A comparative framework of natural nisin variants: linking genetic basis to future bioengineering applications

Abstract

Background

Nisin is a commercially successful antimicrobial peptide (AMP) widely used as a food preservative. As a ribosomally synthesized and post-translationally modified peptide (RiPP), nisin belongs to a diverse family of natural variants. However, despite its importance, a systematic and comprehensive comparison of the biosynthetic gene clusters and resulting structural diversity across the entire nisin family has been a critical gap in the literature.

Results

This review provides the first comprehensive, side-by-side analysis of nisin A and all its known natural variants. We systematically compare their biosynthetic gene clusters (BGCs), post-translational modification pathways, and final amino acid sequences. This comparative analysis reveals direct correlations between the genetic architecture of each variant and its resulting structural and functional properties. By collating and organizing this information, this work establishes a critical resource that clarifies the molecular basis for the functional diversity observed in the nisin family.

Conclusions

This study provides a foundational framework for understanding the natural diversity of nisin. The established links between genotype and phenotype serve as a rational guide for future bioengineering efforts aimed at developing novel nisin derivatives with improved stability, solubility, or specific antimicrobial activity. In the face of growing antibiotic resistance, harnessing this natural diversity holds significant potential for creating new therapeutic agents that extend beyond nisin’s traditional role in food preservation.

Background

The host’s innate defense against a range of pathogens is aided by antimicrobial peptides, which are oligopeptides with varying numbers of amino acids [1–3]. Moreover, there are numerous biological activities, such as wound healing, immunomodulators, and anti-tumor activity [2, 4]. Host defense peptides are the precursors of antimicrobial peptides. Peptides that are ribosomally produced and go through post-translational modifications (RiPPs) after translation are known as antibiotics, and they were originally defined in 1988. They contain meso-lanthionine and 3-methyl-lanthionine structures and have antibacterial properties. Despite being classified as lantibiotics, the majority of ribosomally modified peptides that include lanthionine do not have antibacterial properties. The more general term “lanthipeptides” is used to refer to this tiny class of peptides that include lanthionine but do not have antibacterial activity [5]. Nisin is a prominent example of cyclic antimicrobial peptides. However, beyond natural variants, the field has expanded to include synthetic cyclic peptides and peptoids prepared via polymer chemistry and solid-phase peptide synthesis (SPPS). These synthetic approaches offer opportunities to design mimics with enhanced stability and tailored biological activities [6, 7]. Non-ribosomal peptides are created in the cytoplasm of bacteria and fungi, whereas ribosomal peptides are synthesized in the ribosome of eukaryotes and prokaryotes and are referred to as such [8, 9]. Alexander Fleming identified lysozyme, one of the first antimicrobial peptides, in the late 1920s [8]. There have been reports of differences in the biosynthetic enzymes that synthesize methyl-lanthionine (MeLan) and lanthionine (Lan) among five classes of lanthipeptides. Certain lanthionine synthetases catalyze a series of dehydration and cyclization events that culminate in the formation of lanthionine cross-links. These enzymes vary among the several lantibiotic classes: Specialized enzymes like the cyclase LanC and the dehydratase LanB perform this function in Class I (Nisin-like, Epidermin-like and Pep5-like). Single-chain, multifunctional enzymes, such as LanM for Class II, LanKC for Class III, and LanL for Class IV, carry out these tasks in Classes II, III, and IV. Dehydration is dependent on aminoacyl-tRNA in Class I dehydratases like LanB. First, serine or threonine residues are glutamylated (glutamate is added to their hydroxyl group); next, glutamate is eliminated; and last, dehydroamino acids such dehydroalanine (Dha) or dehydrobutyrine (Dhb) are formed. To carry out the dehydration reaction, multifunctional synthetases (LanM, LanKC, and LanL) need (d)NTPs, which are nucleotide triphosphates like ATP or dATP. To produce Dha (dehydroalanine) or Dhb (dehydrobutyrine), these nucleotides are used to phosphorylate serine (Ser) or threonine (Thr) residues. The phosphate group is then removed. Two domains make up the LanM enzyme in Class II (Lacticin 481-like and Mersacidin-like): a C-terminal cyclase domain and an N-terminal dehydratase domain. On the other hand, Class III (Labyrinthopeptin-like) and Class IV (Venezuelin-like) multifunctional synthetases (LanKC and LanL, respectively) have three domains: The phosphate group is removed following phosphorylation by an N-terminal lyase domain; Ser/Thr is phosphorylated by a central kinase domain; and thioether cross-links are formed by a C-terminal cyclase domain. In Class II synthetases, the N-terminal dehydratase domain is in charge of two primary functions: phosphorylating Ser/Thr residues and removing the phosphate group to generate Dha (dehydroalanine) or Dhb (dehydrobutyrine). In contrast, two distinct domains—the kinase domain for phosphorylation and the lyase domain for phosphate elimination—perform these two actions in Classes III and IV. The cyclization process takes place after serine or threonine has been dehydrated (i.e., converted to Dha or Dhb). This step involves using a Michael-type addition reaction to add the thiol group of a cysteine (Cys) residue to either Dha or Dhb. A lanthionine (Lan: Dha-Cys) or methyl-lanthionine (MeLan: Dhb-Cys) cross-link is then formed as a result of protonating the resultant enolate intermediate [5, 10, 11]. The first known member of the novel RiPP subfamily known as lanthidins, which includes structural components present in lanthipeptides and linaridins, is the antibiotic cacaoidin (Classe V). A strain of Streptomyces cacaoi yielded cacaoidin, which has an unusual N, N-dimethyl lanthionine (NMe2Lan) not present in other lanthipeptides. Although linaridins and LAPs have been shown to exhibit N-terminal bis-N-methylation, lanthionines are absent from these RiPP families. Additionally, the molecule incorporates a number of D-amino acids, including D-2-aminobutyric acid (Abu), and a 6 deoxygulopyranosyl-(rhamnopyranose) disaccharide to O-glycosylate Tyr. No prediction software technique was able to identify the cacaoidin BGC, which was mapped in the area next to the core peptide structural gene. The cluster indicates an altered native RiPP biosynthesis route and exhibits poor similarity with those of other lanthipeptides or linaridins. Because lanthipeptides are identified by lanthionine, linaridins are identified by dehydrobutyrine (Dhb) residues, which are absent from cacaoidin [10]. The most well-known and extensively researched lantibiotic to date is nisin A, the first bacteriocin identified in lactic acid bacteria (LAB). Bacteriocins are cationic and hydrophobic antimicrobial peptides (AMPs) that are produced and secreted ribosomally [12]. They can suppress spoilage/pathogenic bacteria from both Gram-positive and Gram-negative bacteria. They are 20–60 amino acids long [12]. Nisin was first discovered in 1928 in cultures of fermented milk, but Mattick and Hirsch isolated it from strains of L. Lactis subspecies in 1947 [13]. In 1953, it was offered for sale as an antibacterial agent in England. Gross and Morell, revealed its structure in 1971 [14, 15]. The Food and Agriculture Organization (FAO) and the World Health Organization (WHO) jointly approved nisin as a safe food additive in 1969 [16]. The United States Food and Drug Administration (FDA) recognized nisin as generally recognized as safe (GRAS) in 1988 [17].

Nisin is a ribosomally synthesized, cationic, amphiphilic, hydrophobic, five-ring polypeptide having 34 amino acids in its active state and a molecular weight of 3354 Da. Nisin is comprised of several unique amino acids resulting from post-translational modifications, such as dehydroalanine, dehydrobutyrine, one lanthionine residue (the A ring), and four beta-methyllanthionine residues (the B, C, D, and E rings) [18, 19]. The FDA has only authorized nisin as a food additive and preservative [20]. Nisin is the sole bacteriocin. Similar to other antimicrobial peptides, nisin does not destroy food; rather, it maintains its quality without alteration [21]. Nisin is thought to be safe for ingestion by humans due to its low cytotoxicity and gradual rate of resistance development [22]. Nisin’s physicochemical characteristics include being soluble in water and insoluble in non-polar solvents; sensitive to the enzyme alpha-chymotrypsin; resistant to trypsin and pepsin, particularly in acidic conditions; and generally stable against heat. As a result, at pH = 2, nisin’s biological activity is preserved even after autoclaving for a period of time. However, as pH rises, nisin’s stability decreases and its activity decreases noticeably at higher temperatures. Nisin’s solubility also decreases as pH rises. At least 80% of the efficiency of nisin is retained at mild temperatures, such as those experienced during pasteurization [23, 24]. When it comes to a variety of Gram-positive bacteria and their spore forms, nisin exhibits broad inhibitory activity. But it performs poorly against fungi and bacteria that are Gram-negative [23].

14 variants (A, Z, Q, U, U2, O, J, G, F, P, H, E, S, and rombocin A) and two nisin-like bacteriocins (salivaricin D and kunkecin A) have been discovered since the discovery of nisin. The bacterial strains of L. lactis (A, Z, Q, and F), Streptococcus (U, U2, J, G, P, H, E, and salivaricin D), Balutia obeum (O₁₋₃ and O₄), Apilactobacillus kunkeei (kunkecin A), Ligilactobacillus salivarius (S) and the latest natural variant of nisin, named rombocin A, was isolated through genome mining in two strains of Romboutsia sedimentorum, RC001 and RC002. All produce variants of nisin that range in length from 25 to 39 amino acids [25, 26]. Variants of nisin come from closely related or comparable species. In spite of variants in amino acid sequence, all forms of nisin share a common three-dimensional structure made up of five thioether rings (A, B, C, D, and E) arranged between the two dispersed N-terminal (amphiphilic and responsible for recognizing and binding to lipid II) and C-terminal (participant in pore formation) domains. These domains are linked together by a flexible hinge region [25]. It is also worth noting that nisin biosynthesis is tightly regulated by a quorum-sensing mechanism involving a two-component regulatory system. This system consists of a histidine kinase sensor (NisK) and a response regulator (NisR) [27]. In this auto-induction loop, nisin itself acts as a signaling molecule (pheromone); once it reaches a certain threshold concentration externally, it activates NisK, which in turn phosphorylates NisR to upregulate the expression of biosynthetic genes [28, 29]. This signaling architecture ensures that nisin production is synchronized with population density [30].

The desire to develop natural alternatives to chemical preservatives in food is growing as customers become more concerned about the amount of chemicals used in food and the negative effects they can have. However, overuse and long-term use of antibiotics have made pathogenic bacteria resistant to them, making it difficult for doctors to develop substitutes for popular antibiotics. The benefits of nisin in resolving these issues are indicated by its previously mentioned properties, and the significance and goal of this review study are made apparent.

Post-translational modifications and genes cluster

Studies have shown that the production of nisin by L. lactis is dependent on growth, which represents the primary metabolite.

Previous studies have characterized the metabolic kinetics of nisin production as follows: (1) Nisin production is typically low during the initial stages of bacterial growth; (2) Nisin production increases sharply during the exponential growth phase; (3) Nisin production peaks at the end of the phase (i.e., the start of the stationary phase); and (4) Nisin production completely stops when the cells enter the stationary phase [31–35]. Gene clusters comprising conserved genes that codify processes related to production, maturation, immunity, and regulation typically encode the producing of LAB lantibiotics [28]. The genes responsible for immunity and bacteriocin biosynthesis are typically arranged in operon clusters and can be found on plasmids or mobile elements like transposable chromosomes [36]. Bacteriocins are synthesized as inactive precursor peptides and are modified by amino acids coded by gene clusters before export [37]. The significant range of post-translational modifications introduced and the variations in the processing, immunity, and regulatory mechanisms of various lantibiotics explain why different strains that produce lantibiotics do not have the same complement of lan genes [38]. Comparison of bacteriocin gene clusters of variants of nisin is presented in Fig. 1. Nisin A precursor peptide consists of 57 amino acids and is divided into two parts: leader sequence (amino acids 1–23) and mature peptide (amino acids 24–57). Nisin A biosynthesis requires a gene cluster containing 11 genes in three transcription units (Fig. 2). The nisin gene cluster’s arrangement (A) includes nisABTCIPRKFEG, respectively [39], which have been proposed to be nisABTCIP, nisRK and nisFEG transcriptionally arranged and located on a large nisin-sucrose conjugate transposon [30]. nisA is the gene coding for the nisin precursor peptide containing 57 amino acids, which is involved in the process of modifying and targeting the peptide [28]. Membrane-associated proteins involved in intracellular post-translational modification are encoded by nisB and nisC. Serine and threonine residues are dehydrated by NisB to produce Dha and Dhb, respectively. Dehydrated residues are cyclized by the expression of nisC and forms Lan and MeLan from Dha and Dhb, respectively. NisC is a zinc-dependent metalloprotein that reacts dehydrated residues with cysteine, and in this reaction, part of one molecule is connected to another and a closed loop is formed [40, 41]. The biosynthesis of nisin A can be triggered by this peptide by activating the promoters upstream of NisF and NisA via the NisR/NisK two-component regulatory mechanism [29, 42]. The nisin precursor peptide is post-translationally modified after synthesis in the ribosome. Serine and threonine residues are subjected to enzymatic dehydration that leads to the production of Dha and Dhb. Subsequently, thiol groups from adjacent cysteine residues attack Dha and Dhb double bonds, thioether bonds are formed, and lanthionine and beta-methyl lanthionine are formed, respectively. The formation of covalent bonds between these neighboring residues leads to the formation of closed loops in the peptide, which determines the function and structure of the peptide that are necessary for the potent activity of these lantibiotics [43]. Also, from the dehydration of serine residue, D-alanine is also created [44–46]. A putative ABC transporter protein that is involved in moving the fully modified nisin precursor across the cytoplasmic membrane is encoded by the nisT gene [47]. A subtilin-like protease required for extracellular proteolytic activation is encoded by the nisP gene. Protease removes the leader peptide shortly after the nisin precursor is transferred, resulting in the formation of an extracellular mature peptide [27, 48]. The generating cell’s protection against nisin is mediated by two independent mechanisms, nisI and nisFEG. In bacteria that produce protection, nisI encodes a lipoprotein [47, 48], while nisFEG encodes a putative ABC transporter [49, 50].

Fig. 1.

Bacteriocin gene clusters of nisin variants. ORFs are indicated by numbers and those with known function by gene identification and color [25, 26]

Fig. 2.

Amino acid sequence and structural details of nisin A [72]

Lipoprotein NisI is most likely oriented towards the outside of the cytoplasmic membrane, attaches itself to nisin, and stops it from attaching to lipid II and causing pores in the cell membrane and return to the environment. It also maintains the critical threshold of nisin content in the membrane [36, 50, 51]. It is believed that nisin is transported by NisFEG from the cytoplasmic membrane to the extracellular environment, avoiding the build-up of significant nisin concentrations that are required for hole formation [52, 53]. It has been reported that the amount of nisin synthesis is influenced by the immunity level of the producer strain. Both the lipoprotein and the nisin transporter are necessary for the highest level of nisin safety [53, 54]. The two-component regulatory systems represented by nisR and nisK encode a response regulator and a sensor kinase, respectively, from the histidine protein kinase family. It has been revealed that both genes are important for regulating the production of nisin [27, 48]. Nisin biosynthesis is dependent on growth phase and primary metabolite kinetics involving nisin-mediated induction by a two-component system involving a transcriptional response regulator (NisR) and a receptor histidine kinase (NisK)) (to maintain the balance between production and immunity) [38]. The same biosynthetic proteins are used in a similar manner to synthesize other variants of nisin [39]. The gene cluster and arrangement and order of genes in nisin A, Z, Q and F (nisABTCIPRKFEG) are similar. The nucleotide sequence, gene arrangement, mechanism of biosynthesis and regulation and immunity in nisin Z are identical to nisin A, except for a single mutation (His27Asn) in the mature polypeptide [55]. DNA sequencing of the cloned lanQ gene in nisin Q revealed that it encodes a peptide with a determined amino acid sequence with a high similarity to nisin A and Z. which contains 57 amino acids in the precursor peptide, 34 amino acids in the mature peptide, also according to the amino acid sequence at the N-terminal after modifications, was reported to be similar to nisin A and Z [56].

The nisin U gene cluster also included 11 genes similar to the nisin a gene cluster, however different from the nisin in terms of potential function in terms of gene arrangement, a biosynthetic cluster. Cross immunity against nisin A and specific immunity against nisin U were developed in the nisin U producing strain [57].

Molecules of modified nisin A and U act as stimulators of themselves and each other, even when bound to their leader peptides [57]. Probably, the immunity to nisin depends on the expression of both nisI and nsuFEG genes [41]. However, studies showed that nsuI mutants showed more specific sensitivity to exogenous nisin than the nsuFEG mutant [50], which indicates the important role of NsuI in immunity. In nisin U, nsuI encodes a peptide sequence with 55% similarity to nisI and, like nisI [48] and SpaI, also has a lipoprotein signal sequence [57].

The sequence of the amplified fragment from the nisin F producing strain resembles the sequence found for the structural gene that codes for nisin Z, with only one difference of Ile30Val. The plasmid contains the operon that codes for nisin F [58]. The arrangement of genes in the U, U2 and P nisin gene cluster (nsuPRKFEGnsuBTCI) is also similar. The nisin P gene cluster had the same order as the prototype of the nisin 11 genes made up the gene cluster. However, as is also the case in the nisin U gene cluster, the nisPRKFEG genes were found to be more anterior to the nisA structural gene [18]. The nisin-like bacteriocin salivaricin D gene cluster contains 12 genes and includes all functional modules like nisin A. slvN is an additional gene in the salivaricin D gene cluster that encodes the lantibiotic salivaricin N. The way genes are arranged inside its gene cluster is different from nisin A. Salivaricin D consists of two distinct DNA strands containing gene clusters. All ORFs are organized into 14 operons. One strand has the slvANBTCIPRK gene cluster and another cluster (4th operon) containing SlvFEG immune proteins is on the other DNA strand. A gene encoding a transposase-like gene (ORF2) separates these two clusters, indicating that it is located in the genome by a transposon [59].

Nisin H gene cluster consists of 10 genes encoding transposases between nshG/nshK and nshP/nshR. The noteworthy point is lack of a counterpart for the nisin immune gene nisI in its gene cluster. On the other hand, the arrangement and order of genes in its gene cluster is different from nisin A. Studies Siegers, and Entian, showed that a specific immune gene is necessary to create complete immunity [50]. According to Stein et al. nisI and nisFEG can produce immunity in Bacillus subtilis host cells that are vulnerable to nisin [53]. The nisin H producing strain seems to be sufficiently shielded from nisin’s harmful effects by the ABC transporter gene nisFEG [54]. Nisin H lacks the nisI immunity gene, but shows immunity to nisin A and P. NisFEG proteins consists of four operons. While NshF and NshG, nisin H, have relatively little in common with the corresponding proteins in nisin P, A, and U, NisF and NisG proteins are like to nisin P, A, and U counterparts [18].

The nisin O gene cluster putational study revealed 15 ORFs encoding genes associated with this lantibiotic. The orientation of the genes was the same and included four genes encoding nisin O, nsoA (A₁, A₂, A₃ and A₄) (coding the same proteins with only one amino acid difference in the leader peptide) and two double-component nsoRK sets. While no putative protease genes capable of cleaving the leader peptide were found, all the genes found in the nisin A gene cluster were likewise found in this nisin. A region consisting of 506 bp (end of nsoK₁, start of nsoA₁) showed no similarity to more recognized genes common in the lantibiotic cluster. The sequence of the mature protein expressed by nsoA₁ was extremely comparable to other NisA proteins (maintaining positions of serine, threonine and cysteine), and the least similarity was observed in the sequence of the mature protein of nsoA₄ [60].

Three NsoA₁₋₃ precursor peptides, which have 63% similarity with nisin U precursor peptide, were categorized as belonging to the gallidermin/nisin family. The NsoA₄ precursor peptide had a short amino acid equivalent to nisin A. It was showed great likenesses between the nisin O cluster and nisin A, U and Q. Because it is the first known nisin gene cluster to have two sets of nsoRK genes, several copies of the nisin structural gene, and no protease, the nisin O gene cluster is unique. This structural gene triplication occurred by sequential duplication events. The strain that produced nisin O exhibited cross-immunity against nisin A [60].

The order of genes in the gene cluster of nisin J (nsjFEGBTCJP) is different from other variants of nisin and consists of 8 genes against 11 genes of nisin A, and lacks nisRK regulatory genes and nisI immune gene. On a plasmid lies its gene cluster. This strain’s whole genome sequencing revealed that its nisin gene cluster was 9.78 bp longer than its nisin A gene cluster, which is roughly 13.3 bp longer. Nisin J precursor peptide contains 161 amino acids and its leader sequence contains 26 amino acids. The structural peptide of nisin H and the mature peptide of nisin J have 62.5% identity. The nisin J gene cluster contains nsjFEG genes, however nsjI genes (specific immunity) are absent and a regulatory system of nsjRK expression can explain the less protection of the producing cell compared to nisin J. On the other hand, this finding confirms that compared to the nisin A producer strain, the producer strain was more sensitive to the purified nisin J (Contains nisRK, nisI genes). The nisin J gene cluster is located on a plasmid and it is possible that S. Capitis gained its antibacterial capabilities through horizontal gene transfer [61].

Numerous lantibiotics, such as subtilin, nisin A, and U, have been demonstrated to be regulated by a two-component regulatory system that consists of a protein histidine kinase and a response regulator. In addition to being able to induce its own production, nisin can also lead to the production of related immune proteins. Salivaricin D’s nisbox (TCTGA) and the genes of these two regulatory proteins may also function as a signal peptide, controlling salivaricin D’s synthesis and immunity [62, 63]. Identities and similarities exist in the N-terminal, while distinctions were identified near the C-terminal [59]. Genes encoding nisin-like bacteriocin kunkecin A biosynthetic proteins are located in the plasmid, and showed great similarity to biosynthetic nisin A proteins. However, the kunkecin A gene cluster lacked nisI, nisR and nisK genes and had a different arrangement from the nisin A gene cluster. In the cluster, half of the genes have opposite coding directions (unlike nisin A, where all genes are coded in the same direction). Kunkecin A lower immunity in against to the nisin A producing strain is linked to the elevated efficiency of nisin A synthesis and the absence of a protein similar to NisI [64]. Despite NisI and SpaI contribute to overall immunity relative to NisFEG and SpaFEG, respectively, studies have shown that they protect producer strains from the pore-forming function of each corresponding homologous bacteriocin [65, 66]. spaI and nisI both share a very similar structure [67]. The absence of the NisI homologous protein in kunkecin A biosynthesis is probably involved in the C-terminal extension of kunkecin A and its resulting activity. NisFEG alone is known to be sufficient to induce immunity against lantibiotics. This discovery demonstrated that the kunkecin A producing strain may be protected by the KukFEG protein alone, without the assistance of the NisI protein [48, 53].

Compared to nisin A and other nisin variants, the gene organization in the nisin G gene cluster is distinct. However, Genes related to immunity and nisin G production displayed the highest identity with nisin H genes, 89–98%. Also, the cluster of gene nisin G, similar to nisin H, lacks the equivalent of the nisI immune gene. 10 genes and transposases encoded between the structural genes of nisin G, nsgA, and nsgF make up its gene cluster. The nisin G gene cluster is notable for having nsgFEG transcribed in the opposite way from nsgA [68]. Like nisin A and nisin U, the biosynthetic source of nisin E (nmd) contains 11 genes and encodes proteins in several functional modules. However, the order of the genes is different from other nisin (except nisin U and P). The nmd locus organizes similarly to the nsu locus, with the exception that, unlike nsu, which is situated directly upstream of the translocation/transport genes, the nmd gene is situated between the immune and regulatory modules. However, the production of functional nisin E by this peculiar gene arrangement has no effect on the overall expression of nmd. Because the mature nisin E and nisin U peptides differ by only two amino acids, the high homology percentage (92.9% similarity and 9.83% identity) between the precursor peptides of NmdA and NsuA can be attributed to this. In addition, nisin E contains an additional asparagine residue at its C-terminal end [69]. Analysis of the whole genome sequence of Ligilactobacillus salivarius P1CEA3 revealed the presence of two gene clusters [25]. One gene cluster was recognized as the coding sequence for the bacteriocin salivaricin B and the other cluster included a group of genes that code for the production of nisin S (the most recent naturally occurring variant of nisin). One of the shortest nisin gene clusters is the nisin S gene cluster and follows the sequence of nisin A genes, except that it lacks nisI and nisP genes. The nssA gene encodes a structural precursor of 58 amino acids, including a 25 amino acids leader sequence and a cleavage site, after modification may lead to a mature peptide of 34 amino acids. The nisin S cluster’s gene organization like that of the nisin A, Z, Q, and F genes, although they lack genes equivalent to the nisI immune gene and the nisP leader sequence-specific peptidase coding gene [25]. In addition to nisin S, another nisin operon that does not have a gene homologue to nisP is the nisin O₁₋₄ operon [60]. Interestingly, nisins O₁₋₄ and nisin S are the only nisin variants that have lysine rather than arginine at the leader sequence cleavage point (position − 1). As mentioned earlier, the mature bacteriocin is released when the protease NisP cleaves the NisA leader peptide [48]. Most likely as for O₁₋₄ [60], and for other class I bacteriocins, such as subtilin [70], and mutacin I [71], it has been suggested that there may be another protease that is encoded in the genome of the L. salivarius P1CEA3 nisin S producing strain that cleaves the sequence. Its leader, in addition to the transportation and processing of nisin S, is necessary outside the synthesizing cells [25]. The genomes of R. sedimentorum RC001 and RC002 were extracted, and a 12.9 kb type I lantipeptide gene cluster known as rombocin A was found in the genome of R. sedimentorum RC002. 10 genes are present in the gene cluster (lack of the immune gene nisI) as opposed to 11 in the nisin A gene cluster, and the gene order differs from that of nisin A. Among these genes is romPABTCRKFEG, of which romA is the structural gene. Compared to other natural nisin variations, which have 31–35 amino acids, romA encodes a core peptide of 25 amino acids [26].

Structure and sequence of nisin variants

Table 1 displays the strains of nisin that are producing as well as their molecular weight, and sequence of amino acids. The lantibiotics found in nature most likely originate from point mutations in structural genes. Lantibiotics are defined as those that have similar ring patterns and little differences in amino acids, and the strains that produce them must exhibit cross-immunity to other variants [54]. Changes in pH can have an impact on nisin’s stability and activity, which are closely related to its structure. Nisin is more stable at lower pH levels because structural changes brought about by an increase in pH result in a decrease in activity [24]. Lantibiotics include unique amino acids, in contrast to conventional bacteriocins [36].

Table 1.

Amino acids of Nisin variants

Nisin variant	Sequence	Amino acids	MW	Gene clusters	GenBank accession no.	Source
Nisin A	ITSISLCTPGCKTGALMGCNMKTATCHCSIHVSK	34	3354 Da	NisABTCIPRKFEG	HM219853	Lactococcus lactis [12]
Nisin Z	ITSISLCTPGCKTGALMGCNMKTATCNCSIHVSK	34	3331 Da	NisABTCIPRKFEG	ABV64387	Lactococcus lactis NIZO 221 86 [53]
Nisin Q	ITSISLCTPGCKTGVLMGCNLKTATCNCSVHVSK	34	3327 Da	NisABTCIPRKFEG	AB362350	Lactococcus lactis 61−14 [54]
Nisin U	ITSKSLCTPGCKTGILMTCPLKTATCGCHFG	34	3029 Da	NsuPRKFEGBTCIA	DQ146939	Streptococcus uberis 42 [55]
Nisin U2	VTSKSLCTPGCKTGILMTCPLKTATCGCHFG	31	3015 Da	NsuPRKFEGBTCIA	GU384321	Streptococcus uberis D536 [55]
Nisin F	ITSISLCTPGCKTGALMGCNMKTATCNCSVHVSK	31	3315 Da	NisABTCIPRKFEG	EU057979	Lactococcus lactis F10 [56]
Salivaricin D	FTSHSLCTPGCITGVLMGCHIQSIGCNVHIHISK	34	3468 Da	SlvFEGKRPICTBND	JN564797	Streptococcus salivarius 5M6c [57]
Nisin H	FTSISMCTPGCKTGALMTCNYKTATCHCSIKVSK	31	3453 Da	NshHBTCPRKGEF	KP793707	Streptococcus hyointestinalis DPC6484 [52]
Nisin O1−3	YKSKSACTPGCPTGILMTCPLKTATCGCHITGK	34	3259 Da	NsoFEGIRKABTC	KY914474	Blautia obeum A2-162 [58]
Nisin O4	TSQHSFCTPNCLTGFLCPPKTQLTCTCKLKGQ	33	3546 Da	NsoFEGIRKABTC	KY914474	Blautia obeum A2-162 [58]
Nisin P	VTSKSLCTPGCKTGILMTCAIKTATCGCHFG	32	2987 Da	NsuPRKFEGBTCIA	WIDP01000000	Streptococcus agalactiae DPC7040 [16]
Nisin J	ITSKSLCTPGCKTGALQTCFAKTATCHCSGHVHTK	35	3458 Da	NsjFEGBTCJP	MN602039	Staphylococcus capitis APC 2923 [59]
Kunkecin A	ITSYVLCTPGCITGRLMGCNNKTKTCHCHSSNHFSFHAR	39	4218 Da	KukPACTFEGB	AP019008	Apilactobacillus kunkeei FF30-6 [62]
Nisin G	ITSYSLCTPGCKTGVLMACHLKTATCNCSIIVSK	34	3405 Da	NsgGEFABTCPRK	OR885872	Streptococcus salivarius DPC 6487 [66]
Nisin E	ITSKSLCTPGCKTGALMTCPIKTATCGCHFGN	32	3101 Da	NmdPRKAFEGBTCI	JANHMF00000000	Streptococcus equinus MDC1 [67]
Nisin S	ITSYSLCTPGCKTGALMGCTMKTASCGCHVHISK	34	3347 Da	NssABTCRKFEG	CP116812	Ligilactobacillus salivarius P1CEA3 [23]
Rombocin A	ITSISLCTAGCITGVIMTCMSNGCK	25	2380 Da	RomPABTCRKFEG	JASKYN000000000	Romboutisia sedimentorium RC002 [24]

Amino acid residues in bold indicate positions that differ from the nisin A sequence

Nisin Z, produced by L. lactis NIZO 22,186 (1991) isolated from a dairy product. The first and closest variation to nisin A has 34 amino acids and differs from nisin A only at one position: nisin Z has in position 27 asparagine instead of nisin A’s histidine. This substitution impacts solubility; as a result, it has improved solubility and diffusion (permeation) rate in neutral pH conditions. However, it has minimal effect on pH, thermal stability, or antibacterial activity (it has the same antimicrobial qualities as nisin A). In many L. lactis strains, the nisZ gene (nisin Z biosynthesis gene) is widely distributed, and its producers are very common [73].

Nisin Q is an active bacteriocin called nisin Q was isolated from L. lactis 61 − 14 from a river in Japan in 2003. There are four amino acids differences and two leader peptide sites where the mature nisin Q peptide varies from nisin A, and by three amino acids from nisin Z in the mature peptide. Nisin Q also contains asparagine in position 27 like nisin Z [56].

Nisin U was produced by Streptococcus uberis 42 and up to 82% similarity with nisin A and nisin Z, and 85% similarity with nisin Q was observed. The nisin U producing strain, in addition to being immunogenic against nisin U, displays cross-immunity against nisin A as well. One of the primary causes of bovine mastitis is a kind of bacteria that is primarily present on cows’ lips, skin, and mammary tissue in raw milk [74, 75]. The first known nisin variant from a species other than L. lactis is Nisin U [57]. Considering the close identity between the U and A nisins’ amino acid sequences, the obvious similarities in their spectrum of activity, and the mutual immunogenicity, these peptides seem to have the same heterocyclic ring structure. A notable difference in nisin U is the presence of a proline residue at position 20 instead of asparagine in nisin A, Z, and Q.

Nisin U displays characteristics common to lantibiotics, such as exceptional heat stability, a high level of changed amino acids, and an operon made up of necessary and host cell-specific genes [57].

Nisin F, produced by L. lactis F10 which was isolated from the feces of a freshwater catfish in South Africa (2008), produces an active bacteriocin called nisin F. A fifth variant has been discovered. Its molecular mass is 3457 Da and its isoelectric point is 8.73 (derived produced from the structural gene’s DNA sequence, which codes for the mature peptide). Nisin F differs only at position 27 (H27N) from nisin Z and position 30 (I30V) from nisin A, and has structural similarities with other Lactococcus nisins Z and Q as well as nisin A [58].

Salivaricin D, a new lantibiotic from Streptococcus salivarius 5M6c of the natural flora of the mouth, upper respiratory tract and throat with 34 amino acids with a 3468 Da molecular weight was identified in 2012. This bacterium can be used as a potential biological agent to control Streptococci pathogens as well as a probiotic bacterium. Analysis of its amino acid sequence confirmed the lack of amino acids arginine, tyrosine, alanine, serine and threonine. Since serine and threonine residues in lantibiotics are often dehydrated, the lack of these residues offered more proof that salivaricin D is a lantibiotic [59].

Although the mature peptide of salivaricin D was highly similar to the lantibiotic nisin, it differed from nisin Q’s leader sequence by two amino acids and from nisin A, Z, and F’s leader sequence by five alterations. Salivaricin D is encoded by the slvD gene, as evidenced by the mature peptide’s seven hydroxy amino acids, which match the amount of dehydrated residues. On the other hand, the obtained structure indicated that salivaricin D consists of four thioether bridges consisting of two residues lanthionine (Ala-S-Ala), two residues methyllanthionine (Abu-S-Ala) and two residues Dha and Dhb [59].

Despite the significant similarity of salivaricin D to the nisin, it may not be considered a variant because: (1) The amino acid diversity in its sequence is very high, and (2) It possibly lacks the E ring, and according to nisin variant definition previously mentioned, salivaicin D is a novel nisin-like bacteriocin [76]. Salivaricin D has four cysteines at the identical locations as nisin’s five at positions 7, 11, 19, 26, and 28, with the exception of the residue at position 28, which is substituted by valine. Salivaricin D possesses histidine and glycine at positions 25 and 29, respectively, while nisins A, Z, Q, and F have four serine residues at positions 3, 5, 29, and 33 and five threonine residues at locations 2, 8, 13, 23, and 25. This indicates that salivaricin D likewise contains the four rings A (residues 3 and 7), B (residues 8 and 11), C (residues 13 and 19), and D (residues 23 and 26) seen in nisin A, and only E ring (residues 25 and 28) of the last methyllanthionine structure is absent in salivaricin D and contains four thioether rings. Salivaricin D and nisin differ in their antimicrobial activity (killing) resulting in the absence of an E ring in the C-terminal of salivaricin D may be the cause of its incapacity to inhibit Listeria, Staphylococci, and Enterococci. The fact that salivaricin D is inherently resistant to trypsin sets it apart from other nisin variants [77]. Its hinge region consists of His20, Ile21 and Gln22 [59].

Nisin H, produced by Streptococcus hyointestinalis DPC6484 isolated from the pig intestine. It consists of 31 amino acids with a molecular weight of 3453 Da and has a structure similar to nisin A, which differs in five positions. Nisin H is an intermediate betweenLactococcus nisin A and Streptococci nisin U. The first variant isolated from the intestine is of Streptococci origin. Nisin H is intriguing for a number of reasons: (1) Nisin-like bacteriocin produced by an intestinal strain because the production of bacteriocin gives the producing strain a competitive advantage in the intestinal environment. It also probably helps in signaling to the host, (2) It seems to be an evolutionary link between nisins produced by Lactococcus and Streptococci starins, While it differs from nisin A at five positions, it retains an important feature of nisins produced by Lactococcus strains (the C-terminal three amino acid) that is absent in nisin U, (3) The nisin H gene cluster does not have a nisI gene equivalent [54]. It was demonstrated in the study that horizontal gene transfer may have caused nisin-like gene clusters to transfer between Streptococci [78].

The generating strain’s ability to produce bacteriocin is negatively impacted by the lack of the NisI immune protein. The lack of the nisI gene is the reason why the yield of nisin H from the culture supernatant is lower than that of nisin A. Structurally, nisin H is more similar to Lactococcus nisin due to the presence of three amino acids in the C-terminal (nisin P, U and U2 lack these amino acids). The surprising thing is the existence of an aromatic residue in nisin H, which is the first report of the occurrence of such an amino acid in a natural nisin. An uncommon aspect of nisin is the absence of aromatic residues in their sequence (in engineered nisin derivatives containing aromatic residues, activity microbial reduced) [54].

Nisin O was produced by the predominant human intestinal bacteria Blautia obeum A2-162. There is a notable similarity between the amino acid sequences of nisin U and nisin O. A unique leader peptide sequence and four structural peptides were found in a novel lantibiotic cluster from B. obeum A2-162, which was discovered using screening analysis. Nisin O is the first documentation of a Blautia genus cluster resembling nisin. The interesting point was the sensitivity of B. obeum A2-162 to nisin A, which indicates that its immune system probably needs induction [60].

Nisin P is one of the variants of nisin that was previously discovered in the two Streptococcus species’ genomes, but there was no solid proof of its synthesis. In 2020, it was isolated from Streptococcus agalactiae DPC7040, an isolate from human feces. Nisin P has three amino acids less than nisin A in its C-terminal, and next to nisin U and U2, it is the shortest variant of nisin with 31 amino acids and is not like nisin A in 10 positions [18].

Nisin J, is one of the variants of nisin that produced by Staphylococcus capitis APC 2923 was isolated from the toe web space area with a molecular weight of 3458 Da. It is known as the first Staphylococcus species-isolated nisin and the first nisin resistant to NSR (nisin resistance protein). Nisin J has nine amino acids differences with nisin A, and six of these substitutions (I4K, M17Q, G18T, N20F, M21A, I30G, V33H and K34T) are specific to nisin J and absent from other nisin variant. In addition to being the longest version of nisin found so far, it has one extra amino acid at the C-terminal. Natural forms of nisin have a number of N-terminal modifications, the most common being the Ile 4 substitution. An I4K substitution is present in nisin J and is also present in nisin O₁₋₃, P, U, and U2. The remarkable point about nisin J is the presence of the greatest difference in the center and C-terminal of its sequence, relation to other nisin variants, therefore in the center of nisin J there are four amino acids at positions 17 to 21, which are the same as nisin A is different. These changes include a unique M17Q substitution, other naturally occurring forms of nisin with antimicrobial activity contain a methionine at this position. The G18T change, which was also noted in nisin H, U, U2, P, and O₁₋₃, is an interesting substitution because it seems that the dehydrated residue observed in the G18T and M17Q derivative of nisin Z, to Dhb be changed Unlike nisin A, which contains (polar) asparagine, nisin J has a very hydrophobic phenylalanine residue at position 20 [61].

The end of C-terminal of nisin has been reported to improve both membrane penetration ability and ability to prevent the growth of Gram-negative bacteria. Consequently, nisin J’s longer end may be more strongly adsorbed to the negatively charged cell membrane, increasing membrane insertion and potentially explaining its broader host range [79].

Kunkecin A, a nisin-like peptide, produced by Apilactobacillus kunkeei FF30-6 in 2020, apart from honeybees [80]. It is composed of 39 amino acids and has a molecular mass of 4218 Da. Among the bacteriocins that have been identified, kunkecin A has a molecular mass that is very high [81]. The longest and heaviest nisin-like bacteriocin has been identified. Its amio acids have a significant similarity with the biosynthetic protein nisin A. The first nisin-variant lantibiotic in the Lactobacillus family and the first identified bacteriocin in fructophilic lactic acid bacteria (FLAB) is kunkecin A. With the exception of residues 5 and 33, the locations of the changed residues in kunkecin A were reported to be like to those of nisin A, which were replaced by valine and histidine in kunkecin A, respectively. The kunkecin A also contained the conserved motif sequence (FNLD) of the leader sequence in nisin group lantibiotics [82] series of leaders, with the only difference being that it was changed to glycine in the fourth residue. Structural analysis of the peptide demonstrated that the mono sulfide bond pattern shared by nisin A and kunkecin A is comparable. In kunkecin A, the N-terminal amino acid sequence begins with isoleucine and contains a Dhb at position two, also the comparative analysis of relative molecular masses indicated the presence of six dehydrations in this peptide, on the other hand, kunkecin A contains five mono sulfide bonds (one lanthionine and four three-methyllanthionine) that are also present in nisin A.

Nisin G producing Streptococcus salivarius DPC6487 has been isolated from gut microbiome. The seven amino acids that separate nisin G peptide from nisin A and contains three specific substitutions Ala18, His20 and Ile31, which are unique to nisin G. Its mature form showed 78.95% identity with nisin A, 84.21% with nisin H and 70.18% with salivaricin D. Mutation of a locus at position 21 or a histidine at position 20 in the nisin A hinge region (these two substitutions are naturally present in nisin G) results in reduced biological action directed towards Gram-positive bacteria, as a result because histidine is present at position 20 and the leucine at position 21 of this peptide leads to nisin G’s lower antibacterial activity in comparison to nisin A. Nisin G is more similar to nisin Q with only three differences (A15V, M21L, H27N) [68].

Nisin E, an active lantibiotic discovered in 2023, with a potent activity against Micrococcus luteus was isolated from Streptococcus equinus MDC1 from a dog. There is a significant similarity between nisin E and nisin U. The remarkable and unexpected point was nisin U’s partial suppression of S. equinus MDC1, due to the synthesis of bacteriocin U and having immune genes nmdI and nmdFEG, both strains were expected to develop cross-immunity compared to other producing strains. which can be attributed to the significant differences in their immune proteins, which range from 47.1% to 74.8% identity and 68.4%-85.2% similarity for NmdI/NsuI and NmdF/NsuF [69].

Nisin S, was produced by Ligilactobacillus salivarius P1CEA3 from pig gastrointestinal tract in 2023. This is the first documentation of a nisin variant encoded and generated by the probiotic-potent bacterium L. salivarius. The most closely related nisin variant, nisin F, has 82.35% identity to mature nisin S, a 34 amino acids peptide, and 79.41% identity to nisin A [25]. Two distinct amino acids found in positions T20 and S25 of nisin S are not found in other nisin variants. T20 is located in the molecule’s hinge region, which includes positions 20, 21, and 22. This hinge area is likely essential for nisin’s flexibility and reorientation following its attachment to lipid II, which allows it to pass through the target cell membrane [83].

The presence of histidine at position 29 in the mature peptide rather than serine, as is the case with Lactococcus nisins (A, Z, Q, and F), is another intriguing amino acid difference. Considering the resistance of some strains to nisin and encoding a protease named nisin resistance protein (NSR), which can cleave nisin A at position 29 and lead to a decrease in the antimicrobial activity of the peptide, it is clear that amino acid substitutions at this position protect the peptides against the proteolytic activity of NSR [84, 85]. Nisin S, also referred to as a probiotic, is the first lantibiotic to be identified and produced from Ligilactobacillus sp. L. salivarius, a GRAS bacterium with potential and qualified presumption of safety (QPS) [25].

Through genome mining, two strains of Romboutsia sedimentorum isolated from premium packed and refrigerated beef were found to have rombocin A, a natural variation of nisin. Since rombocin A is projected to have just four lanthionine rings and has nine amino acid residues shorter than nisin A, it can be regarded as a naturally occurring short version of nisin (25 amino acids and 2380 Da). Furthermore, the residues have changed in the ways listed below, mostly in loops C and B:

Pro9 → Ala, Lys12 → Ile, Ala15 → Val, Leu16 → Ile, and Gly18 → Thr. It’s interesting to note that rombocin A has alanine and isoleucine in place of the two residues Pro9 and Lys12, which are present in practically all natural forms of nisin. Furthermore, rombocin A has a shorter “hinge region,” which is essential for nisin’s function. Despite these modifications, rombocin A shares a dual mode of action with nisin A. Under a range of pH values (2, 4, 7, and 10) and temperatures (up to 10 h at temperatures between 22 and 50 ◦C), rombocin A is stable. An additional benefit over nisin is that this material exhibits resistance to enzymatic destruction by trypsin, chymotrypsin, and proteinase K. Since the majority of degradations that result in loss of activity take place in the C-terminal region, the absence of this region may be the cause of rombocin’s proteolytic stability. Another possible explanation for rombocin A’s stability is the existence of a Dhb in the C loop [26, 86].

Structure–activity relationship (SAR) in nisin variants

A comparative analysis of nisin variants reveals that specific structural domains dictate their functional properties. The N-terminal region (Rings A, B, and C) is highly conserved across most variants, as it is crucial for binding to the lipid II precursor, the primary cellular target [87–89]. Variations in this region often lead to a loss of activity [43]. In contrast, the flexible “hinge region” (residues 20–22 in nisin A) determines the peptide’s ability to undergo the conformational changes required for pore formation [83, 90]. Variants like nisin Z (His27Asn) show that modifications in the C-terminal region can significantly alter solubility and diffusion rates without compromising lipid II binding [55, 73]. Furthermore, the length and charge of the C-terminal tail (as seen in nisin J and kunkecin A) appear to modulate the initial electrostatic attraction to the bacterial membrane, thereby influencing the host range, particularly against Gram-negative bacteria or strains with modified cell walls [61, 64, 79].

Nisin bioengineering and derivatives

Since nisin is ribosomally synthesized, bioengineering techniques can be employed to create its variations. Nisin bioengineering started more than 20 years ago, but it hasn’t been successful until recently thanks to researchers that have a better grasp of lantibiotic biorecognition and the capacity to carry out bioengineering on a bigger scale [91]. Recent advancements have resulted in the discovery of modifications or mutations that markedly enhance particular action against target cells [92, 93]. In order to increase the production of bioactive peptides, nisin has undergone significant engineering. This approach has given us knowledge regarding substitutions and alterations to amino acids (Table 2). Since the identification and disclosure of nisin’s biological principles in 1992 [94], nisin has been bioengineered by amino acid replacement, yielding several variants with significant utility [17, 93]. Interestingly, the majority of these enhanced variants had different amino acid compositions at the peptide hinge region. It is believed that the hinge region plays a crucial role in providing structural flexibility between the N- and C-terminal functional domains of nisin and connecting them [87, 95]. The hinge region and one or two of its residues have been altered to create the majority of nisin derivatives, which has produced encouraging outcomes in terms of enhanced antibacterial activity. All three of the hinge region’s researchers conducted simultaneous randomization [83]. Using site-directed mutagenesis, the link between nisin’s structure and function was investigated. According to certain reports, lanthionine and three-methyllanthionine residues, as well as dehydrated residues, are required for nisin action [94, 96]. Additionally, the leader peptide sequence’s amino acid substitution was investigated [97]. The findings indicated that while the proline residue in position − 2 was not significant for biosynthesis, the conserved residues in positions − 18 + 0–15 in nisin variants were. Despite being extremely preserved. Nisin is commonly used as a food preservative, but its low stability at physiological pHs and poor solubility make it unsuitable for use as an antibiotic. Since all lantibiotics are genetically encoded, new variants can be produced by manipulating the corresponding gene [83]. Although the identification of nisin with increased specific activity has received particular attention recently, the engineering of nisin bioengineering has the potential to produce peptides with improved stability and solubility [24].

Table 2.

Derivatives characteristics of nisin bioengineering

Changed positions	Nisin variant	Impacts	References
K12A	Nisin A	Increase antimicrobial activity against Staphylococcus, Enterococcus, Bacillus, and Streptococcus spp.	[99]
SVA and NAK	Nisin A	Increase antimicrobial activity against Listeria monocytogenes	[91]
AAK, NAI and SLS	Nisin A	Increase antimicrobial activity against S. aureus, S. agalactiae, L. lactis, and Mycobacterium smegmatis	[83]
Randomizing the positions of the A and B rings and producing truncated nisin (1–22)		Ring A mutants: Enhanced activity. Ring B mutants: Abolished activity. Truncated nisin (1–22): Lost pore-forming ability but inhibited Bacillus spore outgrowth	[43]
Randomizing the A ring and the C-terminal in truncated (1–22) nisin	Nisin A	Mutations at positions − 16, -18, and − 19 (e.g., L-16 A, F-18 H) enhanced nisin production levels. Mutation of the FNLD box abolished post-translational modifications	[82]
M17Q and G18T	Nisin A	S5T mutation: Decreased activity against Micrococcus flavus and Streptococcus thermophilus. M17Q/G18T mutation: Active against Bacillus cereus and M. flavus	[94]
M21A	Nisin A	Increase antimicrobial activity against L. lactis and L. monocytogenes	[100]
M21Y	Nisin A	Decrease antimicrobial activity against L. lactis
Introduction of aromatic residues at any position in the hinge region	Nisin A	Negative effect on activity against L. lactis due to reduced hinge flexibility
N20H, M21L and K22Q	Nisin A	Decrease antimicrobial activity against L. lactis
Introduction of leucine/isoleucine in hinge region		Increase antimicrobial activity against S. aureus and L. monocytogenes
M21V	Nisin A	Increase antimicrobial activity against L. monocytogenes and S. aureus
M20K and N20K	Nisin Z	Gained activity against Gram-negative bacteria (Shigella, Pseudomonas, Salmonella) and increased solubility at pH 8	[90]
N20Q and M21G	Nisin Z	Enhanced thermal and pH stability, despite decreased activity against M. flavus and Streptococcus thermophilus
S29G	Nisin A	Enhanced efficacy against S. aureus and Gram-negative bacteria (E. coli, Salmonella, Cronobacter)	[101]
S29A, S29E and S29D	Nisin A	Increase activity against drug-resistant Gram-positives and Gram-negative pathogens (E. coli, Salmonella, Cronobacter)
DeltaN20/DeltaM21	Nisin Z	Abolished pore-forming ability but retained lipid II binding (inhibition of cell wall synthesis)	[87]
S3T	Nisin A	Reduced lipid II binding affinity and impaired pore formation
Removing the two C-terminal residues of nisin A (nisin (1–32))	Nisin A	Retained potent antimicrobial activity against Gram-positive organisms (comparable to wild-type)	[102]
H31K and N27K	Nisin Z	Significantly increased solubility at neutral pH (pH 7) and improved chemical stability (N27K), while retaining activity against M. flavus and S. thermophilus	[24]
K22G, K22E, K22H and N20F-M21L-K22Q	Nisin A	Predicted decrease in solubility at physiological pH (based on molecular dynamics simulations)	[72]
N20K+−M21K+, N20R and N20R-K22R	Nisin A	Predicted improvement in solubility at physiological pH (potential for clinical application)
S29P	Nisin A	Retained general bioactivity and exhibited a 20-fold increase in specific activity against strains expressing the nisin resistance protein (NSR)	[84]
M17Q, T2L and HTK	Nisin A	Enhanced activity against mastitis-associated Staphylococcus spp., while displaying selectively reduced activity against dairy starter Lactococcus strains	[103]

Heterologous expression and production challenges

While site-directed mutagenesis is a powerful tool for generating novel nisin derivatives, their practical application hinges on efficient and scalable production. Native nisin-producing strains often have complex nutritional requirements or low yields. Consequently, research has expanded to heterologous expression platforms. Food-grade hosts like L. lactis are preferred for direct application in foods, while surrogate hosts such as B. subtilis have been explored to utilize their superior secretion capacities, although this requires the coordinated expression of the entire lan gene cluster (e.g., nisBTCIPRKFEG) to ensure correct post-translational modification [46, 53]. However, industrial production faces regulatory and metabolic bottlenecks. The introduction of large gene clusters can impose a significant metabolic burden on the host, as it must supply precursor amino acids (Ser, Thr, Cys) for both cell growth and high-level peptide synthesis [16, 92]. To overcome these limitations and achieve production optimization, studies have focused on refining fermentation conditions—such as pH control, carbon source selection, and temperature regulation—to maximize yield [31, 32, 34]. Furthermore, addressing scalability challenges involves developing robust downstream processing and rapid purification methods to ensure the economic viability of engineered nisin variants [33, 98].

Mode of action of nisin

Nisin has minimal effect on Gram-negative bacteria and fungus, but exhibits a broad range of inhibitory and antibacterial activity against Gram-positive bacteria and their spore form [104]. Because their cell walls are less permeable than those of Gram-positive bacteria, it can be claimed that Gram-negative bacteria are resistant to nisin [105, 106]. The membranes of Gram-positive bacteria are rich in anionic lipids, and anionic liposomes and positively charged nisin form a strong bond very quickly. Nisin has a dual effect on vegetative cells, killing them while also controlling and inhibiting the growth of new ones. This depends on a number of factors, including the concentration of nisin, the number of bacteria, the environment conditions, and the physiological state of the bacteria. A sporostatic function is spore growth [105, 107].

Bacteriocin mechanism against vegetative cells

One or both of the following mechanisms, as illustrated in (Fig. 3), mediate the antibacterial effect of bacteriocins: (1) suppression of the production of the cell wall; (2) development of pores. Certain bacteriocins can work through both of these methods. According to Jordan et al. [108], this capability stops bacteriocin resistance from developing. These bacteriocins, which include nisin, pediocin, lacticin 3147, epidermin, and gallidermin, have high antimicrobial activity against the microorganisms they target [109]. Bacteriocins produced by LABs can exhibit one or both of the following modes of antimicrobial action: they can inhibit the synthesis of the cell wall by binding to lipid II, a necessary precursor of peptidoglycan. This sequesters the lipid II, thereby stopping the synthesis of the cell wall and causing holes to form in the membrane, which ultimately cause the cell to die [88]. The following are the processes underlying the suppression of pore development and cell wall biosynthesis: Nisin disrupts the cytoplasmic membrane by binding to lipid II (and other anionic phospholipids) through the N-terminal thioether rings (A and B) from the pyrophosphate portion and by forming up to seven hydrogen bonds [89–111].

Fig. 3.

Nisin’s mode of action against bacteria. (a) Nisin interacts with the bacterial cell membrane and (b) specifically targets the pyrophosphate group ofمlipid II through its N-terminal. (c) The C-terminal inserts into the membrane to form a pore. (d) The nisin-lipid II complex pore has a stoichiometry of eight nisin molecules to four lipid II molecules [112]

Other receptors on Gram-positive bacteria that bacteriocins bind to include the mannase PTS system, zn-dependent metallopeptidases, the maltose ABC transporter, and the previously mentioned undecaprenyl pyrophosphate phosphatase. Lipid II is also an important receptor for certain bacteriocins, such as nisin [113]. According to Egan et al. [109], these receptors are thought to be crucial in regulating the action of bacteriocins. As previously noted, nisin’s antimicrobial activity is mediated by pore creation as well as suppression of cell wall production [114]. Two structural domains at the N-terminal (containing three lanthionine rings, A, B, and C) and C-terminal (containing two lanthionine rings, E and D), as well as a flexible hinge region (Asn20, Met21, and Lys22), work together to provide this antibacterial activity form a bond. It has been observed that rings A, B, and C create a “cage-like” compartment that inhibits the formation of the cell wall and enables binding to lipid II pyrophosphate enhances the C-terminal’s capacity to create membrane holes, which in turn raises the pace at which cytoplasmic ions and other solutes move [87]. Combining the two activities results in minimal inhibitory values against sensitive bacteria in the nanomolar range [115]. However, because of the benefits of nisin, they are GRAS and ensure food safety and shelf life., additionally, when nisin is combined with other treatments like heat, high pressure, and organic compounds as part of the preservative and the product packaging, it shows synergistic effects. The same features seem interesting to control spore bacteria, which are common contaminants of food products and the cause of food-borne diseases and spoiling. Because spores are resistant to heat and chemicals when they are latent, it is crucial to eliminate them from food because they can produce harmful toxins and jeopardize food safety [109]. Gram-positive Clostridium and Bacillus species undergo sporulation to produce inactive (dormant) metabolic spores. This mechanism, which entails a complicated series of enzymatic activities, happens in response to cell starvation stress in the absence of nutrients [116].

Bacteriocin–spore interactions

Generally speaking, the action of bacteriocins against vegetative cells is more understood than the interactions between bacteriocins and spores. Spores that are dormant can sprout in a number of ways, including heat. Heat is assumed to increase nutrient accessibility through the germination of receptors (GR), which ultimately triggers the activation of latent spores. Spores are obligated to germinate once GRs and nutrients link together. The process involves multiple stages. Firstly, the release of H⁺, K⁺, and Na⁺ ions occurs, which is followed by the release of Ca²⁺-DPA. DPA is involved in preserving the dormancy of spores and their resistance to agents that damage DNA. It typically binds to divalent cations such as Ca²⁺ in a 1:1 ratio. Actually, the second stage of germination begins with the release of Ca²⁺-DPA. At this point, the shell’s crust has broken down, allowing the cell wall to swell and take in water. The second stage comes to a conclusion and the growth stage begins when the water content increases. At this point, bacteriocins are released to combat latent spores, which were dormant up until this point. They prevent growth and lower the proportion of spore survivors within the germinating spore population [117]. Nisin had no effect on the germination process, according to previous reports about Bacillus anthracis [118, 119], Bacillus licheniformis [120], Clostridium difficile [121], and Clostridium perfringens [122]. This was because germination speed and germination process were not observed to change, initiate, or exhibit inhibition when nisin was present. Interestingly, Aouadhi et al. [123] found that for Clostridium botulinum spores, a concentration of 25 µg/ml increased progenitor activity, resulting in a doubling of the spore germination rate. Conversely, for Bacillus sporothermodurans spores, a concentration of 125 µg/ml. Nisin in the medium decreased spore germination rate.

Safety and applications of nisin

Bacteriocins have minimal toxicity and hemolytic action, according to safety studies [124]. Numerous in vitro and in vivo investigations (on animals and living creatures) have used animal models and non-cancerous mammalian cells to establish the minimal toxicity of nisin [125]. The EFSA panel on food additives, flavorings, processing aids, and materials in contact with food assessed the safety of nisin (E 234) as a food additive in 2006. The panel confirmed an acceptable daily intake (ADI) of 0.13 mg/kg body weight, which is the same amount that the Scientific Committee on Food (SCF) had previously established. In its current review, the panel plans to take into account data from new subchronic toxicity research in addition to the studies that were previously assessed by the European Food Safety Authority (EFSA) in 2006. Mice given nisin A for 90 days showed no negative side effects in a study that looked at oral toxicity with repeated dosages. The largest dose evaluated in this study, 225 mg of nisin A/kg of body weight per day, was found to be the no-observed-adverse-effect level (NOAEL). With this NOAEL, the acceptable daily intake (ADI) for nisin (E 234) was determined to be 1 mg of nisin A/kg of body weight per day, with a default safety factor of 200 applied to account for the conversion from subchronic to chronic exposure as well as interspecies and intraspecies variability [126]. Nisin is currently the only bacteriocin widely approved as a food additive (E234) in over 50 countries [16, 110]. Its application has expanded beyond simple direct addition to more sophisticated methods. For instance, nisin is increasingly utilized in bioactive formulations and packaging strategies to prevent surface contamination of food products [8, 127]. Furthermore, the combination of nisin with other hurdles, such as bioactive molecules or heat treatment, has demonstrated significant synergistic effects, allowing for lower nisin concentrations while maintaining food safety [22, 123]. Despite these advantages, its efficacy can be limited in complex food matrices due to interaction with fat particles or proteolytic degradation, highlighting the need for engineered derivatives or encapsulation strategies [91]. Despite being a GRAS drug, nisin should only be used in certain amounts and never in excess of the recommended dosage, depending on the kind of food product it is added to. Proteins and bioactive peptides that can be broken down in the human digestive system make up nisin, a natural bio-preservative that is generally safe to eat within certain ranges. It has a particular antibacterial activity that prevents the growth of harmful and spoilage bacteria in food when it takes the form of the bioactive peptide nisin. Different countries have different nisin usage restrictions. For instance, the FDA has set a daily maximum of 10,000 IU of pure nisin, whereas the Joint FAO/WHO Expert Committee on Food Additives (JECFA) in the US suggests a daily limit of 60 mg/70 kg of body weight. However, there are no upper restrictions on the amount of nisin that can be used in cheese products in Australia, France, or the UK [127]. The peptide is safe, sensitive to digestive proteases (alpha-chymotrypsin can hydrolyze it into amino acids in the colon), and does not change the organoleptic characteristics of food. These factors have demonstrated that this material works well as a natural food preservative. Nisin is primarily used in food as a natural preservative, but studies have shown that it can also be used therapeutically, particularly to treat immunocompromised patients’ intestinal infections, stomach ulcers, and atopic dermatitis. Furthermore, research has demonstrated that nisin had antibacterial properties in regulating Staphylococcus aureus-induced respiratory infections in a mouse. Nisin is currently used in veterinary medicine as a disinfectant against Streptococcus and Staphylococcus species, which are the pathogens that cause mastitis [16]. The majority of studies show that nisin, both by itself and in conjunction with antibiotics, has a strong inhibitory capacity against microorganisms that are resistant to these drugs. Nisin can suppress bacterial pathogens linked to cancer, restrict the growth of bacteria that cause infections, and control the makeup of the gut and oral microbiome, according to a number of studies. It has been established that nisin has anti-tumor properties and can improve the therapeutic efficacy of chemotherapy medications. Additionally, there is strong evidence of its anti-inflammatory and immunomodulatory properties. Modern biotechnological techniques that enhance nisin’s stability and functional qualities under a range of physiological situations can effectively overcome the issues that have prevented its widespread use in clinical practice. It is important to note that 300–400 µg of nisin/ml can have a contraceptive effect on humans [125].

Future outlook

While the diversity of natural nisin variants offers a rich genetic resource, several challenges remain for their widespread industrial and clinical adoption. Future research must focus on three key areas: (1) Cost-effective production: Optimizing heterologous expression systems to increase yields of novel variants [16, 46]. (2) Stability engineering: Developing variants that resist proteolytic degradation in the human gastrointestinal tract or complex food matrices [24, 77]. (3) Resistance management: Although nisin resistance is rare, the emergence of nisinase-producing strains necessitates the design of “resistance-proof” variants, possibly through modifications in the C-terminal region or ring structures [84, 85]. Addressing these challenges through synthetic biology and protein engineering will be pivotal in transitioning nisin from a food preservative to a viable clinical therapeutic [11, 125].

Conclusions

Natural bacteriocin nisin exhibits potent antibacterial action against a wide range of microorganisms. Because of its special qualities, which include its amphiphilic nature, low cytotoxicity, safety for human consumption, and decreased chance of resistance development, it is a promising antimicrobial agent for the management of pathogenic microbes in the food industry. However, nisin has become a desirable alternative antimicrobial agent due to its capacity to damage bacterial cell membrane integrity, resulting in cell death, as well as its biological activity against their spores and the emergence of microbial resistance to common antibiotics. Unfortunately, its applicability has been limited due to its low solubility, poor stability, and short half-life at neutral pH. Studies have shown that using bioengineering techniques, it is possible to create various recombinant nisin derivatives with enhanced biological activity against target bacteria, improved solubility at neutral pH, and improved and increased overall stability of the peptide. Based on the advancements and achievements made in the last few years, it appears that more effective nisin derivatives with much better uses in food and medicine will be produced in the years to come.

Author contributions

Negin Ghazanfari: Writing – original draft, Investigation. Farideh Tabatabaei Yazdi and Seyed Ali Mortazavi: Writing – review & editing, Supervision. Ali Javadmanesh: Writing – review & editing.

Data availability

No datasets were generated or analysed during the current study.

Declarations

Ethics approval and consent to participate

The authors declare the studies does not involve in humans and animals.

Competing interests

The authors declare no competing interests.