesis is available	Low efficiency for synthesis of long peptides
	High yield and purity
CFPS	Well-established systems such as E. coli	Limited PTM systems in certain platforms
	Simple and fast lysate prepn procedure	Limited platforms
	Simple modification of the reaction system	Development of a new platform is laborious and technically demanding
	Eliminate the cytotoxicity of AMPs
	Easy to scale up
	Available for nonproteinogenic AMPs
	Combination with display technologies
SPI	Fast incorporation of cAA derivatives at multiple sites	Demand for auxotrophic host
	Simple procedure	Cytotoxicity resulting in changes to the whole proteome
		Laborious purification
		Limited types of ncAAs, only cAA derivatives available
GCE	Site-specific incorporation of ncAAs	Low efficiency of incorporation
	Well-established biotechniques in typical hosts	Identification and modification of suitable orthogonal aaRS-tRNA pairs
	Coexpression of valuable products is available	Laborious purification
	Reduced cost through coexpression of synthetic pathways of ncAAs	Heterologous expression of PTM enzymes and immune apparatus

^ancAA, noncanonical amino acid; SPPS, solid-phase peptide synthesis; PTM, post-translational modification; CFPS, cell-free protein synthesis; AMP, antimicrobial peptide; SPI, selective pressure incorporation; cAA, canonical amino acid; GCE, genetic code expansion; aaRS, aminoacyl-tRNA synthetase.

Cell-free protein synthesis.

In some cases, ncAA incorporation in vivo causes cytotoxicity or poor delivery of ncAAs (33). Cell-free protein synthesis (CFPS) has become a promising strategy for producing toxic and membrane proteins which are difficult to express. CFPS was first obtained from Escherichia coli to decipher its genetic code (54) and has allowed successful incorporation of ncAAs into proteins and peptides (55). It is beneficial due to elimination of the cellular barrier, which enables direct access to enzymes, amino acids, and other reaction conditions. CFPS begins with the preparation of crude extracts from chassis strains. After depletion of endogenous DNA and mRNA, the energy sources, cofactors, and other components are added to the lysate to mimic the bacterial environment. Then, a suitable template is added to initiate the transcription and translation reaction at an appropriate temperature (56) (Fig. 1B). A wide range of CFPSs have been reported, including E. coli, Bacillus subtilis, Archaea, fungi, plants, insects, and mammals (56, 57). At present, E. coli without release factor 1 (RF1) is the most efficient platform for ncAA incorporation.

Apparently, this approach is suitable for ribosomally synthesized AMPs. In the natural translation process, an amino acid is charged to the 3′ end of a tRNA by aminoacyl-tRNA synthetase (aaRS) using a specific anticodon. For the nonproteinogenic AMPs, a combination of flexizymes and genetic code reprogramming system, namely, the flexible in vitro translation (FIT) system, was conducted (58). Flexizymes are small (~45 nucleotides) ribozyme aaRSs which facilitate the aminoacylation of tRNAs. Unlike aaRSs, flexizymes can recognize the 3′ end of tRNA without depending on anticodon sequences. Three flexizymes (dFx, eFx, and aFx) have been developed (59). Simple mixing of flexizymes with the desired tRNAs and acid substrates, followed by a few hours’ incubation on ice, can lead to the charged tRNAs.

One obvious advantage of CFPS is its speed. The process of CFPS is faster than that of in vivo protein synthesis because it has no need for cloning steps. Furthermore, CFPS is useful for the incorporation of toxic ncAAs (60). Another advantage of CFPS is the open feature which makes the chemical environment easy to modify. Moreover, the barrier-free system allows lower concentrations of ncAAs, which significantly reduces costs (61). In the envelope-free system, no signal peptides/leader sequences or transporter systems are needed to secrete the mature peptide (62). In some cases, the AMPs’ antibacterial nature makes them potentially fatal to the chassis strains. Fusion strategies with a partner protein (63, 64) or coexpression of immunity proteins (65, 66) were developed to neutralize this toxicity. CFPS, on the other hand, bypasses this limitation. In addition, as a homogeneous catalyzed reaction, CFPS is relatively simple to scale up (67). However, the application of CFPS is limited by finite platforms. Although many platforms have been reported, the E. coli system is most commonly used for ncAA incorporation (68, 69). It is economical and relatively simple to prepare. Recent efforts have further improved the efficiency of CFPS using extracts from a genetically modified E. coli strain, C321.ΔA, in which 321 TAG amber codons were recoded to TAA ochre codons and release factor 1 (RF1) was deleted (55). Nonetheless, E. coli has limited PTMs, which leads to immature or nonactive AMPs. Recently, CFPS has been shown to be successfully applied to AMPs belonging to the lasso peptide class, a class of RiPPs with a topology resembling a threaded lasso or a slipknot, which are very difficult to obtain and impossible to synthesize (70, 71). This allowed the production of a huge amount of sequence-diverse variants affording the proof of concept of using CFPS to create large libraries of difficult RiPPs.

Selective pressure incorporation.

In vivo incorporation of ncAAs has been a topic of interest. One method for in vivo incorporation of ncAAs is selective pressure incorporation (SPI), which relies on competitive incorporation. The endogenous aaRS of cAAs has substrate promiscuity. It can charge structural and chemical analogs to the cognate tRNA and leads to ribosomal incorporation of the ncAAs. SPI relies on the use of an auxotrophic host, whose growth requires the addition of particular amino acids. The auxotrophic host strains are first cultured in defined medium supplied with a limited amount of cAA to be substituted. After depletion of the corresponding cAA, the ncAA is added. Then, production of the target peptide/protein is induced (Fig. 1C). Several studies have been reported to successfully incorporate ncAAs into AMPs using SPI (72–75).

SPI is a residue-specific approach which can easily achieve multi-site incorporation. With the appropriate auxotroph strain, SPI can install ncAAs into AMPs via an endogenous translation apparatus in a relatively simple protocol. However, it is only suitable for ncAAs which share structural and chemical similarities with cAAs, which limits the applicable scope of this method. Furthermore, SPI inevitably results in a proteomic change. Although the enzymes produced during early growth can maintain the growth of the host and expression of the target peptide, SPI is not suitable for AMPs which take a long time to synthesize.

Genetic code expansion.

Another in vivo approach for incorporation of ncAAs is genetic code expansion (GCE), which is in a site-specific manner. This strategy is similar to the natural translation process. An aaRS charges an ncAA onto the tRNA with a specific anticodon. Then, the charged tRNA is delivered to the ribosome, leading to the incorporation of the corresponding amino acid (Fig. 1D). GCE relies on the use of an orthogonal aaRS-tRNA pair. Four aaRS-tRNA pairs are most common used for ncAA incorporation: the tyrosyl-tRNA synthetase (MjTyrRS)-tRNA_CUA pair from Methanococcus jannaschii, tyrosyl-tRNA synthetase (EcTyrRS)-tRNA_CUA pair from E. coli, leucyl-tRNA synthetase (EcLeuRS)-tRNA_CUA pair from E. coli, and pyrrolysyl-tRNA synthetase (PylRS)-tRNA_CUA pair from Methanosarcina spp. (76). Each of these orthogonal pairs can be used in certain organisms. To date, the suppression of nonsense codons, usually the amber codon TAG, has been successfully applied to multiple organisms (77–82). Although stop-codon suppression (SCS) has become increasingly sophisticated, its competition with RF1-mediated termination of peptide chain and low heterogeneous enzyme activity result in low integration efficiency. Several studies have been performed on improving its efficiency, such as elimination of RF1 (83, 84), directed evolution of aaRSs (85, 86) and ribosome (87), optimization of the orthogonal system (88), and modification of elongation factor Tu (89). A recent study reported a genetically recoded E. coli C321.ΔA in which all 321 amber codons were recoded to ochre codons and RF1 was deleted (90). Two synonymous serine codons were further removed to create a new chassis using 59 codons to encode the 20 cAAs (91). The reassignment of sense codons allows incorporation of multiple ncAAs. In addition, quadruplet codons have been utilized to encode ncAAs, which, in principle, might provide 256 bland codons (92, 93). Rodriguez et al. (94) successfully introduced multiple ncAAs in a neuroreceptor expressed in vivo through stop codon and quadruplet codon suppression. Quadruplet codons have also been applied to Caenorhabditis elegans, paving the way toward in vivo multi-incorporation of ncAAs in a multicellular organism (95). Over the past 2 decades, GCE has been extensively used to incorporate ncAAs into AMPs. For example, four ncAAs have been used to replace four positions in the lasso peptide microcin J25, a 21-residue RiPP produced by E. coli strains (96), via SCS (97). Using this approach, ncAAs have also been incorporated into lanthipeptides (78, 98), thiopeptides (a group of sulfur-containing macrocyclic peptides [81, 99]), and cyanobactins (a group of ribosomal cyclic peptides produced by cyanobacteria [100, 101]). These efforts led to a batch of AMP derivatives with novel chemical and/or functional activities.

As described above, the incorporation efficiency of GCE is dependent on several parameters and requires specific genetic engineering of the host strain. At present, E. coli is the most investigated platform for GCE, and is available for heterologous expression of ncAA-incorporating AMPs. However, it is still challenging because the corresponding PTM enzymes and immune proteins must be coexpressed. Moreover, some proteases in the E. coli cytoplasm can degrade heterologous proteins, which may impede proper folding and biological activity of the proteins (102, 103). A recent study reported the incomplete dehydration of the heterologously expressed NisA in E. coli BL21(DE3). The endonuclease rne and the proteases ompT and lon were deleted to ensure the complete modification of NisA (104). Because AMPs have been successfully heterologously expressed in different host strains, such as Lactococcus, Bacillus, and yeast, some of which showed better AMP expression than E. coli (105–107), it is necessary to develop new chassis, based on the principles of GCE, for suitable bioproduction of ncAA-incorporating AMPs. In addition, the in vivo GCE approaches are only available for non-toxic ribosomal synthesized peptides. To address this limitation, in vitro platforms combining CFPS and GCE have been developed (108).

RECENT DEVELOPMENTS OF NCAAS INCORPORATION INTO AMPS

Expansion of the physicochemical diversity.

In nature, the diversity of AMPs is limited due to the conservative set of amino acids and modification systems. With the development of ncAA incorporation strategies, a large number of studies have been performed to expand the repertoire of AMPs (Table 2). One example is the incorporation of the α-chloroacetamide-containing ncAA into nisin A, a well-known lanthibiotic, which resulted in novel macrocyclic topologies. Although no variants retained antimicrobial activity, this strategy could be extended to other ring junctions for structural diversification (109). Furthermore, ncAAs have also been successfully incorporated into other AMPs, such as lasso peptides (110), macrocyclic peptides (111), thiopeptides (81), and the tetracyclic polypeptide cinnamycin (112).

TABLE 2.

Recent applications of ncAA incorporation into AMPs^a

Application and AMP	Class(es)	Strategy	Modifications and consequences	Reference
Expansion of physicochemical diversity
Nisin A	Lanthipeptide isolated from bacteria	SPI	Incorporation of 6 proline analogs into nisin A; obtained 5 variants with retained bioactivity.	75
		GCE	Incorporation of several ncAAs, including an α-chloroacetamide-containing ncAA which enabled nisin A variants with novel macrocyclic topologies.	109
Macrocyclic peptide	Macrocyclic α/β-peptide foldamer	CFPS/FIT	Incorporation of consecutive stereoisomeric cyclic β2,3-amino acids and construction of a macrocyclic peptide library.	115
	Exotic macrocyclic peptide	CFPS/FIT	Assembly of a macrocyclic peptide library with 28 ncAAs, obtained at least 2 high-affinity ligands of IL-6.	175
Alteration/enhancement of bioactivity or stability
Amphipathic peptidomimetic	Peptidomimetic	SPPS	Synthesis of several amphipathic peptidomimetics containing Fmoc-triazine amino acids; obtention of a trimer, BJK-4, with enhanced proteolytic stability and no hemolytic activity.	49
Pep05	Histatin from human saliva	SPPS	Incorporation of d-AA and ncAAs enhanced stability toward proteases.	50
Coralmycin	Nonproteinogenic acyl pentapeptide	SPPS	Incorporation of an unusual 4-amino-2-hydroxy-3isopropoxybenzoic acid motif, obtained a desmethoxy analog with higher potent antimicrobial activity.	121
Cinnamycin	Lanthipeptide isolated from bacteria	GCE	Incorporation of 3 pyrrolysine analogues into cinnamycin, obtention of 5 deoxycinnamycin derivatives. Incorporation of H-Lys-Alloc-OH (Alk) in position 2 increased bioactivity.	112
Nisin A and lacticin 481	Lanthipeptide isolated from bacteria	GCE	Incorporation of 4 phenylalanine derivatives at 3 positions of lacticin 481, and 2 phenylalanine derivatives at one position of nisin A. Five lacticin 481 variants were obtained with slightly higher activities.	104
HPA3NT3-A2	Cecropin-like bactericidal peptide isolated from insects	SPPS	Replacement of l-Lys with d-enantiomer increased stability in serum.	137
W3R6	Chensinin-1 family peptide isolated from amphibian skin	SPPS	Replacement of l-Arg with d-enantiomer increased resistance to proteases, but slightly reduced antimicrobial activity.	176
Polybia-CP	Novel peptide isolated from wasp venom	SPPS	Replacement of all the l-amino acids with d-enantiomers increased resistance to trypsin and chymotrypsin; partial d-Lys-substituted derivative was resistant to trypsin only.	177
P-113	Histatin from human saliva	SPPS	Incorporation of ncAAs into P-113 altered the antimicrobial activity, bacterial membrane permeability, antibiotic synergism, and supernatant LPS-neutralizing activities.	178
Anti-Zika peptides	Macrocyclic peptide	SPPS	Incorporation of 3-(2-cyano-4-pyridyl)-alanine facilitated the condensation reaction with cysteine, resulting in a cyclized anti-Zika peptide with high affinity and proteolytic stability.	179
AMP screening through in vivo fluorescent imaging
Nisin A	Lanthipeptide isolated from bacteria	SPI	Incorporation of 4 methionine analogues with various chemical handles, allowing “click” reactions for fluorescent label. Some variants displayed increased selectivity.	73
LL37 and melittin	Cathelicidin isolated from human, peptide isolated from bee venom	SPPS	Incorporation of an azido-lysine at C terminus of LL37 and melittin enabled single-molecule imaging of AMP-bacterium interactions.	163
LL37, cecropin B, and melittin	Cathelicidin isolated from human, cecropin isolated from insects, peptide isolated from bee venom	SPPS	Incorporation of an azido-lysine to measure the affinity of 3 AMPs to lipid A extracted from 4 Gram-negative bacteria.	162
BODIPY-cPAF26	Rationally designed cyclic fluorogenic peptide	SPPS	Incorporation of a Fmoc-Trp (C2-BODIPY)-OH amino acid into AMP; real-time imaging of Aspergillus fumigatus.	180
Apo-15	Rationally designed cyclic amphipathic peptide	SPPS	Incorporation of a Trp-BODIPY fluorophore into Apo-15 allowed quantification and imaging of drug-induced apoptosis.	181
Teixobactin	Novel RiPP isolated from E. terrae	SPPS	Incorporation of an amine-reactive rhodamine fluorophore into teixobactin enabled fluorescent imaging of Gram-positive bacteria.	182

^ancAA, noncanonical amino acid; AMP, antimicrobial peptide; SPI, selective pressure incorporation; GCE, genetic code expansion; CFPS, cell-free protein synthesis; FIT, flexible in vitro translation; SPPS, solid-phase peptide synthesis; LPS, lipopolysaccharide; RiPP; post-translationally modified peptide.

The development of the FIT system expands the generation of numerous peptide analogues with diverse structural features. In particular, integration of the FIT system and display technologies has been used for the high-throughput discovery of ligands with high target affinities, especially for macrocyclic peptides (113, 114). For example, Katoh et al. (115) reported the incorporation of consecutive derivatives from 2-aminocyclohexanecarboxylic acid and 2-aminocyclopentanecarboxylic acid and construction of a macrocyclic peptide library containing 10¹² members via the FIT system. By using the random nonstandard peptide integrated discovery (RaPID) system, they identified a variation in mRNA display against human factor XIIa and interferon gamma receptor 1, an anti-hFXIIa macrocyclic peptide with high inhibitory activity and serum stability.

Alteration/enhancement of bioactivities.

The potential for alternatives to antibiotics has always been the most popular topic concerning AMPs. A large number of studies have been performed to alter/enhance their antimicrobial activity, selectivity, and antibacterial spectrum (Table 2). Previous reports have shown that modification at certain sites with certain types of amino acids could alter the bioactivity of AMPs (116). For example, saturation mutagenesis of the lantibiotics nisin A, mersacidin, and nukacin allowed access to several derivatives with enhanced activity (117–119). Furthermore, site-directed mutagenesis of nisin Z generated two mutants with enhanced activity against Gram-negative bacteria (120). Based on this preliminary work, researchers have been committed to incorporating ncAAs into AMPs (32, 42). Recently, Deng et al. (73) incorporated four Met analogs into four sites of nisin A. Several variants showed improved activity against a specific target strain with reduced activity against other strains, suggesting increased antimicrobial selectivity. Another example is coralmycin, a nonproteinogenic acyl pentapeptide produced by the myxobacterium Corallococcus coralloides and exhibiting potent activity against a range of Gram-negative bacteria via inhibition of DNA gyrase (121). The incorporation of the unusual 4-amino-2hydroxy-3isopropoxybenzoic acid motif enabled the variant to have higher potent bioactivity.

As previously mentioned, the type of amino acid is an important factor in AMP modification. The charges of naturally identified AMPs vary from 0 to +20, facilitating interaction with negatively charged bacterial membranes (122). Studies have shown that enhancing cationicity without drastically altering other physicochemical parameters increases the antimicrobial activity and spectrum (123, 124). Almaaytah et al. (125) designed a scorpion venom-derived analog (A3) with enhanced cationicity. Because bacterial membranes display a negative charge while normal mammalian cells are usually zwitterionic, the derived peptide exhibited increased antimicrobial activity and reduced cytotoxicity due to its enhanced selectivity against bacterial membranes. Among the 20 cAAs, lysine and arginine are commonly used for AMP modification due to their cationicity (126). Besides the net charge, the hydrophobicity of AMPs is also a key factor in bioactivity (127, 128). The incorporating site is another factor. For example, consider the well-studied AMP nisin A. Nisin A is composed of five lanthionine rings and a hinge region which is located between the first three and the last two rings (129). Previous studies have shown that the hinge region is a more promising site for alteration of bioactivity (130). However, for AMPs with few fundamental studies, the biosynthesis pathways, structures, and antibiotic mechanisms still need further exploration.

Improvement of proteolytic resistance.

Another area of research interest involving AMP modification is increasing their proteolytic resistance. Susceptibility to host and microbial proteolytic degradation is a great challenge in transforming AMPs into therapeutics. Many efforts have been taken to improve the proteolytic resistance of AMPs, such as encapsulation, pegylation, acetylation, amidation, cyclization, and the substitution of ncAAs (131, 132). Among these, ncAA substitution (including d-amino acids) and cyclization via reactive side chains of ncAAs are potential strategies to improving the stability of AMPs.

Replacement of some l-amino acids with d-enantiomer residues is a common strategy to increase proteolytic resistance, as d-residues are not recognized by proteases which only catalyze bonds formed by l-residues. For example, MSI-238, an all-d analogue of the amphibian AMP magainin 2 isolated from the African clawed frog (Xenopus laevis), exhibited increased proteolytic resistance (133–135). Another example is the modification of HPA3NT3-A2, an analog of the cecropin-like bactericidal peptide HP (2-20) derived from Helicobacter pylori; cecropin is a positively charged AMP originally isolated from insects and cecropia moths (136). Substitution of l-lysine in HPA3NT3-A2 with d-residues increased its half-life and maintained its antimicrobial activity (137). Likewise, incorporation of certain ncAAs can also enhance the stability of AMPs. Lu et al. (50) demonstrated that installation of some d- and unnatural amino acids could enhance the proteolytic stability of the peptides. Furthermore, the incorporation of a single alpha-isobutyric acid residue significantly increased plasma stability and activity. Another example is that of Gunasekaran et al. (49), who successfully synthesized a series of amphipathic peptidomimetics containing Fmoc-triazine amino acids and obtained a trimer, BJK-4, with enhanced proteolytic stability. Furthermore, incorporation of N-alkyl amino acids, α-substituted α-amino acids, β-substituted α-amino acids, β- (and γ-) amino acids, and proline analogues have also been reported to improve peptide stability (138). Generally speaking, decreasing peptide-like features leads to enhanced stability toward proteases.

Cyclization is another potential strategy to improve the proteolytic stability of AMPs (139). The most popular connections for peptide cyclization are lactam, lactone, and disulfide bonds. Other covalent connections, such as thioether bonds and carbon-carbon bonds, have also been used for peptide cyclization (140–144). Cross-linking of amino acid side chains is an effective method to cyclize peptides. Cysteine, lysine, and tyrosine are widely used cAAs for such conjugation (145, 146). ncAAs with reactive side chains have also been used for cyclization of AMPs (147, 148) (Table 2). A recent study reported that cyclization of buforin II was facilitated by the introduction of aldehyde and hydrazide groups. A series of peptides with single-, double-, and triple-loop was obtained with increased activity (149). Cyclic efficiency may be affected by the residues around the cyclization site. Amino acids with more flexibility (such as Gly) and smaller space requirements are preferred (150). Cyclization may also influence the activity, selectivity, and hemolytic activity of AMPs (140, 141, 151–153), although the relation is complex. In summary, the introduction of ncAAs provides increased potential for AMP cyclization, which is an effective strategy to improve the stability of linear AMPs. The effects of ring size, amphiphilicity, and peptide sequence, and the potential side effects, still require more investigation.

Screening of AMPs through in vivo fluorescent imaging.

Fluorescent imaging of AMPs is of great contribution in understanding their in vivo antimicrobial mechanisms, and requires precise recognition, localization, and quantification (154, 155). The common labeling method of fluorescent protein fusion may disturb the structure and activity of short peptides. Conjugation with fluorophores through click chemistry is a promising tool for in vivo molecule imaging due to its high specificity, quick reaction rate, and stability (156). Incorporation of ncAAs enables the site-specific installation of a suitable handle/arm, allowing bio-orthogonal labeling of AMPs by taking advantage of specific reactive groups (such as azides, alkynes, and alkenes) of ncAAs. A recent study by Deng et al. (73) successfully installed Met analogs with bio-orthogonal reactive handles into nisin A and enabled fluorescent detection. Advances in biomolecule imaging technology have now enabled the investigation of subcellular protein organization (157). Since the first reported single-molecule imaging of fluorescently labeled ncAA done by Pantoja et al. (158), super-resolution techniques utilizing ncAA incorporation have been applied to several proteins and AMPs (159–163) (Table 2). These advances pave the way to investigating interactions between AMPs and pathogens at the molecular level.

In some cases, fluorescent labeling can also alter the conformation of peptide biological properties. To diminish this effect, some fluorescent ncAAs have been developed. Analogues of tryptophan, tyrosine, and phenylalanine are common candidates because they are themselves fluorescent. A range of non-natural fluorescent amino acids was also generated by either appending fluorescent moieties to amino acids or de novo construction with integrated chromophores. For detailed information on fluorescent amino acids, researchers are referred to a recent review by Cheng et al. (164).

Conclusion and future prospects.

Over the past few years, intensive research has been conducted to introduce novel properties into AMPs through ncAA installation (Table 2). This review provides a brief overview of common strategies for ncAA incorporation into AMPs and recent applications. Each of these technologies has different features and suitabilities (Table 1). At present, SPPS is still the primary approach for peptide synthesis, allowing multi-type and multi-site incorporation of ncAAs. In particular, SPPS is a more appropriate strategy for synthesizing toxic AMPs or ncAAs with ambiguous permeability and solubility. CFPS is another efficient platform to synthesize toxic AMPs. Specifically, with the development of flexizymes and display techniques, CFPS has a unique advantage in construction and screening of AMP libraries. However, with the available chassis, ncAAs, and/or orthogonal pairs, in vivo SPI or GCE is preferred due to their relatively simple procedures.

One important value of a promising biotechnology is the implementation of industrial application. Currently, ncAA modification of AMPs is more of a theoretical verification or mechanistic investigation. One obstacle is the high costs of most ncAAs. Although many ncAAs are commercially available, their cultural expenses are still high because a high concentration of ncAAs is required for efficient incorporation. In addition, many ncAAs are not yet commercialized and rely on chemical synthesis, resulting in even higher costs. Coupling GCE and certain biosynthetic pathways, through which ncAAs can be produced from inexpensive substrates, offers a promising way to reduce the cost of ncAA incorporation (165, 166). In addition, for industrial production of potent variants, a standardized and relatively simple protocol, inexpensive substrates, safety, and efficiency are essential. Over the past few decades, massive research has been done to synthesize AMPs through biological approaches. Microbial engineering has been done via (i) heterologous expression of orthogonal aaRS-tRNA pairs for ncAA incorporation (78, 104, 109); (ii) reprogramming the genetic code of the microbial chassis to improve incorporation efficiency (83, 84, 90, 91); (iii) directed evolution of the translational apparatus (85–89); and (iv) mining and overexpressing the synthesis pathways of ncAAs to reduce production costs (167, 168). While the most-understood model organism E. coli has become an optimal chassis for heterologous expression, the engineering of the original production strains is still necessary, considering some characteristics of the original hosts. For example, we previously reported the co-production of nisin Z and γ-aminobutyric acid in the nisin Z production strain Lactococcus lactis, which exhibited a synergistic effect of food preservation. Because L. lactis is a probiotic strain, its freeze-dried product can be directly used, eliminating the process of purification (169). In addition, it also excludes the trouble of heterologous expression of multiple sets of synthetic and immune enzymes. Indeed, complete reprogramming of every host is an impractical task. It is a compromise strategy for prior modification of essential or growth-related genes which may reduce the effect of ncAA introduction. Furthermore, the identification of new orthogonal pairs which can recognize novel ncAAs is desired.

With the development of next-generation sequencing and computational tools, various algorithms have been established to discover novel AMPs (170–172). It is foreseeable that more AMP-related synthesis pathways, physiochemical properties, and structures could be discovered. The machine-learning technique has been used to guide the optimization and designation of AMPs with ncAAs (173, 174). Given advances in computational system biology and structural biology, rational design and prediction of novel AMPs will become practical in the coming years.