As the global population rises, safeguarding food safety has become a worldwide priority[1]. While conventional chemical preservatives effectively prolong shelf life, their extensive use raises concerns about allergenicity, antimicrobial resistance, and possible carcinogenicity. Consequently, the food industry is shifting toward natural, safe, and eco-friendly alternatives.Among these, lysozyme and antimicrobial peptides stand out for their potent antimicrobial activity and excellent safety profile. Combining the two promises synergistic efficacy and reduced reliance on traditional chemicals, thereby enhancing both food safety and freshness[2-5]. The main goal of this project is to develop a novel food preservative technology to improve the shelf life and safety of food through the combination of lysozyme and antimicrobial peptides. We finally screened out 1 lysozyme and 9 antimicrobial peptides. The lysozyme and antimicrobial peptide genes were then cloned into expression vectors and transformed into Escherichia coli DH5α and Pichia pastoris GS115 respectively. Recombinant protein expression was carried out in Pichia pastoris GS115 to construct the genetic system of lysozyme and antimicrobial peptides. Finally, the antibacterial effect of lysozyme and antimicrobial peptides was verified by qualitative characterization and judgment, as well as quantitative characterization and quantitative analysis.pPICZα A/antisepsis mix, pPICZα A/Hepcidin, pPICZα A/Buforin II, pPICZα A/Piscidin-1, pPICZα A/LL-37, pPICZα A/Cecropin A, pPICZα A/Melittin, pPICZα A/Plectasin, pPICZα A/hLYZ, pPICZα A/Lactoferricin
During our experiment, we added some new parts for iGEM part and new information to an existing part (Table 1).
Table 1. Part contributions
| Part number | Part name | Contribution type | Part type |
| BBa_252QZKWP | hLYZ | new part | Basic part |
| BBa_25W3IV6V | Cecropin A | new part | Basic part |
| BBa_25KMWWNF | Melittin | new part | Basic part |
| BBa_255VLZAT | Buforin II | new part | Basic part |
| BBa_25F0UF1O | LL-37 | new part | Basic part |
| BBa_253TY9SH | Plectasin | new part | Basic part |
| BBa_25DXW7OG | Hepcidin | new part | Basic part |
| BBa_256I2SA1 | Piscidin-1 | new part | Basic part |
| BBa_2518A7VZ | Lactoferricin | new part | Basic part |
| BBa_25YA2AQF | antisepsis mix | new part | Composite part |
| BBa_25A3W2FJ | pPICZα A/antisepsis mix | new part | Composite part |
| BBa_25QHRFNC | pPICZα A/Hepcidin | new part | Composite part |
| BBa_25QC1Y0D | pPICZα A/Buforin II | new part | Composite part |
| BBa_259RA27S | pPICZα A/Piscidin-1 | new part | Composite part |
| BBa_25SIVGES | pPICZα A/LL-37 | new part | Composite part |
| BBa_256BXWDZ | pPICZα A/Cecropin A | new part | Composite part |
| BBa_256Y7M7S | pPICZα A/Melittin | new part | Composite part |
| BBa_25Y5S7FX | pPICZα A/Plectasin | new part | Composite part |
| BBa_25I2WEW7 | pPICZα A/hLYZ | new part | Composite part |
| BBa_25L12B4Z | pPICZα A/Lactoferricin | new part | Composite part |
1.1 hHLZ, BBa_252QZKWP
Name: Hlyz
Base Pairs: 455 bp
Origin: human
Usage and Biology
This gene encodes human lysozyme, whose natural substrate is the bacterial cell wall peptidoglycan (cleaving the beta[1-4]glycosidic linkages between N-acetylmuramic acid and N-acetylglucosamine). Lysozyme is one of the antimicrobial agents found in human milk, and is also present in spleen, lung, kidney, white blood cells, plasma, saliva, and tears. The protein has antibacterial activity against a number of bacterial species. Missense mutations in this gene have been identified in heritable renal amyloidosis[1].
1.2 Cecropin A, BBa_25W3IV6V
Name: Cecropin A
Base Pairs: 122 bp
Origin: Hyalophora cecropia
Usage and Biology
Cecropin A is a linear peptide containing 37 amino acids, which mainly interacts with the negative charge on the bacterial plasma membrane through its cationic properties, and then inserts and destroys the lipid bilayer structure. This destruction leads to the leakage of bacterial contents and eventually kills bacteria[2].
1.3 Melittin, BBa_25KMWWNF
Name: Melittin
Base Pairs: 89 bp
Origin: Synthetic construct
Usage and Biology
Melittin is the main active ingredient in bee venom, which has many biological activities, including anti-inflammatory, analgesic, immunomodulation, cardiovascular effect, antibacterial and anticancer potential. Melittin shows its medical effects by inhibiting the release of inflammatory mediators, directly relieving pain, regulating the immune system, dilating blood vessels, improving blood circulation, inhibiting the growth of various bacteria and possibly resisting cancer[3].
1.4 Buforin II, BBa_255VLZAT
Name: Buforin II
Base Pairs: 74 bp
Origin: Asian toad Bufo bufo gargarizans
Usage and Biology
Buforin II, derived from buforin I, a protein isolated from the stomach of the Asian toad Bufo bufo gargarizans, is a potent antimicrobial peptide. Buforin II has antimicrobial activity against a broad spectrum of Gram-positive and Gram-negative bacteria[4].
1.5 LL-37, BBa_25F0UF1O
Name: LL-37
Base Pairs: 122 bp
Origin:Human
Usage and Biology
LL-37 is the main protein in neutrophils, and widely exists in neutrophils, bone marrow, cervical squamous epithelium and vaginal squamous epithelium. The precursor of LL-37 is composed of a signal peptide, cathelin conserved region and 37 amino acid residues. When the cell is activated, it is cleaved by serine protease 3 and other proteolytic enzymes to produce bioactive LL-37, which has antibacterial, antifungal and antiviral functions, as well as chemotaxis and immune stimulation/regulation[5].
1.6 Plectasin, BBa_253TY9SH
Name: Plectasin
Base Pairs: 131 bp
Origin: Pseudoplectania nigrella
Usage and Biology
Plectasin adopts a compact cysteine-stabilized fold that is representative of a large family of host defence peptides in invertebrates. The peptide and its variants show high activity against pathogens such as methicillin-resistant Staphylococcus aureus, Streptococcus pneumoniae or Mycobacterium tuberculosis, including in animal infection models. Plectasin exerts its bactericidal activity by targeting the peptidoglycan precursor lipid II in the plasma membrane, blocking the cell wall biosynthesis[6].
1.7 Hepcidin, BBa_25DXW7OG
Name: Hepcidin
Base Pairs: 71 bp
Origin: Human
Usage and Biology
Hepcidin is a cysteine-rich antibacterial polypeptide synthesized and secreted by the liver, which can be expressed in large quantities during the immune process and participate in the immune response. It plays a negative role in the regulation of iron balance in the body, and it has certain curative effect in the related clinical application of iron metabolism diseases[7].
1.8 Piscidin-1, BBa_256I2SA1
Name: Piscidin-1
Base Pairs: 77 bp
Origin: Fish
Usage and Biology
Piscidin-1 is a cationic antimicrobial peptide primarily isolated from the mast cells of fish, such as bass. It exhibits potent activity against bacteria, fungi, viruses, and parasites, serving as a key component of the innate immune system in fish[8].
1.9 Lactoferricin, BBa_2518A7VZ
Name: Lactoferricin
Base Pairs: 158 bp
Origin: Human
Usage and Biology
Lactoferricin is not directly secreted as a peptide, but rather is a bioactive fragment released from lactoferrin through proteolytic hydrolysis (e.g., by pepsin) in the gastric acidic environment. It exhibits significantly more potent antimicrobial activity than its parent protein (lactoferrin), demonstrating efficacy against Gram-positive bacteria, Gram-negative bacteria, fungi, viruses, and even certain cancer cells[9].
Figure 1. PCR results of the fragments antisepsis mix, Hepcidin, Buforin II, Piscidin-1, LL-37, Cecropin A, Melittin, Plectasin, hLYZ, Lactoferricin synthesized by a biotech company.
1. Add new Composite part partName: pPICZα A/antisepsis mix(BBa_25A3W2FJ), pPICZα A/Hepcidin(BBa_25QHRFNC), pPICZα A/Buforin II(BBa_25QC1Y0D), pPICZα A/Piscidin-1(BBa_259RA27S), pPICZα A/LL-37(BBa_25SIVGES), pPICZα A/Cecropin A(BBa_256BXWDZ), pPICZα A/Melittin(BBa_256Y7M7S), pPICZα A/Plectasin(BBa_25Y5S7FX), pPICZα A/hLYZ(BBa_25I2WEW7), pPICZα A/Lactoferricin(BBa_25L12B4Z)
Construction DesignWe use pPICZα A plasmid as our vector. On the basis of the vector, we introduced the antimicrobial peptide or lysozyme gene, and added genes such as AOX1 promoter and bleomycin resistance gene. Based on this, a complete plasmid gene with function was constructed. We also design a antisepsis mix protein, which connecting lysosome and 9 AMPs by flexible linker (GGGGS).
Figure 2. The plasmid maps. A: pPICZα A/Plectasin; B: pPICZα A/LL-37; C: pPICZα A/Melittin; D: pPICZα A/Hepcidin; E: pPICZα A/antisepsis mix; F: pPICZα A/Piscidin-1; G: pPICZα A/Lactoferricin; H: pPICZα A/hLYZ; I: pPICZα A/Buforin II; J: pPICZα A/Cecropin A.
Experimental Approach
A. Construction of plasmids
(1) Amplification of target genes and vector fragment
We obtained target genes for constructing plasmid pPICZα A/antisepsis mix, pPICZα A/Hepcidin, pPICZα A/Buforin II, pPICZα A/Piscidin-1, pPICZα A/LL-37, pPICZα A/Cecropin A, pPICZα A/Melittin, pPICZα A/Plectasin, pPICZα A/hLYZ, pPICZα A/Lactoferricin using PCR technology, as shown in Figure 2A. We obtained fragments of approximately 1600bp, 80bp, 80bp, 80bp, 130bp, 130bp, 90bp, 130bp, 460bp, 160bp in length, which are consistent with our expected sizes.
Subsequently, we selected EcoRI and NotI restriction sites for enzymatic digestion of the target genes and vector fragment (as shown in Figure 3B). We obtained fragments of approximately 3600bp in length, which are consistent with our expected sizes.
Figure 3. Gel electrophoresis validation. A: PCR results; B: vector enzyme digestion results.
(1) Recombination reaction and transformation
The digested PCR fragment and vector were ligated using T4 DNA ligase and then we transformed them into Escherichia coli DH5α, as shown in Figure 4A. The results preliminarily proved that the strain construction was successful. In order to further confirm the result, we sequenced the DNA (Figure 4C). The results of DNA sequencing are completely consistent with expectations, demonstrating that all ten target gene fragments were precisely integrated into pPICZα A.
Compared with bacteria, yeast is a eukaryote, which is more closely related to human beings, and the protein expressed by yeast may be safer for human beings. Therefore, we chose Pichia pastoris as the strain for protein expression. The verified plasmids were then extracted from E. coli and transformed into Pichia pastoris GS115 (Figure 4D). Further, in order to verify the correctness of the strain construction, we selected Single colony from the plate for colony PCR and observed the results by gel electrophoresis. Consistent with expectations, all candidates are the same size as expected (Figure 4B). In order to determine whether antibacterial peptides and lysozyme can be expressed normally, we want to induce the strain and verify whether the protein can be expressed normally after being transformed into yeast by SDS-PAGE.
Figure 4. Construction results of plasmid pPICZα A/antisepsis mix, pPICZα A/Hepcidin, pPICZα A/Buforin II, pPICZα A/Piscidin-1, pPICZα A/LL-37, pPICZα A/Cecropin A, pPICZα A/Melittin, pPICZα A/Plectasin, pPICZα A/hLYZ, pPICZα A/Lactoferricin. A: Transformation into DH5α after ligation; B: Colony PCR results; C: Sequencing results; D: Successful transformation of correct plasmid into Pichia pastoris GS115.
B. Protein expression
(1) SDS-PAGE
Pichia pastoris GS115 strains carrying the correct plasmid were cultured individually at 30℃ and induced protein expression with 10 g/L methanol, and the proteins were purified using His-Tag affinity chromatography. SDS-PAGE was used to verify the protein expression of the target gene. As shown in Figure 5, different bands corresponding to the expected molecular weight were clearly observed in the purified components, confirming the accurate expression of each protein.
Figure 5 The SDS-PAGE results
(1) Growth curve
The expression of protein may affect the growth level of the strain. Therefore, in order to determine whether the protein expression of antimicrobial peptides and lysozyme affects the normal growth of the strain, we tested the growth curve of the strain with different antimicrobial peptide protein expressions. As shown in Figure 6, all engineered strains exhibited good growth kinetics under the test conditions. This result shows that yeast strains can grow normally and express the required antimicrobial peptides and lysozyme proteins under this culture condition.
Figure 6 Growth curves of 10 recombinant strains.
C. Functional TestMinimum Inhibitory Concentration (MIC) determination
The minimum antibacterial concentration (MIC) of biological preservatives was determined by Broth microdilution method to evaluate their antibacterial activity. Single colonies of the test strains E. coli and Bacillus subtilis were inoculated into LB medium for overnight culture, and the resulting suspension was adjusted to 106 CFU mL-1 in fresh medium. Dispense 100 μL of the purified protein, from 128 to 2 μg·mL-1, into a 96-well microplate. Subsequently, 100 μL of standardized bacterial suspension was added to each well and the plates were incubated at 37℃ for 24 h. Wells containing only sterile LB medium can serve as negative controls, while wells containing bacteria serve as positive controls to confirm bacterial viability.
The MIC50 of the purified protein is shown in Table 1. From MIC results, we can see that Plectasin has the strongest antibacterial effect on both Escherichia coli and Bacillus subtilis. When the concentration of Plectasin is 4 μg/ml, 50% bacteria can be killed. The MIC50 values of all antimicrobial peptides are below 16 μg/ml, which indicates that all antimicrobial peptides involved in the experiment have certain antibacterial activity. However, the MIC50 value of lysozyme is higher, this means the bacteriostatic effect of lysozyme is slightly weak, but the MIC50 value is also below 32 μg/ml.Thus lysozyme also has certain bacteriostatic effect.
Table 1. The Inhibitory concentration curve results.
| Drug | MIC50 | |
| E. coli | Bacillus subtilis | |
| Cecropin A | 8 μg/ml | 16 μg/ml |
| Melittin | 8 μg/ml | 16 μg/ml |
| LL-37 | 8 μg/ml | 4 μg/ml |
| Plectasin | 4 μg/ml | 4 μg/ml |
| Piscidin-1 | 8 μg/ml | 4 μg/ml |
| Hepcidin | 8 μg/ml | 16 μg/ml |
| Lactoferricin | 8 μg/ml | 16 μg/ml |
| Buforin II | 8 μg/ml | 16 μg/ml |
| HLYZ | 16 μg/ml | 32 μg/ml |
| Antisepsis mix | 16 μg/ml | 16 μg/ml |
The growth of bacteria at different time points was monitored by optical density method (OD600). By drawing the growth curve of bacteria treated with antimicrobial peptides, we can know the inhibitory effect of antimicrobial peptides on bacteria and how long it takes for bacteria to recover their growth ability after antimicrobial peptides treatment. Equal volumes of biopreservatives and bacterial suspensions were mixed and incubated at 30℃ for 24 h, and absorbance was measured at 0, 2, 4, 6, 8 and 24 h to determine the growth curve of bacteria under the condition of adding different biopreservatives.
As shown in Figure 7, the addition of biological preservatives inhibited the growth of bacteria to varying degrees. From the curve results, the inhibitory effect of some antibacterial peptides will gradually weaken with the increase of time. In the experiment of inhibiting Escherichia coli, four antimicrobial peptides(Cecropin A, Lactoferricin, LL-37 and Plectasin) showed strong activity. With the increase of culture time, the growth of bacteria was still inhibited to a great extent. In order to explore whether there is a combined effect between antibacterial peptides and lysozyme, we mixed all antibacterial peptides with lysozyme and determined the antibacterial activity of the mixed samples. The results show that the mixed samples have certain antibacterial activity, but the combined effect is not significant, which may be because we mixed all antibacterial peptides, some of which have poor antibacterial activity or antagonistic effect, resulting in poor mixed effect. Later, we will consider the combination of two, so as to find the best combination scheme with antibacterial effect. Compared with Escherichia coli, most antimicrobial peptides have better antibacterial effect on Bacillus subtilis. Notably, Plectasin showed the most significant inhibitory effects against E. coli and Bacillus subtilis.
Figure 7. Time-Kill curve. A: E. coli; B: Bacillus subtilis.
Laser Scanning Confocal Microscope observationThe bacterial suspension was incubated with different biopreservatives for a total of 10 h at 37℃, followed by double staining of live and dead cells using Calcein-AM/PI. Calcein-AM is hydrolyzed by intracellular esterase after entering the cell to produce a strong anionic green fluorescence product, thereby labeling the living cells with green fluorescence. Propidium iodide (PI) cannot cross the cell membrane of living cells and only enters when the membrane is damaged; It inserts into the DNA, producing a red fluorescence that labels dead cells. This differential staining protocol labels both live and dead cells in all samples, and their visualization under confocal microscopy provides a rapid quantitative assessment of the antimicrobial efficacy of bio preservatives.
As shown in Figure 8, all tested bio preservatives exerted bactericidal activity against both Escherichia coli and Bacillus subtilis, although the extent of killing varied markedly among treatments. Notably, exposure to Plectasin or the LL-37 resulted in a pronounced reduction in B. subtilis viability, indicating potent bactericidal efficacy against this species. Conversely, Cecropin A left a substantially higher residual viable count of B. subtilis, revealing a comparatively weak activity against this bacterium. In contrast, Plectasin was less effective against E. coli than against B. subtilis, whereas LL-37 displayed superior bactericidal performance against E. coli relative to its effect on B. subtilis. Through the observation under confocal microscope, we confirmed that our antimicrobial peptide protein has certain killing ability to bacteria. Moreover, our lysozyme also has a certain bactericidal effect on Bacillus subtilis. Through the screening of the above experiments, we determined that Plectasin and LL-37 are the best antibacterial peptides we have obtained at present, and further we want to observe the morphology of the treated bacteria by scanning electron microscope.
Figure 8. Laser Scanning Confocal Microscope results. A: E. coli; B: Bacillus subtilis.
Morphological observation of scanning electron microscopeFurther, we want to observe the morphology of bacteria treated with antibacterial peptides by scanning electron microscope. We chose two antibacterial peptides (Plectasin and LL-37) which had the most remarkable effect in the previous antibacterial experiment and chose AMP as the positive control. By scanning electron microscope, we can see that compared with the bacteria not treated with antimicrobial peptides, the surfaces of Escherichia coli and Bacillus subtilis were damaged to varying degrees after treated with antimicrobial peptides, which means that our antimicrobial peptides may damage the cell wall or cell membrane structure of bacteria, thus causing bacterial death. The specific bactericidal mechanism of antimicrobial peptides needs further analysis.
Figure 9. Scanning electron microscope observation results. A: E. coli; B: Bacillus subtilis.
This study provides an important reference for other iGEM teams. Other teams can carry out further exploration based on our research results, mainly proposing to clone lysozyme and antimicrobial peptide genes into expression vectors, respectively, and transform them into Escherichia coli DH5α and Pichia GS115. By expressing recombinant proteins in Pichia yeast, the genetic system of lysozyme and antimicrobial peptides was successfully constructed. Finally, the antibacterial effects of lysozyme and antimicrobial peptides were verified by qualitative characterization and judgment, quantitative characterization and quantitative analysis. In this study, the expression of lysozyme and antimicrobial peptides was improved, but the yield was still unsatisfactory, which was probably related to improper promoter selection and insufficient codon optimization. To this end, subsequent teams can increase product concentration and biological activity by implementing codon-promoter comediation, co-expression of chaperones, and high-density cell culture strategies. In addition, functional tests showed that the antibacterial efficacy of the obtained biopreservatives was limited, suggesting that the expression environment may affect the conformation or stability of the fusion protein. Therefore, other iGEM team can also adopt a multi-expression system combination strategy: by comparing different host strains, the best expression system that can maintain the natural fold structure and maximize the antibacterial effect can be screened.
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