Antimicrobial Peptides Research Articles

Antimicrobial peptide GL13K-Modified titanium in the epigenetic regulation of osteoclast differentiation via H3K27me3

Antimicrobial peptide GL13K-Modified titanium in the epigenetic regulation of osteoclast differentiation via H3K27me3

Luteibacter sahnii sp. nov., A Novel Yellow-Colored Xanthomonadin Pigment Producing Probiotic Bacterium from Healthy Rice Seed Microbiome.

To explore the rice seed microbiome, our objective was to isolate novel strains of Xanthomonas, a plant-associated bacterium with diverse lifestyles. Four isolates, anticipated to be Xanthomonas based on morphological features of yellow colonies, were obtained from healthy rice seeds. Phylo-taxono-genomic analysis revealed that these isolates formed monophyletic lineages belonging to a novel species within the genus Luteibacter. Pairwise ortho Average Nucleotide Identity and digital DNA-DNA hybridization confirmed their distinct species status. We propose Luteibacter sahnii sp. nov. as a novel species, with PPL193T = MTCC 13290T = ICMP 24807T = CFBP 9144T as the type strain and PPL201, PPL552, and PPL554 as other constituent members. The fatty acid profile of the type strain is dominated by branched fatty acids like Iso-C15:0, consistent with other members of the genus. The novel species displays non-pathogenic attributes and exhibits plant probiotic properties, protecting rice plants from the leaf blight pathogen X. oryzae pv. oryzae. Production of Indole-3-Acetic Acid (IAA) and genomic regions encoding anti-microbial peptides emphasize its potential contributions to plant hosts. This study underscores the importance of employing a combination of phenotypic and genotypic methods in culturomics to enhance our understanding of rice seed microbiome diversity.

Caffeic acid phenethyl ester ameliorates colistin-induced nephrotoxicity in rats via modulation of FOXO1/Nrf2/Sirt1 axis.

Colistin (Cst) is one of the antimicrobial peptides and is reserved for use against multi-drug-resistant Gram-negative bacteria. However, the clinical value of Cst is limited by its nephrotoxic adverse effects. Caffeic acid phenethyl ester (CAPE) is a honeybee propolis flavonoid recognised for its diverse pharmacological potential. It has demonstrated d antioxidant and anti-inflammatory properties, as well as protective effects against chemically induced toxicity in variuos biological systems. This study aimed to investigate the impact of CAPE on nephrotoxicity induced in rats by Cst. Animals were randomly divided into five groups. Group 1 served as control, group 2 received CAPE (10 mg/kg) orally, group 3 received Cst IP, group 4 received Cst + CAPE (5 mg/kg) and group 5 received Cst + CAPE (10 mg/kg). All treatments were given daily for 10 consecutive days. CAPE notably attenuated Cst-inducednephrotoxicity as shown by reducing urea serum levels, creatinine, cystatin C, urinary protein contents and urinary N-acetyl-β-D-glucosaminidase (NAG). This was confirmed by histological investigations that indicated amelioration of histopathological changes in the kidney architecture as well as the deposition of collagen in renal tissues. CAPE exhibited antioxidant effects supported by the prevention of rise in Cst-induced lipid peroxidation and depletion of superoxide dismutase and catalase enzymatic activities. In addition, CAPE inhibited the expression of the inflammatory markers including tumour necrosis factor-α, nuclear factor kappa B and interleukin-6. These actions were associated with modulation of messenger ribonucleic acid (mRNA) expression of Bax and Bcl-2 in favour of anti-apoptosis. CAPE inhibited Cst-induced rise in forkhead box O1 (FOXO1) expression and downregulation of nuclear factor erythroid 2-related factor 2 (Nrf2) and Sirtuin 1 (Sirt1) immune-expression. CAPE protects against nephrotoxicity induced by Cst in ratsprimarily through its antioxidant, antiinflammatory and antiapoptotic activities. These pritective effects are mediatedvia modulation of FOXO1/Nrf2/Sirt1 axis.

Alteration of Cecal Microbiota by Antimicrobial Peptides Enhances the Rational and Efficient Utilization of Nutrients in Holstein Bulls.

We previously observed that supplementation with antimicrobial peptides facilitated the average daily weight gain, net meat, and carcass weights of Holstein bulls. To expand our knowledge of the possible impact of antimicrobial peptides on cecum microbiota, further investigations were conducted. In this study, 18 castrated Holstein bulls with insignificant weight differences and 10 months of age were split randomly into two groups. The control group (CK) was fed a basic diet, whereas the antimicrobial peptide group (AP) was supplemented with 8 g of antimicrobial peptides for 270 days. After slaughter, metagenomic and metabolomic sequencing analyses were performed on the cecum contents. The results showed significantly higher levels of amylase, cellulase, protease, and lipase in the CK than in the AP group (P ≤ 0.05). The levels of β-glucosidase and xylanase (P ≤ 0.05), and acetic and propionic acids (P ≤ 0.01), were considerably elevated in the AP than in the CK group. The metagenome showed variations between the two groups only at the bacterial level, and 3258 bacteria with differences were annotated. A total of 138 differential abundant genes (P < 0.05) were identified in the CAZyme map, with 65 genes more abundant in the cecum of the AP group and 48 genes more abundant in the cecum of the CK group. Metabolomic analysis identified 68 differentially expressed metabolites. Conjoint analysis of microorganisms and metabolites revealed that Lactobacillus had the greatest impact on metabolites in the AP group and Brumimicrobium in the CK group. The advantageous strains of the AP group Firmicutes bacterium CAG:110 exhibited a strong symbiotic relationship with urodeoxycholic acid and hyodeoxycholic acid. This study identified the classification characteristics, functions, metabolites, and interactions of cecal microbiota with metabolites that contribute to host growth performance. Antimicrobial peptides affect the cecal microorganisms, making the use of nutrients more efficient. The utilization of hemicellulose in the cecum of ruminants may contribute more than cellulose to their production performance.

A Review of Antimicrobial Peptides: Structure, Mechanism of Action, and Molecular Optimization Strategies

Antimicrobial peptides (AMPs) are bioactive macromolecules that exhibit antibacterial, antiviral, and immunomodulatory functions. They come from a wide range of sources and are found in all forms of life, from bacteria to plants, vertebrates, and invertebrates, and play an important role in controlling the spread of pathogens, promoting wound healing and treating tumors. Consequently, AMPs have emerged as promising alternatives to next-generation antibiotics. With advancements in systems biology and synthetic biology technologies, it has become possible to synthesize AMPs artificially. We can better understand their functional activities for further modification and development by investigating the mechanism of action underlying their antimicrobial properties. This review focuses on the structural aspects of AMPs while highlighting their significance for biological activity. Furthermore, it elucidates the membrane targeting mechanism and intracellular targets of these peptides while summarizing molecular modification approaches aimed at enhancing their antibacterial efficacy. Finally, this article outlines future challenges in the functional development of AMPs along with proposed strategies to overcome them.

MdSVWC1, a new pattern recognition receptor triggers multiple defense mechanisms against invading bacteria in Musca domestica

BackgroundSingle-domain von Willebrand factor type C (SVWC) constitute a protein family predominantly identified in arthropods, characterized by a SVWC domain and involved in diverse physiological processes such as host defense, stress resistance, and nutrient metabolism. Nevertheless, the physiological mechanisms underlying these functions remain inadequately comprehended.ResultsA massive expansion of the SVWC gene family in Musca domestica (MdSVWC) was discovered, with a count of 35. MdSVWC1 was selected as the representative of the SVWC family for functional analysis, which led to the identification of the immune function of MdSVWC1 as a novel pattern recognition receptor. MdSVWC1 is highly expressed in both the fat body and intestines and displays acute induction upon bacterial infection. Recombinant MdSVWC1 binds to surfaces of both bacteria and yeast through the recognition of multiple pathogen-associated molecular patterns and exhibits Ca2+-dependent agglutination activity. MdSVWC1 mutant flies exhibited elevated mortality and hindered bacterial elimination following bacterial infection as a result of reduced hemocyte phagocytic capability and weakened expression of antimicrobial peptide (AMP) genes. In contrast, administration of recombinant MdSVWC1 provided protection to flies from bacterial challenges by promoting phagocytosis and AMP genes expression, thereby preventing bacterial colonization. MdSPN16, a serine protease inhibitor, was identified as a target protein of MdSVWC1. It was postulated that the interaction of MdSVWC1 with MdSPN16 would result in the activation of an extracellular proteolytic cascade, which would then initiate the Toll signaling pathway and facilitate the expression of AMP genes.ConclusionsMdSVWC1 displays activity as a soluble pattern recognition receptor that regulates cellular and humoral immunity by recognizing microbial components and facilitating host defense.

Type VI secretion systems promote intraspecific competition and host interactions in a bee gut symbiont

The Type VI Secretion System (T6SS) is a sophisticated mechanism utilized by gram-negative bacteria to deliver toxic effector proteins into target cells, influencing microbial community dynamics and host interactions. In this study, we investigated the role of T6SSs in Snodgrassella alvi wkB2, a core bacterial symbiont of the honey bee gut microbiota. We generated single- and double-knockout mutants targeting essential genes (tssD and tssE) in both T6SS-1 and T6SS-2 and assessed their colonization and competition capabilities in vivo. Our results indicate that T6SSs are nonessential for colonization of the bee gut, although T6SS-2 mutant strains exhibited significantly lower colonization levels compared to the wild-type (WT) strain. Further, a defined community experiment showed that S. alvi wkB2 T6SSs do not significantly impact interspecific competition among core gut bacteria. However, cocolonization experiments with closely related S. alvi strains demonstrated that T6SS-1 plays a role in mediating intraspecific competition. Transcriptomic analysis of bee guts monocolonized by WT or T6SS mutants revealed differential expression of host immunity-related genes relative to microbiota-deprived bees, such as upregulation of the antimicrobial peptide apidaecin in the presence of WT S. alvi and the antimicrobial peptide defensin in the presence of T6SS-2 mutant S. alvi, suggesting that T6SSs contribute to shaping host immune responses. These findings provide insight into the ecological roles of T6SSs in the honey bee gut microbiota, emphasizing their importance in maintaining competitive dynamics and influencing host-bacterial interactions.

Time-Responsive Activity of Engineered Bacteria for Local Sterilization and Biofilm Removal in Periodontitis.

Periodontitis is a highly prevalent and common condition in people of all ages, however, existing drugs to treat periodontitis have difficulty penetrating complex biofilms. Here, we report a biofilm-penetrating probiotic hybrid strategy for the treatment of periodontitis. It consists of therapeutic probiotics of E. coli Nissle 1917, which can produce antimicrobial peptides and hydrogen, and is coated with D-amino acids that can penetrate biofilms. After the fusion of D-amino acids with the biofilm, EcN entered the plaque biofilm and produced antimicrobial peptides to kill porphyromonas gingivalis and eliminate periodontitis under the action of hydrogen. The efficacy of EcN@DA-D in biofilm penetration and treatment of periodontitis was demonstrated in a rat model of periodontitis. In addition, the clinical combination to construct a rat periodontitis model by using clinical tissue has a significant therapeutic effect. In conclusion, EcN@DA-D offers a promising topical treatment for periodontitis without developing detectable pathogen resistance and side effects.

A novel broad-spectrum antibacterial and anti-malarial Anopheles gambiae Cecropin promotes microbial clearance during pupation.

Anophelinae mosquitoes are exposed to a variety of microbes including Plasmodium parasites that cause malaria. When infected, mosquitoes mount versatile immune responses, including the production of antimicrobial peptides. Cecropins are one of the most widely distributed families of antimicrobial peptides in insects and all previously studied Anopheles members are playing roles in adult mosquito immunity. We have identified and characterized a novel member of the Anopheles gambiae cecropin family, cecropin D (CecD), that is uniquely expressed and immune-responsive at late larval stages to promote microbial clearance through its broad-spectrum antibacterial activity during larval-pupal developmental transition. Interestingly, Cecropin D also exhibited highly potent activity against Plasmodium falciparum sporozoites, the malaria parasite stage that is transmitted from mosquitoes and infects humans and thereby holds promise as a malaria transmission-blocking agent. Finally, we have defined unequivocal cecropin-specific molecular signatures to systematically organize the diversity of the cecropin family in malaria vectors.

Photocatalytic Degradation of Bacterial Lipopolysaccharides by Peptide-Coated TiO2 Nanoparticles.

In this study, we report the degradation of smooth and rough lipopolysaccharides (LPS) from Gram-negative bacteria and of lipoteichoic acid (LTA) from Gram-positive bacteria by peptide-coated TiO2 nanoparticles (TiO2 NPs). While bare TiO2 NPs displayed minor binding to both LPS and LTA, coating TiO2 NPs with the antimicrobial peptide LL-37 dramatically increased the level of binding to both LPS and LTA, decorating these uniformly. Importantly, peptide coating did not suppress reactive oxygen species generation of TiO2 NPs; hence, UV illumination triggered pronounced degradation of LPS and LTA by peptide-coated TiO2 NPs. Structural consequences of oxidative degradation were examined by neutron reflectometry for smooth LPS, showing that degradation occurred preferentially in its outer O-antigen tails. Furthermore, cryo-TEM and light scattering showed lipopolysaccharide fragments resulting from degradation to be captured by the NP/lipopolysaccharide coaggregates. The capacity of LL-37-TiO2 NPs to capture and degrade LPS and LTA was demonstrated to be of importance for their ability to suppress lipopolysaccharide-induced activation in human monocytes at simultaneously low toxicity. Together, these results suggest that peptide-coated photocatalytic NPs offer opportunities for the confinement of infection and inflammation.

Fluorescent Antimicrobial Peptides Based on Nile Red: Effect of Conjugation Site and Chemistry on Wash-Free Staining of Bacteria.

Fluorescent probes for bacterial detection can be obtained by conjugating antimicrobial peptides with fluorescent dyes. However, little is known about the effect of the conjugation site and linker chemistry on staining efficiency. We synthesized three conjugates of the antimicrobial peptide ubiquicidin with the environmentally sensitive fluorophore Nile Red that differed by the attachment site and the chemical composition of the linker. We showed that incorporating fluorophore as a minimalistic non-natural amino acid resulted in a superior probe compared with the typically used bioconjugation approaches. The new peptide-based probe named UNR-1 displayed red fluorescence and enabled robust wash-free staining of Gram-positive and Gram-negative bacteria. The probe exhibited selectivity over mammalian cells and enabled rapid fluorescence detection of bacteria by fluorescence microscopy and flow cytometry in an add-and-read format. Our results may foster the development of next-generation fluorescent AMPs for clinical laboratory diagnostics and medical imaging.

Single-Cell Transcriptomics Unveils Skin Cell Specific Antifungal Immune Responses and IL-1Ra- IL-1R Immune Evasion Strategies of Emerging Fungal Pathogen Candida auris.

Candida auris is an emerging multidrug-resistant fungal pathogen that preferentially colonizes and persists in skin tissue, yet the host immune factors that regulate the skin colonization of C. auris in vivo are unknown. In this study, we employed unbiased single-cell transcriptomics of murine skin infected with C. auris to understand the cell type-specific immune response to C. auris. C. auris skin infection results in the accumulation of immune cells such as neutrophils, inflammatory monocytes, macrophages, dendritic cells, T cells, and NK cells at the site of infection. We identified fibroblasts as a major non-immune cell accumulated in the C. auris infected skin tissue. The comprehensive single-cell profiling revealed the transcriptomic signatures in cytokines, chemokines, host receptors (TLRs, C-type lectin receptors, NOD receptors), antimicrobial peptides, and immune signaling pathways in individual immune and non-immune cells during C. auris skin infection. Our analysis revealed that C. auris infection upregulates the expression of the IL-1RN gene (encoding IL-1R antagonist protein) in different cell types. We found IL-1Ra produced by macrophages during C. auris skin infection decreases the killing activity of neutrophils. Furthermore, C. auris uses a unique cell wall mannan outer layer to evade IL-1R-signaling mediated host defense. Collectively, our single-cell RNA seq profiling identified the transcriptomic signatures in immune and non-immune cells during C. auris skin infection. Our results demonstrate the IL-1Ra and IL-1R-mediated immune evasion mechanisms employed by C. auris to persist in the skin. These results enhance our understanding of host defense and immune evasion mechanisms during C. auris skin infection and identify potential targets for novel antifungal therapeutics.

Interferon lambda 4 is a gut antimicrobial protein

To withstand complex microbial challenges, the mammalian gut largely depends on the secretion of diverse antimicrobial proteins. Type III interferons (IFNλs) are ordinarily considered inducible antiviral cytokines involved in intestinal immunity. Unlike other IFNλs, we found that newly identified IFNλ4 is an intestinal antibacterial protein. Large amounts of natural IFNλ4 are present in the secretory layer of the intestinal tracts of healthy piglets, which suggests that IFNλ4 is in direct physiological contact with microbial pathogens. We also identified two biochemical functions of mammalian IFNλ4, the induction of bacterial agglutination and direct microbial killing, which are not functions of the other IFNλs. Further mechanistic investigations revealed that after binding to the carbohydrate fraction of lipopolysaccharide, mammalian IFNλ4 self-assembles into bacteria-surrounding nanoparticles that agglutinate bacteria, and that its unique cationic amphiphilic molecular structure facilitates the destruction of bacterial membranes. Our data reveal features of IFNλ4 distinct from those of previously reported IFNλs and suggest that noncanonical IFNλ4 is deeply involved in intestinal immunity, beyond simply cytokine signaling.

Investigating the Inhibitory Effect of Animal Antimicrobial Peptides on Gram-Positive and Gram-Negative Bacteria in Diabetic Foot Ulcers

Investigating the Inhibitory Effect of Animal Antimicrobial Peptides on Gram-Positive and Gram-Negative Bacteria in Diabetic Foot Ulcers

The Wheel of Fortune: Helical Wheel Alanine Scanning of a Spider Venom Antimicrobial Peptide Reveals Residues Involved in Antimicrobial and Cytotoxic Activity.

A preference for several amino acids is observed to occur at particular positions of cationic α-helical antimicrobial peptides (AMPs), which ensures the formation of amphipathic regions once they assume their correct secondary structure in membranes or membrane-mimicking environments and makes them active against pathogens. This study determined the effect of alanine mutations on the secondary structure and bioactivity of lyp1987 (GRLQAFLAKMKEIAAQTL-NH2), a cationic α-helical AMP obtained from the venom of Lycosa poonaensis which exhibits broad range activity against Gram-positive and Gram-negative bacteria with micromolar minimum inhibitory concentrations (MIC). CD spectroscopy revealed no significant difference in the secondary structure, with all alanine-substituted analogs exhibiting predominantly α-helical structure in buffered 2,2,2-trifluoroethanol solution. Alanine substitution at Glu12 and Thr17 increased the activity of lyp1987 against Gram-positive and -negative bacteria, while alanine substitution at Lys9 increased its selectivity against Gram-positive bacteria. Further investigation can be done to determine positions and substitutions that will give less cytotoxic analogs.

Comb-like dynamic antimicrobial peptides for serum-resistant intracellular protein delivery and antibacterial treatment

Comb-like dynamic antimicrobial peptides for serum-resistant intracellular protein delivery and antibacterial treatment

Liver-expressed antimicrobial peptide 2 is a hepatokine regulated by ghrelin, nutrients, and body weight.

Liver-expressed antimicrobial peptide 2 (LEAP2) is a peptide that counteracts the hunger hormone ghrelin-induced functions. Recently, we showed that vertical sleeve gastrectomy (VSG) did not alter the serum LEAP2 concentration in individuals with obesity. Here, we investigated the effects of VSG in both chow diet (CD)-fed and high-fat diet (HFD)-fed mice. In CD-fed mice, VSG increased plasma LEAP2 levels and hepatic Leap2 mRNA levels while decreasing body weight, blood glucose levels, and ghrelin levels. Intraperitoneal (ip) administration of ghrelin reversed these changes. These effects were found in both male and female mice. In contrast, VSG or weight loss in HFD-induced obese mice decreased LEAP2 levels. After fasting, the plasma LEAP2 concentration was in the following order: hepatic vein > abdominal aorta > portal vein. A high glucose concentration robustly increased the plasma LEAP2 concentration in the hepatic vein and abdominal aorta but not in the portal vein. In addition, corn oil or palmitate increased LEAP2 expression and secretion. The increase in LEAP2 levels after the meal tolerance test was delayed in the human subjects with diabetes. Our data suggest that various factors (metabolic, hormonal, and nutritional) regulate LEAP2, and the liver is the predominant site for the production and secretion of LEAP2. Furthermore, the interaction between ghrelin and LEAP2 is involved in the pathogenesis of obesity and diabetes.

Characterization of Kunitz-Domain Anticoagulation Peptides Derived from Acinetobacter baumannii Exotoxin Protein F6W77.

Recent studies have revealed that the coagulation system plays a role in mammalian innate defense by entrapping bacteria in clots and generating antibacterial peptides. So, it is very important for the survival of bacteria to defend against the host coagulation system, which suggests that bacterial exotoxins might be a new source of anticoagulants. In this study, we analyzed the genomic sequences of Acinetobacter baumannii and a new bacterial exotoxin protein, F6W77, with five Kunitz-domains, KABP1-5, was identified. Each Kunitz-type domain features a classical six-cysteine framework reticulated by three conserved disulfide bridges, which was obviously similar to animal Kunitz-domain peptides but different from plant Kunitz-domain peptides. Anticoagulation function evaluation showed that towards the intrinsic coagulation pathway, KABP1 and KABP5 had apparently inhibitory activity, KABP4 had weak inhibitory activity, and KBAP2 and KABP3 had no effect even at a high concentration of 20 μg/mL. All five Kunitz-domain peptides, KABP1-5, had no inhibitory activity towards the extrinsic coagulation pathway. Enzyme-inhibitor experiments showed that the high-activity anticoagulant peptide KABP1 had apparently inhibitory activity towards two key coagulation factors, Xa and XIa, which was further confirmed by pull-down experiments that showed that KABP1 can bind to coagulation factors Xa and XIa directly. Structure-function relationship analyses of five Kunitz-type domain peptides showed that the arginine of the P1 site of three new bacterial anticoagulants, KABP1, KABP4 and KABP5, might be the key residue for their anticoagulation activity. In conclusion, with bioinformatics analyses, peptide recombination, and functional evaluation, we firstly found bacterial-exotoxin-derived Kunitz-type serine protease inhibitors with selectively inhibiting activity towards intrinsic coagulation pathways, and highlighted a new interaction between pathogenic bacteria and the human coagulation system.

Identification of Multifunctional Putative Bioactive Peptides in the Insect Model Red Palm Weevil (Rhynchophorus ferrugineus)

Innate immunity, the body’s initial defense against bacteria, fungi, and viruses, heavily depends on antimicrobial peptides (AMPs), which are small molecules produced by all living organisms. Insects, with their vast biodiversity, are one of the most abundant and innovative sources of AMPs. In this study, AMPs from the red palm weevil (RPW) Rhynchophorus ferrugineus (Coleoptera: Curculionidae), a known invasive pest of palm species, were examined. The AMPs were identified in the transcriptomes from different body parts of male and female adults, under different experimental conditions, including specimens collected from the field and those reared in the laboratory. The RPW transcriptomes were examined to predict antimicrobial activity, and all sequences putatively encoding AMPs were analyzed using several machine learning algorithms available in the CAMPR3 database. Additionally, anticancer, antiviral, and antifungal activity of the peptides were predicted using iACP, AVPpred, and Antifp server tools, respectively. Physicochemical parameters were assessed using the Antimicrobial Peptide Database Calculator and Predictor. From these analyses, 198 putatively active peptides were identified, which can be tested in future studies to validate the in silico predictions. Genome-wide analysis revealed that several AMPs have predominantly emerged through gene duplication. Noticeably, we detect a newly originated defensin allele from an ancestral defensin via the deletion of two amino acids following gene duplication in RPW, which may confer an enhanced resilience to microbial infection. Our study shed light on AMP gene families and shows that high duplication and deletion rates are essential to achieve a diversity of antimicrobial mechanisms; hence, we propose the RPW AMPs as a model for exploring gene duplication and functional variations against microbial infection.

Boosting Expression of a Specifically Targeted Antimicrobial Peptide K in Pichia pastoris by Employing a 2A Self-Cleaving Peptide-Based Expression System.

Background/Objectives: The current epidemic of drug-resistance bacterial strains is one of the most urgent threats to human health. Antimicrobial peptides (AMPs) are known for their good activity against multidrug resistance bacteria. Specifically targeted AMPs (STAMPs) are a fraction of AMPs that target specific bacteria and maintain the balance of the healthy microbiota of a host. We reported a STAMP Peptide K (former name: peptide 13) for E. coli. The aim of this study was to effectively produce peptide K using methylotrophic yeast Pichia pastoris. Methods: Three inserts (sequence of peptide K (K), two copies of peptide K fused with 2A sequence (KTK), and two copies of peptide K fused with 2A and an extra α mating factor (KTAK)) were designed to investigate the effect of the number of repeats and the trafficking of peptide on the yield. Results: The yield from KTK was the highest-more than two-fold higher compared with K-implying the role of the 2A sequence in heterologous peptide expression apart from the co-translation. Then, the fermentation condition for KTK was optimized. The optimized yield of KTK was 6.67 mg/mL, suggesting the efficiency of the expression system. Selectivity, antibacterial activity, biocompatibility, and the stability of the fermentation product were equivalent to the chemically synthesized peptide. The actional mechanism of the fermentation product included membrane permeabilization and ROS induction. Conclusions: Together, our work provided a new perspective to augment the yield of the antimicrobial peptide in the microbial system, building a technological foundation for their large-scale production and expanding the market application of AMPs.