Biosynthesis and Production of Class II Bacteriocins of Food-Associated Lactic Acid Bacteria

: Bacteriocins are ribosomally synthesized peptides made by bacteria that inhibit the growth of similar or closely related bacterial strains. Class II bacteriocins are a class of bacteriocins that are heat-resistant and do not undergo extensive posttranslational modiﬁcation. In lactic acid bacteria (LAB), class II bacteriocins are widely distributed, and some of them have been successfully applied as food preservatives or antibiotic alternatives. Class II bacteriocins can be further divided into four subcategories. In the same subcategory, variations were observed in terms of amino acid identity, peptide length, pI, etc. The production of class II bacteriocin is controlled by a dedicated gene cluster located in the plasmid or chromosome. Besides the pre-bacteriocin encoding gene, the gene cluster generally includes various combinations of immunity, transportation, and regulatory genes. Among class II bacteriocin-producing LAB, some strains/species showed low yield. A multitude of fermentation factors including medium composition, temperature, and pH have a strong inﬂuence on bacteriocin production which is usually strain-speciﬁc. Consequently, scientists are motivated to develop high-yielding strains through the genetic engineering approach. Thus, this review aims to present and discuss the distribution, sequence characteristics, as well as biosynthesis of class II bacteriocins of LAB. Moreover, the integration of modern biotechnology and genetics with conventional fermentation technology to improve bacteriocin production will also be discussed in this review.


Introduction
Lactic acid bacteria (LAB) constitute a ubiquitous bacterial group that is widespread in niches of fermented food and gastrointestinal tracts of humans and many animals [1].LAB are especially known for their ability to produce lactic acid as the main end-product.These microorganisms also possess the ability to synthesize a wide variety of bioactive metabolites, belonging to different classes of chemicals including diacetyl, hydrogen peroxide, antibiotics, and bacteriocins.Bacteriocins are ribosomally synthesized, extracellularly released bioactive peptides which have a bactericidal or bacteriostatic effect on other closely related species [2].Bacteriocins from LAB have been used as food preservatives due to their heat stability and safety.Nisin is the most famous and best-studied bacteriocin, and it has high antibacterial activity against a wide range of Gram-positive bacteria.Nisin has received considerable attention in the food industry because it is the only purified bacteriocin approved for food use by the FDA and EU.However, several restrictions to the efficacy of nisin limit its range of practical applications.For instance, nisin has weak inhibition activity against Gram-negative bacteria, and its biological activity is reduced at an elevated pH.Moreover, spontaneous nisin resistance may occur in target bacteria.

Classification of LAB Bacteriocins
There are several classification schemes based on the biochemical and structural features of LAB bacteriocins.In 1993, Klaenhammer et al. suggested a classification system that divides LAB bacteriocins into four groups [6].The class I bacteriocins are lantibiotics, which are small membrane-active peptides (<5 kDa) containing uncommon amino acids such as lanthionine, β-methyl lanthionine, and dehydrated residues.Class II includes small heatstable peptides without lanthionine residues.Class III comprises large heat-labile proteins, while class IV is composed of large peptides complexed with carbohydrates or lipids.Cotter et al. (2005) performed a thorough modification of Klaenhammer's classification scheme and they grouped bacteriocins into just two categories: lantibiotics (class I) and nonlanthionine-containing bacteriocins (class II).They also suggested that the high-molecular weight thermolabile peptides (previously class III) should be designated as "bacteriolysins", and the previous class IV should be extinguished [7].Cotter's classification scheme was broadly accepted for a long time, and has continuously been modified by researchers, since the repertoire of bacteriocins is rapidly growing [8][9][10].
In general, class I bacteriocins are produced as precursor peptides that undergo extensive post-translational modifications.The mature peptides contain unusual amino acids, such as 2,3-didehydroalanine, D-alanine, and 2,3-didehydrobutyrine, as well as characteristic lanthionine rings that result from thioether formation between the side chains of cysteine and serine or threonine.Class Ia bacteriocins, which include nisin, consist of cationic and hydrophobic peptides that form pores in target membranes and have a flexible structure compared to the more rigid class Ib.Class Ib bacteriocins, which are globular peptides, have no net charge or a net negative charge.Class Ic is a growing class of two-component lantibiotic systems that utilize two peptides that are each posttranslationally modified to an active form and act in synergy to provide antibacterial activity.More detailed information on the structure and biosynthesis of lantibiotics is presented in previous reviews [11][12][13].
Class II bacteriocins are a class of small non-lanthionine-containing peptides.Unlike the lantibiotics described above, class II bacteriocins are less modified; a disulfide bridge and some N-terminal modifications are known to exist in some class II bacteriocins.Class II bacteriocins are divided into four subclasses, IIa, IIb, IIc, and IId.Subclass IIa bacteriocins are the most thoroughly studied.They are also known as pediocin-like peptides with antilisterial activity [14].The class IIb bacteriocins (two-peptide bacteriocins) require two different peptides for optimal activity [15].Class IIc bacteriocins are referred to as circular bacteriocins whose ring structure is formed in a head-to-end fashion [16].Class IId bacteriocins are categorized as bacteriocins that have no significant sequence similarity to the other class II bacteriocins [17].

Sequence Properties of Identified Class II Bacteriocins
Class II is the largest group of bacteriocins and includes a range of small peptides with antimicrobial activity.Great efforts have been made to identify the sequence of class II bacteriocins that LAB can produce and to further recognize the bases of their antibacterial activity.Nonetheless, sequence diversification can be found among class II bacteriocins.On the one hand, some motifs are conserved throughout evolution, such as the "YGNGV" motif in class IIa and the "GXXXG-like" motif in class IIb (Table 1).On the other hand, class IIc bacteriocins have poor sequence similarity but share similar structural patterns of globular arrangement of four or five helices (Table 1 [18]).Gray letters represent leader sequence.
It has been shown that the gene content and organization of bacteriocin are conserved among phylogenetically different bacteria.For instance, most class II bacteriocin precursors have the double-glycine motif in their leader sequence.This motif serves as a processing site by dedicated downstream transport machinery to cleave off the leader peptide concomitant with transport [103].Thus, putative bacteriocins can be identified by comparing new genome sequences against well-characterized biosynthetic genes or gene clusters.This "genome mining" approach allows for the discovery of potentially novel bacteriocins in a complete culture-independent fashion, with the potential to reduce the rediscovery rate of known molecules [104].Various powerful tools with broad databases have been created for the automated screening of bacteriocin gene clusters.BAGEL4 (http://bagel4.molgenrug.nl/)(accessed on 15 January 2022) is a versatile fast genome-mining tool valid for modified-and non-modified bacteriocins [105].BAGEL4 uses DNA nucleotide sequences as the input, which are analyzed in parallel via two different approaches; one is the context of bacteriocin-or RiPP (ribosomally synthesized and post-translationally modified peptides) gene-based mining, the other is the precursor (structural gene)-based mining directly by Glimmer, which increases the success rate and lowers the need for the manual evaluation of results [105].By genome mining of 1011 LAB strains (including WGS and complete genomes) of 82 species using BAGEL4, we shed light on the diversity of food-associated LAB that processes biosynthetic genes of class II bacteriocin at the species level (Table 3, Supplementary Table S1).
The genus Enterococcus is the most controversial group of LAB that comprises both pathogenic and commensal microorganisms; some strains of Enterococcus spp.are highly adapted to several food systems, and they are also involved in the fermentation activity of traditionally manufactured cheese, dry sausages, and olives [110].The production of bacteriocins by enterococci is well documented.Our analysis shows that Enterococcus faecium and E. faecalis are predominant species that producing class II bacteriocins, which is in accordance with previous reports [111].E. durans and E. mundtii also have great potential for class II bacteriocin screening.More than 50% of investigated strains have at least one bacteriocin encoding genes (Table 3).

Lactobacilli Group
The genus Lactobacillus used to comprise over 200 species, making it the largest and most diverse genus of LAB [112].In 2020, a taxonomic reorganization of the lactic acid bacteria reclassified the genus Lactobacillus into 25 genera.The lactobacilli group occupies a variety of niches, including milk and plant surfaces, as well as the gastrointestinal tract of humans and animals [113].Lactobacillus spp.has been deployed and studied extensively as fermentation starter cultures and as probiotics, of which bacteriocin production has been considered an important trait.However, the ability to produce class II bacteriocins varied among different species.Notably, almost all L. acidophilus strains contain IIa, IIb and IId bacteriocins.L. casei, L. paracasei and L. rhamnosus stand out as rich sources of IIa and IId bacteriocin.

Lactococci
Due to their particular ability to ferment lactose, members of the genus Lactococcus are widely used as starter cultures in the dairy industry.Some strains of lactococci of human and milk origin are reported to have probiotic properties [114].This genus is well known for its ability to produce class I bacteriocin nisin.Our analysis showed that some species also have considerable potential for class II bacteriocin.Specifically, 85% of tested Lc. lactis subsp.cremoris strains and 50% of tested Lc. lactis subsp.lactis strains harbor class IId bacteriocin.Class IIa and IId bacteriocins were found in some Lc.piscium and Lc.raffinolactis strains.

Leuconostoc Spp.
The genus Leuconostoc naturally exists in vegetables and some fermented dairy products [115].Some species of this genus were considered major microorganisms responsible for food spoilage [116].Class II bacteriocin genes were mainly detected in L. gelidum, L. lactis, and L. mesenteroides, of which the class IId was the dominant subgroup (Table 3).The genus Pediococcus has a negative role in the spoilage of beer/wine and a positive role in many fermented foods.Most species of this genus are used in the food industry as probiotic products and starter cultures for fermentation [117].

Pediococci
The genus Pediococcus consists of eight species and various species and strains differ in tolerance to oxygen, pH, temperature, and NaCl [118].P. acidilactici and P. pentosaceus take place in food fermentations either as indigenous microflora or in starters, and both have been used in natural and controlled fermentations of vegetables and sausages.Studies have shown that non-starter and adjunct Pediococcus spp.impart desirable attributes to cheese [119].P. pentosaceus, P. damnosus, and P. acidilactici are major species of this genus that are capable of producing class II bacteriocins, especially pediocin and other class IIa bacteriocins.

Others
Streptococcus thermophilus is the only streptococcal species widely used in food fermentations, especially for yogurt manufacturing [120].Several S. thermophiles strains can produce thermophilin 13, a two-peptide class IIb bacteriocin.Most S. thermophiles strains contain genes encoding bacteriocin-like peptide (blp), which was also frequently detected in other pathogenic streptococci strains [121].The genus Oenococcus plays an important role in wine fermentation, while the species Weissella have been described to be associated with vegetable fermentations [122].However, no class II bacteriocin encoding genes were detected in these two genera.

IIa
Class IIa bacteriocin is first ribosomally synthesized as a prebacteriocin, which contains an N-terminal leader sequence to keep the peptide inactive (Figure 1).The leaders contain 15 to 30 residues, most of which are featured for the double-glycine residues upstream of the cleavage site.The leader is believed to serve as a signal sequence for the processing and secretion of bacteriocins by a dedicated system comprising an ABC transporter and an accessory protein.ABC-transporter protein contains the C-terminal ATP-binding domain and the N-terminal transmembrane domain embedded in the membrane bilayer.The Nterminal region can cleave the leader peptide at the double-glycine motif.The binding of prebacteriocin with N-terminal proteolytic domain triggers the ATP hydrolysis and subsequent conformational changes of the transporter, resulting in leader cleavage and translocation of the mature bacteriocin across the membrane [14].The accessory proteins are postulated to facilitate the membrane translocation and/or help in the processing of the leader peptide [14].For some class IIa bacteriocins, the accessory protein ensures the formation of correct disulfide bond formation [123].However, not all class IIa bacteriocins are transported via ABC-transporter.Some bacteriocins including enterocin P, bacteriocin 31, enterocin SE-K4 lack the double-glycine motif in their leaders and are exported by sec-dependent translocation system [20,29,124].These bacteriocins have a hydrophobic N-terminal sec-dependent leader to direct the secretion of the prebacteriocins.The leader was removed by a signal peptidase during translocation.The synthesis of class IIa bacteriocins is typically regulated by a quorum sensing (QS) system that consists of three components, an inducing peptide, a membrane-associated histidine protein kinase (HPK) and a cytoplasmic response regulator (RR).The inducing peptide is initially synthesized as a prepeptide with N-terminal leader sequence, which is cleaved upon secretion by the ABC-transporter.The concentration of inducer peptide increased along with cell growth.An excess in inducer peptide concentration activates the three-component system by triggering the autophosphorylation of HPK, which transfers a phosphate group to its cognate RR.The phosphorylated RR acts as a transcriptional activator and activates the expression of biosynthetic gene clusters (Figure 1).Moreover, environmental parameters may influence the production of class IIa bacteriocin by acting on the bacteriocin regulatory system or affecting the binding of the induction peptide to HPK [125].
The bacteriocin-producing bacteria avoid killing by their own bacteriocins through The synthesis of class IIa bacteriocins is typically regulated by a quorum sensing (QS) system that consists of three components, an inducing peptide, a membrane-associated histidine protein kinase (HPK) and a cytoplasmic response regulator (RR).The inducing peptide is initially synthesized as a prepeptide with N-terminal leader sequence, which is cleaved upon secretion by the ABC-transporter.The concentration of inducer peptide increased along with cell growth.An excess in inducer peptide concentration activates the three-component system by triggering the autophosphorylation of HPK, which transfers a phosphate group to its cognate RR.The phosphorylated RR acts as a transcriptional activator and activates the expression of biosynthetic gene clusters (Figure 1).Moreover, environmental parameters may influence the production of class IIa bacteriocin by acting on the bacteriocin regulatory system or affecting the binding of the induction peptide to HPK [125].
The bacteriocin-producing bacteria avoid killing by their own bacteriocins through the co-expression of immunity proteins.Immunity proteins for the class IIa bacteriocins range from 81 to 115 amino acids in length and display substantial variation in their sequences.The C-terminal region is involved in specific recognition of their related bacteriocins.However, "cross-immunity" against other class IIa bacteriocins was observed [126].The immunity protein folds into a globular protein in an aqueous solution and contains an antiparallel four-helix bundle [127].There are currently two models being proposed regarding the mechanism of immunity protein: (i) the immunity protein directly interacts with the bacteriocin to obstruct pore formation; (ii) the immunity protein binds to the cytoplasmic side of the receptor and blocks the receptor's ability to interact with the bacteriocin (Figure 1).Although direct evidence of contact between the immunity protein and bacteriocin has not been obtained, there are experimental data to support the first model.The expression of MunC protein (enterocin CRL35 immunity protein) in E. coli is sufficient to prevent the lethal effects of the hybrid suicide probe EtpM-enterocin CRL35.E. coli is naturally insensitive to enterocin CRL35, since it does not express the receptor.These results prove that the immunity protein MunC can protect bacterial cells in the absence of the receptor [27].The second model of "indirect immunity protein and bacteriocin binding" was recently proved experimentally.When the bacterocin targets the membrane from the outside, it gets locked onto the receptor by its immunity protein by forming a ternary complex.For pediocin PA-1, both IIC and IID components of the man-PTS play an important role in the specific recognition between the bacteriocin-receptor complex and the immunity protein PedB [128].
Production of class IIa bacteriocins is often associated with the presence of a plasmid.In some cases, the biosynthetic gene clusters could be located in the chromosome, as exemplified by enteriocin A, divercin V41, sakacin P and canobacteriocin B2, etc.The genetic organization of this class shows considerable conservation.The gene cluster encoding class IIa bacteriocins usually also contain an operon that encodes ABC-transporters and their accessory proteins.The bacteriocin structural genes are generally located in a small operon with the immunity genes, with the exception of divercin V41, mundicin KS and mundicin L (Figure 2).For most class IIa bacteriocins, three genes encoding the regulatory system are located in the same operon, in which the two genes encoding HPK and RR follow the gene encoding the inducing peptide (Figure 2).Intriguingly, the sakacin G gene cluster contains duplicated structural genes skgA1 and skgA2 that encode essentially identical bacteriocin.The leader sequence of SkgA1 and SkgA2 differed only by three residues [39].with the immunity genes, with the exception of divercin V41, mundicin KS and mundicin L (Figure 2).For most class IIa bacteriocins, three genes encoding the regulatory system are located in the same operon, in which the two genes encoding HPK and RR follow the gene encoding the inducing peptide (Figure 2).Intriguingly, the sakacin G gene cluster contains duplicated structural genes skgA1 and skgA2 that encode essentially identical bacteriocin.The leader sequence of SkgA1 and SkgA2 differed only by three residues [39].

IIb
Similar to some class IIa bacteriocins, class IIb bacteriocins are initially synthesized as precursor peptides containing N-terminal extensions (leader peptides) which are cleaved off during maturation.All class IIb bacteriocins identified so far contain a dou-

IIb
Similar to some class IIa bacteriocins, class IIb bacteriocins are initially synthesized as precursor peptides containing N-terminal extensions (leader peptides) which are cleaved off during maturation.All class IIb bacteriocins identified so far contain a double-glycinetype leader.The ATP-binding cassette (ABC) transporter and an accessory protein lead to the cleavage of inactive pre-peptide with the concomitant export of the mature bacteriocin across the cytoplasmic membrane.The accessory protein may be involved in immunity against the bacteriocin or required for secretion of the bacteriocin.However, for some twopeptide bacteriocins such as sakacin T, the processing and secretion are solely dependent on the ABC-transporter since the gene encoding the accessory protein is absent [129].
The production of class IIb bacteriocins was commonly regulated by a three-component regulatory system.The inducing peptide acts as an indicator of the cell density, which is sensed by the corresponding HK, resulting in the activation of the RR, which then activates the expression of all operons necessary for bacteriocin synthesis, transport, and regulation.The best example of such a regulatory system is the production of plantaricin E/F and plantaricin J/K by L. plantarum C11.The inducing peptide plantaricin A is secreted at low basal levels, thus enabling the bacterium to sense its own growth.At a certain threshold level of plantaricin A, an autoinduction loop is triggered, which leads to massive production of plantaricin E/F and plantaricin J/K [130].Notably, there are two RRs encoded by L. plantarum C11, PlnC and PlnD.It has been shown that PlnC activates while PlnD represses the genes involved in bacteriocin synthesis [131] (Figure 3).However, truncated versions of the activator PlnC, resulting from the translation from alternative start codons within plnC, were found to exhibit repression on the bacteriocin biosynthesis operon [132].Moreover, L. lactis MG1363 produced supernatants acting as environmental signals which can switch on bacteriocin production in L. plantarum NC8 via a quorum-sensing mechanism mediated by the inducing peptide PLNC8IF [133].The mechanism of how immunity proteins protect producing cells from class IIb bacteriocins was not fully elucidated.Some immunity proteins, including plnI for plantaricin EF and plnLR for plantaricin JK, show homology to the Abi family of proteins, which are putative membrane-bound metalloproteases characterized by three conserved motifs.These immunity proteins probably function by proteolytically degrading their cognate bacteriocins [134].Other immunity proteins, including the immunity protein for lactococcin G, likely interact directly both with the bacteriocin and its cellular receptor [135].So far, all the immunity proteins for class IIb bacteriocins are predicted to contain transmembrane domains (TMD).However, they range in length, number of TMDs, and orientation across the membrane.The smallest immunity protein, CbnZ for carnobacteriocin XY, has just 42 amino acids and contains as few as one TMD, while LagC is a membrane-associated protein with four TMDs [136].The wide structural variety of immunity proteins may be attributed to the fact that class IIb bacteriocins adopt different receptors as targeting molecules.
A typical gene set for class IIb bacteriocin production comprises five to eight genes (Figure 4) [137][138][139].These include two bacteriocin encoding genes, whose closely adjacent gene encodes the immunity protein.The genes encoding a three-component regulatory system may locate up-or downstream of the bacteriocin structural genes.Most class IIb gene clusters also have two genes encoding an ABC transporter complex.The mechanism of how immunity proteins protect producing cells from class IIb bacteriocins was not fully elucidated.Some immunity proteins, including plnI for plantaricin EF and plnLR for plantaricin JK, show homology to the Abi family of proteins, which are putative membrane-bound metalloproteases characterized by three conserved motifs.These immunity proteins probably function by proteolytically degrading their cognate bacteriocins [134].Other immunity proteins, including the immunity protein for lactococcin G, likely interact directly both with the bacteriocin and its cellular receptor [135].So far, all the immunity proteins for class IIb bacteriocins are predicted to contain transmembrane domains (TMD).However, they range in length, number of TMDs, and orientation across the membrane.The smallest immunity protein, CbnZ for carnobacteriocin XY, has just 42 amino acids and contains as few as one TMD, while LagC is a membrane-associated protein with four TMDs [136].The wide structural variety of immunity proteins may be attributed to the fact that class IIb bacteriocins adopt different receptors as targeting molecules.
A typical gene set for class IIb bacteriocin production comprises five to eight genes (Figure 4) [137][138][139].These include two bacteriocin encoding genes, whose closely adjacent gene encodes the immunity protein.The genes encoding a three-component regulatory system may locate up-or downstream of the bacteriocin structural genes.Most class IIb gene clusters also have two genes encoding an ABC transporter complex.
all the immunity proteins for class IIb bacteriocins are predicted to contain transmembrane domains (TMD).However, they range in length, number of TMDs, and orientation across the membrane.The smallest immunity protein, CbnZ for carnobacteriocin XY, has just 42 amino acids and contains as few as one TMD, while LagC is a membrane-associated protein with four TMDs [136].The wide structural variety of immunity proteins may be attributed to the fact that class IIb bacteriocins adopt different receptors as targeting molecules.
A typical gene set for class IIb bacteriocin production comprises five to eight genes (Figure 4) [137][138][139].These include two bacteriocin encoding genes, whose closely adjacent gene encodes the immunity protein.The genes encoding a three-component regulatory system may locate up-or downstream of the bacteriocin structural genes.Most class IIb gene clusters also have two genes encoding an ABC transporter complex.

IIc
Three key steps are involved in the biosynthesis of circular bacteriocins: cleavage of the leader, circularization, and exportation of the mature bacteriocin.Leader cleavage is believed to be the first step in the maturation and a requirement for further processing into the mature bacteriocins.The leader peptides ranging between 2 and 35 amino acids share no sequence similarity and the function of the leaders awaits further investigation.Unlike class IIa and IIb bacteriocins, whose leader was generally cleaved at the double-glycine site, there is no common recognition site for leader cleavage of circular bac-

IIc
Three key steps are involved in the biosynthesis of circular bacteriocins: cleavage of the leader, circularization, and exportation of the mature bacteriocin.Leader cleavage is believed to be the first step in the maturation and a requirement for further processing into the mature bacteriocins.The leader peptides ranging between 2 and 35 amino acids share no sequence similarity and the function of the leaders awaits further investigation.Unlike class IIa and IIb bacteriocins, whose leader was generally cleaved at the double-glycine site, there is no common recognition site for leader cleavage of circular bacteriocins.Moreover, the enzymes responsible for the cleavage of the leader peptide have not yet been identified.
The exact mechanism of the circularization reaction for circular bacteriocins is not fully understood.The ligation sites of all circular bacteriocins are located within a helical structure, consisting mainly of stretches of hydrophobic residues.It was suggested that the hydrophobic environment is essential for the circularization reaction [16].The properties of both the N-and C-terminal residues are critical to the efficiency of the circularization process.In the case of AS-48, the substitution of Met1 to Ala lowered the circularization efficiency significantly, whereas the substitution of Trp70 (last residue) to Ala resulted in the production of both circular and linear forms of the bacteriocin [140].Mutational analysis at the Leu1 position of enterocin NKR-5-3B revealed that only mutations with helix structure-promoting hydrophobic residues (Ala, Ile, Val or Phe) were able to yield the mature Ent53B derivative [141].These results highlight the importance of the hydrophobic nature of ligation points for the circularization mechanism.Most of the proteins encoded by the biosynthetic gene clusters contain multiple putative membrane-spanning domains and are probably associated with the membrane (Figure 5).The circularization reaction may be catalyzed by a membrane-located protein complex [142].Such a complex may be also responsible for exporting circular bacteriocins in a manner of coupling circularization and secretion reactions.
Several proteins have been identified to be involved in immunity to circular bacteriocins.As-48D1, GaaI, and CclI are the dedicated immunity proteins for AS-48, gassericin A, and carnocyclin A, respectively [143][144][145].These immunity proteins are small (49-56 amino acids), cationic (high pI), and contain one or two transmembrane domains, suggesting that they may be located in the cell membrane.These immunity proteins can provide a certain level of immunity to their cognate bacteriocins.Full immunity requires the combined activity of several other proteins.
yield the mature Ent53B derivative [141].These results highlight the importance of the hydrophobic nature of ligation points for the circularization mechanism.Most of the proteins encoded by the biosynthetic gene clusters contain multiple putative membrane-spanning domains and are probably associated with the membrane (Figure 5).The circularization reaction may be catalyzed by a membrane-located protein complex [142].Such a complex may be also responsible for exporting circular bacteriocins in a manner of coupling circularization and secretion reactions.Several proteins have been identified to be involved in immunity to circular bacteriocins.As-48D1, GaaI, and CclI are the dedicated immunity proteins for AS-48, gassericin A, and carnocyclin A, respectively [143][144][145].These immunity proteins are small (49-56 amino acids), cationic (high pI), and contain one or two transmembrane domains, suggesting that they may be located in the cell membrane.These immunity proteins can provide a certain level of immunity to their cognate bacteriocins.Full immunity requires the combined activity of several other proteins.
Circular bacteriocin gene clusters often consist of overlapping genes, demonstrating a tight organizational structure or genes which depend upon the ribosomal binding site of upstream genes (Figure 5).This indicates that expression is regulated by translational coupling.The minimal set of genes required for bacteriocin production and immunity, in general, comprises 5 to 10 genes [146].Interestingly, the bacteriocin structural genes are not adjacent to the immunity genes, some of them are located in different operons (Figure 5).The transportation system of class IIc bacteriocins is usually more complex than other class II bacteriocins.They have an accessory operon (cclEFGH, as-48EFGH, garEFGH) encoding an ABC transporter complex, consisting of a permease, an ATPase, and an extracellular protein (Figure 5).Circular bacteriocin gene clusters often consist of overlapping genes, demonstrating a tight organizational structure or genes which depend upon the ribosomal binding site of upstream genes (Figure 5).This indicates that expression is regulated by translational coupling.The minimal set of genes required for bacteriocin production and immunity, in general, comprises 5 to 10 genes [146].Interestingly, the bacteriocin structural genes are not adjacent to the immunity genes, some of them are located in different operons (Figure 5).The transportation system of class IIc bacteriocins is usually more complex than other class II bacteriocins.They have an accessory operon (cclEFGH, as-48EFGH, garEFGH) encoding an ABC transporter complex, consisting of a permease, an ATPase, and an extracellular protein (Figure 5).

IId
Most of the leaderless bacteriocins remain to be studied in more detail regarding the biosynthetic mechanism.The leader sequences of other general bacteriocins play an important role in the recognition by transporters.Moreover, the leader sequences keep the precursor peptides inactive during biosynthesis inside the host until the appropriate time for secretion.How leaderless bacteriocins are recognized by transporter protein and secreted remains elusive.A distinguishing feature of leaderless bacteriocins is the presence of a formylated N-terminal methionine residue.Interestingly, lacticins Q expressed in E. coli BL21(DE3) has unformylated methionine at the N-terminal.Nevertheless, the peptide demonstrated antimicrobial activity against several of the indicator strains tested [147].Thus, leaderless bacteriocins may not require a formylated N-terminus for full activity.However, more studies are needed to decipher the importance of the formylated methionine at the N-terminus for the biosynthesis of leaderless bacteriocins.The leaderless bacteriocins are active immediately after their translation process.The transport and immunity of leaderless bacteriocins may be carried out by one protein or protein complex.LmrB, an ABC-type multidrug resistance transporter, has been shown to be involved in both the secretion and self-immunity of this leaderless bacteriocin [148].The secretion of lacticin Q is strictly controlled by the presence of LnqBCDEF complex, whereas immunity is flexible in that LnqEF (ABC transporter) is the minimal unit required for sufficient immunity and LnqBCD could be considered an accessory protein that supports the activity of LnqEF [149].This may indicate that leaderless bacteriocins have in common the feature of having one dedicated ABC transporter mediating both secretion and immunity.However, a recent study showed that the ABC transporter is only involved in the transport but not the immunity of enterocin DD14, a leaderless two-peptide bacteriocin.The intracellular enterocin DD14 plays a role in its own immunity system [150].
Similar to class IIa and IIb bacteriocins, the non-pediocin liner bacteriocins are synthesized as biologically inactive pre-peptides consisting of an N-terminal leader peptide.Following synthesis of the pre-peptide, cleavage of the N-terminal leader sequence generally occurs at the double glycine site by means of a dedicated membrane protein from the ATP-binding cassette transporter family.In addition, a number of non-pediocin liner bacteriocins including lactococcin 972 and divergicinA are secreted through a general sec-dependent pathway and their leaders are cleaved by extracellular signal peptidase.Most LAB have a dedicated immunity protein to protect the cells from their own nonpediocin liner bacteriocins.It is not clear if there is a common mechanism of immunity for non-pediocin liner bacteriocins.For Lactococcin A, its immunity protein LciA has a similar four-helix bundle fold with the immunity proteins of the pediocin-like bacteriocins.Interestingly, LciA and the pediocin-like immunity proteins function in a similar manner.They bind to the bacteriocin-man-PTS complex and prevent membrane leakage [151].
The gene clusters for most of the leaderless bacteriocins have been identified.Genes involved in transport and immunity are often closely associated with bacteriocin structural genes (Figure 6).Leader-containing bacteriocins need an accessory protein function together with the cognate ABC transporter to mediate bacteriocin secretion.Such an accessory protein is not required for transporting leaderless bacteriocins.Moreover, genes related to formylase synthesis were not found in the vicinity of the bacteriocin structural gene, indicating that the N-terminal formylation of leaderless bacteriocins may be carried out by a host-encoded formylase that exists outside of the biosynthetic gene cluster [16,152].Interestingly, the structural genes of two leaderless bacteriocins weissellicin Y and weissellicin M produced by Weissella hellenica QU 13 are located in the same locus [87] (Figure 6).The structural genes of multi-peptides leaderless bacteriocins are co-transcribed.For instance, there is only one promoter has been detected upstream of the ddA gene, and a clear processing site motif of 48 bp was detected between the ddB and ddC genes [150].The genes responsible for regulation were only found in the biosynthetic gene cluster of lacticinQ/Z, whose production was positively regulated by LnqR, a TetR-family transcriptional regulator [153] (Figure 6).The regulation of most leaderless bacteriocins was associated with environmental stimuli.The production of Enterocin L50, Enterocin P, and Enterocin Q by Enterococcus faecium L50 was temperature-dependent [154].The production of weissellicin Y and weissellicin M by Weissella hellenica QU 13 was nutrition-adaptive and thiamine addition decreases weissellicin Y production [155].Pasteurized milk supplemented with tryptone significantly improved the production of garvicin KS [156].

Production of Class II Bacteriocins by Microbial Fermentation
The biosynthesis of bacteriocins can be influenced by various culture conditions, such as the composition of the medium, pH, temperature, and growth kinetics of the microorganisms [157].Fermentation studies on the class II bacteriocin production indicate that it follows primary metabolite kinetics producing the bacteriocin during the growth phase and declines completely after entering the stationary phase [158].The commercial availability of bacteriocins is still limited due to the low yield of the product.Moreover, bacteriocin-producing LAB needs complex nutrition to grow, and this not only increases the production cost but also gives rise to the difficulties related to their purification.The effi- The regulation of most leaderless bacteriocins was associated with environmental stimuli.The production of Enterocin L50, Enterocin P, and Enterocin Q by Enterococcus faecium L50 was temperature-dependent [154].The production of weissellicin Y and weissellicin M by Weissella hellenica QU 13 was nutrition-adaptive and thiamine addition decreases weissellicin Y production [155].Pasteurized milk supplemented with tryptone significantly improved the production of garvicin KS [156].

Production of Class II Bacteriocins by Microbial Fermentation
The biosynthesis of bacteriocins can be influenced by various culture conditions, such as the composition of the medium, pH, temperature, and growth kinetics of the microorganisms [157].Fermentation studies on the class II bacteriocin production indicate that it follows primary metabolite kinetics producing the bacteriocin during the growth phase and declines completely after entering the stationary phase [158].The commercial availability of bacteriocins is still limited due to the low yield of the product.Moreover, bacteriocin-producing LAB needs complex nutrition to grow, and this not only increases the production cost but also gives rise to the difficulties related to their purification.The efficient use of these compounds requires various approaches to overcome the low yield and the high production costs.In this regard, different studies have examined the effects of various media compositions and culture conditions on the yield of bacteriocins [159].In recent years, growing knowledge of the genetics and biosynthesis of class II bacteriocins has enabled researchers to quickly manufacture and engineer LAB strains for improved bacteriocin production.LAB strains developed by genetic engineering can not only be used to enhance yields but also for increased tolerance to various biotic and abiotic stresses during fermentation.

Natural Fermentation
Various media are used to cultivate the bacteriocin-producer such as CM, SM8, M17, and MRS media.These media are good for neutralizing lactic acid and improving cell growth, but do not consider the accumulation of bacteriocin and high content of nitrogen sources, especially proteins and peptides, that may bring about the difficulties of bacteriocin purification [160].Avonts et al. compared the bacteriocin production of seven Lactobacillus strains during fermentation in MRS medium and milk medium [161].Their results showed that L. acidophilus IBB 801 and L. gasseri K7 performed better than L. casei complex strains including L. rhamnosus GG.Although natural fermentation only reached a limited bacteriocin yield, the preservation potential of bacteriocins could be achieved by applying a bacteriocin-producing strain as starter culture.
Most class II bacteriocins are regulated by a quorum sensing (QS) system whose initiation can be induced by environmental factors and other bacterial strains.The production of class IIb bacteriocin plantaricin NC8 by L. plantarum NC8 is inducible by co-culture with Gram-positive bacterial strains and requires cell-to-cell contact with the inducer bacteria [61].This activates the expression of the operon plNC8IF-plNC8HK-plnD encoding a three-component regulatory system (TCRS) formed by an autoinducer peptide (PLNC8IF), a histidine protein kinase (PLNC8HK), and a response regulator (PlnD), which is indispensable for bacteriocin production by NC8 and is thought to be involved in quorum sensing [162].The bacteriocin synthesis of L. plantarum NMD-17 in co-cultivation has a close relationship with LuxS-mediated quorum sensing system [163].Enhanced bacteriocin production in a co-culture system has also been reported in Pediococcus pentosaceus and Enterococcus faecium [164,165].

Improving Class II Bacteriocin Production by Optimizing Fermentation Conditions
Similar to other metabolites, the yield of class II bacteriocins was strongly influenced by medium compositions and fermentation factors.The optimization of fermentation conditions is a complex approach but critically essential for high-performance bacteriocin production at a commercial scale [158].The culture medium is one of the key factors that need to be considered in the enhancement of any fermentation processes.The properties of the growth media including amino acid composition, carbon/nitrogen ratio, pH and lactose levels have a great influence on the change in biomass of the culture and the corresponding change in the level of bacteriocin production.In many cases, the optimal growth medium does not reflect the optimal productivity of bacteriocins by strain producers.The production of bacteriocins by L. lactis Gh1 in soytone was 1.28-times higher as compared to that of organic nitrogen sources ((NH 4 ) 2 SO 4 ) [166].The addition of certain amino acids in the fermentation medium stimulates bacteriocin production.For instance, glycine and cystine could stimulate the production of certain bacteriocins, while no stimulus effect was observed for alanine, tyrosine, and glutamic acid [167].
It was reported that some stressful environments could enhance bacteriocin production.For instance, a downward temperature shift stimulated amylovorin L471 production [168].
Nutrient stress is known to increase the bacteriocin production capability of L. plantarum B21 during industrial processing.Further investigation revealed that unstressed L. plantarum B21 cells use glucose as their primary energy source with high concentrations of metabolites involved in glycolysis and organic acid synthesis.In contrast, large numbers of metabolites involved in amino acid metabolism were upregulated in glucose-stressed cells, indicating that they were using amino acids as their main source of energy, which may favor the synthesis of bacteriocin [169].The effects of NaCl on bacteriocin production are controversial.It has been reported that the supplementation of appropriate NaCl could enhance the growth and bacteriocin production of Lactobacillus amylovorus DCE 471 [170], while optimum bacteriocin production by L. plantarum 17.2b requires the absence of NaCl [168].Other inorganic ions including KH 2 PO 4 , CaCl 2 and NH 4 PO 4 also have a profound influence on bacteriocin production [171].
Given the high number of influencing factors, the application of an adequate experimental design (optimization) is the best strategy to obtain maximum information with a minimum number of experiments.Response surface methodology is a powerful tool used for building models and evaluating the effects of factors and searching for optimum conditions of factors for bacteriocin production [172].For Latilactobacillus curvatus P99, RSM analysis revealed that the optimum production of bacteriocin was obtained at pH 6.22 and 30.6 • C for 17.9 h.Suganthi and Mohanasrinivasan reported a 20-fold increase in bacteriocin for Pediococcus pentosaceus KC692718 by using the RMS tool.The optimum conditions were soytone (1.03%), sucrose (2.4%), pH (5.5) and temperature (34.5 • C) [173].

Improving Class II Bacteriocin Production by Genetic Engineering
Besides the optimization of fermentation conditions, bacteriocin production can be increased by genetic approaches either by engineering the producer cells or using various heterologous expression systems.The entire gak locus including the genes involved in immunity and transport of class IId bacteriocin garvicin KS was cloned into a plasmid and transformed into native producers Lactococcus garvieae KS1546.The bacteriocin of the engineered KS1546 in optimized medium is about 2000-fold higher compared to that initially achieved by wild-type strain in GM17 [156].A green fluorescent protein (gfp)-based promoter-trap reporter system was used to screen conditions with enhanced bacteriocin production by Companilactobacillus crustorum MN047 [174].
Recombinant bacteriocin production was also widely investigated by using bacteria and yeast cells as hosts.Heterologous expression of three different class II bacteriocins, sakacin P, pediocin PA-1 and piscicolin 61, was successful in L. sake Lb790 (pSAK20).Bacteriocin enterocin A and its immune protein Ent I from E. faecium T136 were cloned for co-expression under Lc.lactis MG1363 Usp45 protein signal peptide [175].Arbulu et al. reported the use of synthetic genes designed from the published amino acid sequence of the mature bacteriocins SRCAM 602, OR-7, E-760, and L-1077 and with adapted codon usage for successful expression by Pichia pastoris [176].The class IIb two-peptide bacteriocins plantaricin EF (composed of PlnE and PlnF) and plantaricin NC8 (composed of PLNC8α and PLNC8β) were successfully heterologously expressed in E. coli BL21 cells to enhance bacteriocin production yield [177].Yu et al. constructed recombinant plasmids harboring genes encoding bacteriocin lactocinAB and expressed in E. coli BL21 cells with high yield [178].The enterocin P signal peptide was used to facilitate the secretion of the munA-cvaC hybrid bacteriocin in Lc. lactis NZ9000.The engineered hybrid bacteriocin was produced in situ in food products to effectively control Gram-negative and Gram-positive foodborne pathogens [179].

Conclusions
Class II bacteriocins of LAB with broad-spectrum antibacterial activity are expected to play a major role in many fields.Bacteriocins have great potential for use as biopreservatives, antibiotic alternatives, health-promoting gut modulators, and animal growth promoters [180].Class II bacteriocins can be directly applied as food preservatives.Moreover, some bacteriocins, e.g., AS-48, become alternative antibiotics through the development of bacteriocin-based therapies and offer promising revenue to address the problem of antibiotic resistance.
In general, LAB strains have a system to coordinate the production of class IIa and IIb bacteriocins at an adequate stage of growth, which is called a quorum-sensing system.The regulation mechanisms of the genes encoding class IIc and IId bacteriocin biosynthesis need to be investigated further.The potential application of bacteriocin as natural food preservatives depends on the capacity of expression of bacteriocin genetic determinants by genetically modified heterologous host strains at the industrial level.At present, commercial-scale bacteriocin production is still hampered by high costs and low yield.Overcoming this task will be unimaginable without a deep understanding of the bacteriocins' genetics and biosynthesis.In the last few decades, our understanding of bacteriocins' biosynthesis and regulation has considerably increased, which provides opportunities for the development of more advanced systems for the cost-effective production of bacteriocins.

Fermentation 35 Figure 1 .
Figure 1.A schematic diagram of the biosynthesis of class IIa bacteriocins.

Figure 1 .
Figure 1.A schematic diagram of the biosynthesis of class IIa bacteriocins.

Figure 2 .
Figure 2. Organization of biosynthetic gene clusters of class IIa bacteriocins of LAB.

Figure 2 .
Figure 2. Organization of biosynthetic gene clusters of class IIa bacteriocins of LAB.

Fermentation 35 Figure 3 .
Figure 3.A schematic diagram of the biosynthesis of plantaricinE/F and plantaricinJ/K.

Figure 3 .
Figure 3.A schematic diagram of the biosynthesis of plantaricinE/F and plantaricinJ/K.

Figure 4 .
Figure 4. Organization of biosynthetic gene clusters of class IIb bacteriocins of LAB.

Figure 4 .
Figure 4. Organization of biosynthetic gene clusters of class IIb bacteriocins of LAB.

Figure 5 .
Figure 5. Organization of biosynthetic gene clusters of class IIc bacteriocins of LAB.

Figure 5 .
Figure 5. Organization of biosynthetic gene clusters of class IIc bacteriocins of LAB.

Figure 6 .
Figure 6.Organization of biosynthetic gene clusters of class IId bacteriocins of LAB.

Figure 6 .
Figure 6.Organization of biosynthetic gene clusters of class IId bacteriocins of LAB.

Table 1 .
Amino acids sequences of class II bacteriocins.

Table 2 .
Characteristics of the amino acid sequence of class II bacteriocins of LAB.

Table 3 .
Distribution of class II bacteriocin in some food associated LAB.