Abstract
Lactoferricin, a multifunctional peptide located in the N-terminal region of lactoferrin, has a broad-spectrum bacteriostatic activity. It is a promising candidate as a food additive and immune fortification agent and does not have the risks associated with drug residues and drug resistance. First, we performed promoter and host cell screening to achieve the recombinant expression of lactoferricin in Pichia pastoris, showing an initial titer of 19.5 mg/L in P. pastoris X-33 using PAOX1 promoter. Second, we constructed a 0030-α hybrid signal peptide by fusing the 0030 signal peptide with the pro-sequence of α-factor secretory signal peptide. This further increased the production of lactoferricin, with a titer of 28.8 mg/L in the fermentation supernatant in the shaking flask. Next, we increased the expression of lactoferricin by fusing it with anionic antioxidant peptides. The neutralization of positive charges yielded a titer of 55.3 mg/L in the shaking flask, and a highest titer of 193.9 mg/L in a 3-L bioreactor. The antimicrobial activity analysis showed that recombinant-expressed lactoferricin exhibited potent antibacterial activity against Escherichia coli, Bacillus subtilis, and Staphylococcus aureus. This study provides a reference for the construction of microbial cell factories capable of efficiently synthesizing antimicrobial peptides.
Keywords: Antimicrobial peptide, Lactoferricin, Recombinant expression, Pichia pastoris
Graphical abstract
1. Introduction
Antimicrobial peptides (AMPs) are small peptides that exhibit broad-spectrum antimicrobial activities against bacteria and fungi [1,2]. These unique naturally occurring antimicrobial compounds can be used as antioxidants, food additives, and immune fortification agents regardless of the problems related to drug residues and drug resistance [3]. AMPs have been identified in diverse organisms, from bacteria to plants to humans. Some of the most well-known AMPs are lactoferrin peptides, which are short-functional peptides released from lactoferrin by the action of proteolytic enzymes in the digestive environment [4]. It has been reported that lactoferrin peptides exhibit antibacterial effects that are 200-400-fold more potent than lactoferrin [5].
Many antimicrobial lactoferrin peptides have already been isolated and characterized, and the N-terminal peptides of lactoferrin have stronger antibacterial activities than its C-terminal peptides [6]. Lactoferricin, a multifunctional peptide located in the N-terminal region at amino acids 17–41 of the lactoferrin protein, is characterized by a disulfide bond, an abundance of basic and hydrophobic amino acids, and a net positive charge of 8+ [1]. Lactoferricin exhibits multiple biological activities, including bacteriostatic, antiviral, antiprotozoal, tumor-suppressive, and cell apoptosis induction properties [1,7].
Recombinant peptide expression in microbial cells has attracted widespread attention due to its low cost and simple preparation process [3,4]. For example, the pPIC9K-His plasmid has been used to express the bovine lactoferricin-derived peptide in Pichia pastoris GS115, with methanol as the inducer [4]. Moreover, Lee et al. used the promoter PtrnQ to express six tandem repeats of lactoferricin in Bacillus subtilis [8]. However, the levels of lactoferricin produced via recombinant expression in microbial cells are relatively low, and further research is needed to develop more efficient production methods.
P. pastoris has clear advantages in the protein processing, extracellular secretion, post-translational modifications, and glycosylation, which is now widely used for the expression of exogenous proteins or peptides [9,10]. In this study, we first performed host cell selection and signal peptide (SP) screening to express and extracellularly secrete the lactoferricin peptide in P. pastoris. Then, the fusion of lactoferricin with anionic antioxidant peptides alleviated its positive charge and enhanced lactoferricin synthesis, achieving a titer of 55.3 mg/L. The antibacterial activity of the recombinant lactoferricin peptide against Escherichia coli, Staphylococcus aureus, and Bacillus subtilis was assessed using the Oxford cup method. Our results demonstrated that recombinant lactoferricin exhibited a potent antibacterial activity, suggesting that our approach has potential for industrial production of antimicrobial peptides.
2. Materials and methods
2.1. Chemicals and reagents
Unless otherwise specified, all chemicals and the bicinchoninic acid (BCA) Protein Assay Kit were purchased from Sangon Biotech Co., Ltd. (Shanghai, China). The plasmid extraction and DNA gel purification kits were purchased from Thermo Scientific (Waltham, USA). The Gibson Assembly Kit was purchased from New England Biolabs (Beijing, China). The primers used in this study were synthesized by GENEWIZ (Suzhou, China).
2.2. Plasmids and strains
The strains and plasmids used in this study are listed in Table 2, while the primers are listed in Table S1. The E. coli DH5α strain was used for plasmid construction, and P. pastoris GS115, X-33, SMD1163, and SMD1168 strains were used as the host cells for recombinant expression of the lactoferricin. P. pastoris was transformed via electrotransformation [11].
Table 2.
Strains and plasmids used in this study.
| Strains/Plasmids | Characteristic | Sourcea |
|---|---|---|
| Strains | ||
| E. Coli DH5α | F−, φ80, lacZΔM15, Δ(lacZYA-argF)U169, recA1, endA1, hsdR17(rk-, mk+), phoA, supE 44, thi-1, gyrA 96, relA1, λ- | Lab stock |
| P. pastoris SMD1163 | his4 pep4 prb 1 | Lab stock |
| P. pastoris SMD1168 | his4, pep4 | Lab stock |
| P. pastoris GS115 | Wild type | Lab stock |
| P. pastoris X-33 | Wild type | Lab stock |
| GS115-PAOX1-lactoferricin | GS115 strain with pPICZɑA-lactoferricin | This study |
| GS115-PGAP-lactoferricin | GS115 strain with pGAPZɑA-lactoferricin | This study |
| X33-PAOX1-lactoferricin | X-33 strain with pPICZɑA-lactoferricin | This study |
| SMD1163-PAOX1-lactoferricin | SMD1163 strain with pPICZɑA-lactoferricin | This study |
| SMD1168-PAOX1-lactoferricin | SMD1168 strain with pPICZɑA-lactoferricin | This study |
| 0030-lactoferricin | X-33 strain with pPICZɑA-0030-lactoferricin | This study |
| INU1-lactoferricin | X-33 strain with pPICZɑA-INU1-lactoferricin | This study |
| MEL1-lactoferricin | X-33 strain with pPICZɑA-MEL1-lactoferricin | This study |
| OST-lactoferricin | X-33 strain with pPICZɑA-OST-lactoferricin | This study |
| PHO11-lactoferricin | X-33 strain with pPICZɑA-PHO11-lactoferricin | This study |
| SCW10-lactoferricin | X-33 strain with pPICZɑA-SCW10-lactoferricin | This study |
| SP-lactoferricin | X-33 strain with pPICZɑA-SP-lactoferricin | This study |
| SUC2-lactoferricin | X-33 strain with pPICZɑA-SUC2-lactoferricin | This study |
| UTH1-lactoferricin | X-33 strain with pPICZɑA-UTH1-lactoferricin | This study |
| 0030-α-lactoferricin | X-33 strain with pPICZɑA-0030-α-lactoferricin | This study |
| INU1-α-lactoferricin | X-33 strain with pPICZɑA-INU1-α-lactoferricin | This study |
| MEL1-α-lactoferricin | X-33 strain with pPICZɑA-MEL1-α-lactoferricin | This study |
| OST-α-lactoferricin | X-33 strain with pPICZɑA-OST-α-lactoferricin | This study |
| PHO11-α-lactoferricin | X-33 strain with pPICZɑA-PHO11-α-lactoferricin | This study |
| SCW10-α-lactoferricin | X-33 strain with pPICZɑA-SCW10-α-lactoferricin | This study |
| SP-α-lactoferricin | X-33 strain with pPICZɑA-SP-α-lactoferricin | This study |
| SUC2-α-lactoferricin | X-33 strain with pPICZɑA-SUC2-α-lactoferricin | This study |
| UTH1-α-lactoferricin | X-33 strain with pPICZɑA-UTH1-α-lactoferricin | This study |
| PEP1-lactoferricin | X-33 strain with pPICZɑA-0030-α-PEP1-lactoferricin | This study |
| PEP2-lactoferricin | X-33 strain with pPICZɑA-0030-α-PEP2-lactoferricin | This study |
| PEP1/2-lactoferricin | X-33 strain with pPICZɑA-0030-α-PEP1/2-lactoferricin | This study |
| Plasmids | ||
| pPICZαA | PAOX1 promoter:, α-factor secretory signal peptide, 6-His tag ，Zeocin | Lab stock |
| pGAPZαA | PGAP promoter:, α-factor secretory signal peptide, 6-His tag ，Zeocin | Lab stock |
2.3. Cultivation conditions
Luria–Bertani (LB) medium (10 g/L tryptone, 10 g/L NaCl, 5 g/L yeast extract) was used to culture E. coli, and yeast extract peptone dextrose (YPD) medium (20 g/L tryptone, 20 g/L glucose, 10 g/L yeast extract) was used to culture P. pastoris. Ampicillin (100 μg/mL) was used to screen E. coli transformants, while zeocin (100 μg/mL) were used as selection reagents of P. pastoris transformants.
Shake flask fermentation was performed using BMGY (Buffered Glycerol-complex Medium) medium (20 g/L peptone, 10 g/L yeast extract, 0.1 M potassium phosphate buffer [pH 6.0], 1.34% yeast nitrogen base, 4 × 10−5% biotin, and 1% glycerin). BMMY (Buffered Methanol-complex Medium) medium can be produced by replacing 1% glycerin in BMGY medium with 0.5%–1% methanol. BSM medium (26.7 ml/L 85% phosphoric acid, 40.0 g/L glycerin, 14.9 g/L magnesium sulfate-7H2O, 18.2 g/L potassium sulfate, 4.13 g/L potassium hydroxide, and 0.93 g/L calcium sulfate) was used for fed-batch culture. PTM1 consisted of 65.0 g/L ferrous sulfate-7H2O, 20.0 g/L zinc chloride, 6.0 g/L copper sulfate-5H2O, 3.0 g/L manganese sulfate-H2O, 0.08 g/L sodium iodide, 0.5 g/L cobalt chloride, 0.2 g/L biotin, 0.2 g/L sodium molybdate-2H2O, 0.02 g/L boric acid, and 5.0 ml/L sulfuric acid.
The single colonies of P. pastoris were cultured using YPD medium at 30 °C and 220 rpm in a shaken test tube for 16 h. The seed culture bacteria were further inoculated into a 500 mL baffled flask containing 50 mL BMMY medium at a 10% ratio and incubated for 36 h. Subsequently, the cells were harvested, washed, and inoculated into BMGY medium at an equivalent volume. The medium was supplemented daily with 1% methanol. Cell suspension samples were collected every 24 h to measure optical density at 600 nm (OD600) and to perform SDS-PAGE analysis.
For fed-batch culture, the seed culture was performed in baffled 500-mL shake flasks containing 150 mL YPD medium and then incubated for 16 h. The seed culture was inoculated into a 3-L fermenter (T&J Bio-engineering Co., Ltd., Shanghai, China) containing 1 L of fermentation medium. The pH was maintained at 5.5 by adding NH3 H2O. A total of 12 ml/L PTM1 was added to the additional glycerol and methanol, and the methanol concentration was adjusted based on dissolved oxygen levels.
2.4. Antibacterial activity assay
E. coli, B. subtilis, and S. aureus colonies were inoculated into tubes containing LB medium, and cells were grown for 16 h at 37 °C and 220 rpm. The antibacterial activity of the samples was analyzed using the agar diffusion method. Petri dishes were coated with LB agar medium and 100 μL of 16 h culture of the indicator strain. After punching 6-mm-diameter wells into agar, 100 μL of the culture supernatant was loaded into each well, and the plates were incubated at 37 °C for 16 h.
2.5. SDS-PAGE and western blotting
After 24 h of fermentation, 1 mL of each of the culture samples was collected and used to perform centrifugation at 12,000×g and 4 °C for 8 min. The resulting fermentation broth supernatant was treated with 4× sample buffer and boiled for 10 min. The prepared samples and markers were separated via SDS-PAGE on a 10% Bolt Bis-Tris Plus gel using MES SDS running buffer (Thermo Scientific, Waltham, USA). Subsequently, the proteins with different molecular weights were visualized via Coomassie Brilliant Blue R250 staining.
For Western blot analysis, the separated recombinant proteins were transferred from gels to 0.2-μm polyvinylidene difluoride membranes. The membranes were blocked for 1 h in QuickBlock™ Blocking Buffer (Beyotime, Shanghai, China), and then incubated overnight at 4 °C with primary mouse monoclonal antibody against His tag (1:1000 dilution). After washing three times in Tris-Buffered Saline Tween-20, the membranes were incubated with a goat antimouse secondary antibody (1:1000 dilution). The results of the Western blot analysis were visualized via color-developing using the ECL Color Development Kit.
2.6. Lactoferricin purification
The supernatant of the fermentation broth was collected via centrifugation at 6000×g and then purified using the Ni Beads 6FF Gravity column (Smart-Lifesciences). First, the column was washed with 10 column volumes of phosphate-buffered saline (PBS), and the supernatant was loaded onto the pre-balanced gravity column. Next, 10 column volumes of washing buffer with 5 mM imidazole in PBS were added to wash the column. Subsequently, the His-tagged proteins were eluted using 4 column volumes of an elution buffer with 250 mM imidazole in PBS, collecting fractions with each column volume in individual tubes. Afterward, the purity of the lactoferricin was assessed using SDS-PAGE. If only a single target band was observed (Fig. S1), quantification of the purified lactoferricin was performed using BCA Protein Assay Kit.
2.7. Statistical analysis
All experiments were independently repeated at least three times, and the results were expressed as mean ± standard deviation. Statistical comparisons were performed using the two-sided t-test in Excel. P values of <0.05 were considered statistically significant.
3. Results and discussion
3.1. Recombinant expression and host selection of lactoferricin
The recombinant expression of the lactoferricin was achieved by inserting a 75-bp DNA fragment encoding the codon-optimized lactoferricin into the plasmid pPICZɑA containing the inducible promoter PAOX1, α-factor secretory SP and 6-His tag (Fig. 1A). The inducible promoter PAOX1 was then replaced by the constitutive promoter PGAP to compare the relative levels of lactoferricin under inducible and constitutive conditions, respectively. The recombinant plasmids were transformed into P. pastoris GS115 to generate recombinant GS115-PAOX1-lactoferricin and GS115-PGAP-lactoferricin strains. The cell density of GS115-PAOX1-lactoferricin and GS115-PGAP-lactoferricin strains, as measured at OD600, did not show significant changes compared to the control strains expressing the pPICZɑA plasmid (Fig. 1B), suggesting that low doses of lactoferricin peptide did not inhibit the growth of P. pastoris. After 144 h of fermentation in shaking flasks, the supernatants of the fermentation broth were harvested for Western blot analysis. As shown in Fig. 1C, a weak band was detected in the supernatant of the GS115-PGAP-lactoferricin strain, and the band in the supernatant of the GS115-PAOX1-lactoferricin strain was much stronger than that of the GS115-PGAP-lactoferricin strain, with a titer of 11.5 mg/L (Fig. 1C and D). These results indicate that lactoferricin was successfully expressed and secreted using the inducible PAOX1 promoter. Therefore, PAOX1 was selected to carry out the following experiments.
Fig. 1.
Lactoferricin expression in Pichia pastoris. (A) Structure of the recombinant plasmid. (B) Comparison between growth curves of GS115-PAOX1-lactoferricin and GS115-PGAP-lactoferricin strains and the GS115-pPICZɑA strain in the baffled flask. (C) Western blot analysis was performed to detect the expression of lactoferricin. (D) The production of GS115-PAOX1-lactoferricin was detected via the bicinchoninic acid (BCA) protein assay.
The ability of different genotypes to express exogenous proteins can be highly variable [12,13]. Therefore, we examined the expression of lactoferricin in four P. pastoris strains, including GS115, X-33, SMD1163, and SMD1168. The recombinant pPICZɑA-lactoferricin plasmid was transferred into X-33, SMD1163, and SMD1168 strains to obtain X33-PAOX1-lactoferricin, SMD1163-PAOX1-lactoferricin, and SMD1168-PAOX1-lactoferricin strains, respectively. As shown in Fig. 2A, except for the SMD1163-PAOX1-lactoferricin strain, which hardly grew, the OD600 of the X33-PAOX1-lactoferricin, SMD1168-PAOX1-lactoferricin, and GS115-PAOX1-lactoferricin strains did not show significant changes compared to the control strains. The Western blot analysis revealed that the lactoferricin level in the X33-lactoferricin strain was significantly higher than that in SMD1168-PAOX1-lactoferricin and GS115-PAOX1-lactoferricin strains (Fig. 2B). The BCA analysis indicated that the X33-PAOX1-lactoferricin strain produced 19.3 mg/L of lactoferricin, which is 1.7 fold more than the titer produced by the GS115- PAOX1-lactoferricin strain (11.5 mg/L) (Fig. 2C). The above results demonstrate that, for lactoferricin expression, the X-33 strain is a host with a significant advantage. The speculated main reason is that the wild-type X-33 strain has better resistance, such as its enhanced ability to resist metabolic stress or toxicity caused by the overexpression of proteins or peptides. Additionally, similar conclusions were drawn in the expression of HIV-negative factor and human interleukin-1 beta proteins [12,13]. Therefore, the X33-lactoferricin strain was selected for further studies.
Fig. 2.
Comparative analysis of lactoferricin expression profiles in different strains. (A) Comparison of the growth curve among different strains. (B) Western blot analysis was conducted to detect the effects of different genotypes on lactoferricin synthesis. (C) The BCA protein assay was used to detect the synthesis of lactoferricin in the X-33-PAOX1-lactoferricin strain.
3.2. SP screening for lactoferricin secretion
SPs are short, N-terminal peptides ubiquitously present in eukaryotic and prokaryotic cells, which play a role in marking the protein for secretion and target to specific cellular locations [14]. We screened out the most suitable SP for lactoferricin by comparing the secretion of lactoferricin with different SPs, including 0030, INU1, MEL1, OST, PHO11, SCW10, SP, SUC2, and UTH1 (Table 1). The α-factor secretory SP of the pPICZɑA-lactoferricin plasmid was sequentially replaced with these SPs to generate 0030-lactoferricin, INU1-lactoferricin, MEL1-lactoferricin, OST-lactoferricin, PHO11-lactoferricin, SCW10-lactoferricin, SP-lactoferricin, SUC2-lactoferricin, and UTH1-lactoferricin strains. As shown in Fig. 3A, the production of lactoferricin was significantly reduced when the nine SPs were used to replace the α-factor secretory SP, and no immunoblot band was observed.
Table 1.
The sequences and source of signal peptides.
| signal peptides | Amino acid sequences | Source |
|---|---|---|
| 0030 | MKFAISTLLIILQAAAVFAAF | PAS_chr3_0030 signal peptide from P. pastoris. |
| INU1 | MKLAYSLLLPLAGVSASVINYKR | Inulinase signal peptide from Kluyveromcyes maxianus. |
| MEL1 | MRAFLFLTACISLPGVFGVNET | α-galactosidase signal peptide from Saccharomyces cerevisiae. |
| OST1 | MRQVWFSWIVGLFLCFFNVSSA | Ost1 signal peptide from S. cerevisiae. |
| PHO11 | MLKSAVYSILAASLVNA | Acid phosphatase signal peptide from S. cerevisiae. |
| SCW10 | MQVKSIVNLLLACSLAVA | SCW10 signal peptide from P. pastoris |
| SP | MKLVFLVLLFLGALGLCLA | Lactoferrin signal peptide from Bos taurus |
| SUC2 | MLLQAFLFLLAGFAAKISA | Invertase signal peptide from S. cerevisiae. |
| UTH1 | MKSQLIFMALASLVAS | UTH1 signal peptide from K. marxianus. |
Fig. 3.
Signal peptide screening for lactoferricin secretion. (A) Western blot analysis was performed to detect the effects of different signal peptides on lactoferricin synthesis. (B) The schematic diagram of hybridization of different signal peptides. (C) The Western blot analysis was used to detect the effects of hybridized signal peptides on lactoferricin synthesis. (D) The BCA protein assay was used to compare the production of lactoferricin in α-lactoferricin and 0030-α-lactoferricin strains.
Conventional SPs consist solely of the pre sequence, while the α-factor secretory SP is comprised of both pre- and pro-sequences, with the latter containing an endoplasmic reticulum (ER) export signal that is specifically recognized by the transmembrane Erv29 receptor [15]. The receptor-mediated export likely contributes to the efficacy of the α-factor secretory SP in facilitating heterologous protein production [15]. But a limitation of the α-factor secretory SP is that the signal sequence portion directs posttranslational translocation across the ER membrane, as a result, if the α-factor secretory SP is fused to a protein that can fold in the yeast cytosol, the protein may be unable to cross the ER membrane to initiate the secretory pathway. Replacing the pre-sequence of α-factor secretory SP with the Ost1 SP could increase protein expression [16]. For example, the secretion levels of E2-Crimson and lipase BTL2 were significantly increased 20 and 10-fold compared to the original α-factor secretion SP, respectively, when a hybrid SP consisting of OST1 and the α-factor pro-sequence was used [16]. We attempted to hybridize the α-factor secretory SP by replacing its pre-sequence with that of 0030, INU1, MEL1, OST, PHO11, SCW10, SP, SUC2, and UTH1 SPs (Fig. 3B). The resulting strains were named as follows: 0030-α-lactoferricin, INU1-α-lactoferricin, MEL1-α-lactoferricin, OST-α-lactoferricin, PHO11-α-lactoferricin, SCW10-α-lactoferricin, SP-α-lactoferricin, SUC2-α-lactoferricin, and UTH1-α-lactoferricin. The titers of lactoferricin in 0030-α-lactoferricin, OST-α-lactoferricin, UTH1-α-lactoferricin strains were significantly higher than that in the X33-PAOX1-lactoferricin strain, with the highest titer observed in the 0030-α-lactoferricin strain (28.8 mg/L) (Fig. 3C and D). These results suggest that hybrid SPs can combine the properties of different SPs to enhance the secretion of recombinant proteins or peptides.
3.3. Fusion of anion antioxidant peptides promotes lactoferricin synthesis
Our experimental results show that the increase in lactoferricin synthesis was correlated with a decrease in OD600 (Fig. 4A), suggesting that high doses, especially those exceeding 20 mg/L, lactoferricin has an inhibitory effect on P. pastoris. It has been shown that the antibacterial activity of antimicrobial peptides is positively correlated with the number of net positive charges [1]. For example, the net positive charge of bovine lactoferrampin (5+) has been associated with higher antibacterial activity than that of human lactoferrampin, which has a reduced positive charge (2+) [17]. Indeed, the antibacterial activity of human lactoferrampin can be significantly improved by artificially increasing its net positive charge [18]. Because the net positive charge of lactoferricin is 8+ [1], we attempted to alleviate the antimicrobial effect by fusing lactoferricin with anionic antioxidant peptides, thereby neutralizing the positive charges.
Fig. 4.
The fusion of anion antioxidant peptides promotes the synthesis of lactoferricin. (A) Effects of lactoferricin expression on strain growth. (B) Structure of lactoferricin fused with anionic antioxidant peptides. Anionic antioxidant peptides were ligated to the N-terminus of the gene via hydrolysis. (C) SDS-PAGE was used to detect the effects of antioxidant peptides on the synthesis of lactoferricin. (D) A BCA protein assay was performed to compare lactoferricin production in 0030-α-lactoferricin and PEP1/2-lactoferricin strains. (E) Growth curve and lactoferricin production of the PEP1/2-lactoferricin strain during fermentation, with methanol added after 24 h of culture.
The anionic antioxidant peptides PEP1 and PEP2, which consist of DAQEKLEIEAEGE and EELDNALN amino acid sequences, respectively, were selected from the porcine myofibrillar protein. PEP1 and PEP2 peptides have five and three net negative charges, respectively [3]. Single PEP1 and PEP2 peptides and the tandem PEP1-PEP2 dipeptide were fused at the N-terminal region of lactoferricin (Fig. 4B), and the resulting strains were named PEP1-lactoferricin, PEP2-lactoferricin, and PEP1/2-lactoferricin, respectively. SDS-PAGE analysis after 6 days of fermentation showed that the PEP1/2-lactoferricin strain exhibited the highest titer (55.3 mg/L). These results suggest that neutralizing the positive charge may enhance lactoferricin expression (Fig. 4C and D). The lactoferricin synthesis ability of the PEP1/2-lactoferricin was further validated in a 3-L bioreactor. The titer of lactoferricin produced by the PEP1/2-lactoferricin was monitored at 24 h intervals following the addition of methanol. The BCA protein assay revealed that after 96 h of fermentation, the titer of lactoferricin reached a maximum of 193.9 mg/L. With the extension of fermentation time, lactoferricin underwent partial degradation, and by 120 h of fermentation, the titer decreased to 150.3 mg/L (Fig. 4E). To the best of our knowledge, this is the highest titer of lactoferricin produced by recombinant expression.
3.4. Antimicrobial activity of recombinant-expressed lactoferricin
The antimicrobial activity of recombinant lactoferricin was assessed in E. coli, B. subtilis, and S. aureus test strains. The fermentation supernatants of the strains with the empty pPICZɑA plasmid and 100 μg/L of ampicillin were used as negative and positive controls, respectively. As shown in Fig. 5A and B, the negative control did not exhibit antibacterial activity. The 0030-α-lactoferricin strain showed potent antibacterial activity against E. coli, B. subtilis, and S. aureus, as evidenced by the diameters of the inhibition zone (20, 20, and 19 mm, respectively). In contrast to 0030-α-lactoferricin, the antibacterial activity of the PEP1/2-lactoferricin strain was significantly weak, as evidenced by the almost complete absence of an inhibition zone (Fig. 5A and B). The lower antibacterial activity of PEP1/2-lactoferricin may be attributed to the decrease in its positive charge, which is consistent with previous reports [1]. These results suggest that the net positive charge of lactoferricin plays a role in its antimicrobial activity, which is supported by the observation that recombinant lactoferricin exhibits significantly higher antibacterial activity than fusion peptides.
Fig. 5.
Antimicrobial activity of recombinant-expressed lactoferricin. (A) The inhibition zones of the 0030-α-lactoferricin strain and PEP/1/2-lactoferricin strain against Escherichia coli, Staphylococcus aureus, and Bacillus subtilis are shown. References: 0030-α: sixth-day culture supernatant of the 0030-α-lactoferricin strain. PEP1/2: sixth-day culture supernatant of the PEP/1/2-lactoferricin strain. Ampicillin: 100 μg/mL, positive control. X-33-pPICZɑA: sixth-day culture supernatant of the X-33-pPICZɑA strain, negative control. (B) The inhibition zones formed by the culture supernatants of the 0030-α-lactoferricin strain, PEP1/2-lactoferricin strain, the positive control, and the negative control were measured and compared. The diameter of the Oxford cup hole was 8 mm.
4. Conclusions
In this study, host cell selection, SP screening, and fusion expression of anionic antioxidant peptides enabled efficient recombinant expression of lactoferricin to maximum titers of 55.3 mg/L and 193.9 mg/L in shaking flask and a 3-L bioreactor, respectively. The agar diffusion assay showed that recombinant lactoferricin exhibited potent antimicrobial activity against gram-negative and gram-positive bacteria, indicating that it is an efficient broad-spectrum AMP. The findings of this study provide valuable information for the development of cell factories for AMPs.
CRediT authorship contribution statement
Xueqin Lv: Conceptualization, Investigation, Methodology, Visualization, Writing – original draft. Yuting Zhang: Investigation, Methodology. Lingrui Wang: Methodology. Shixiu Cui: Investigation. Yanfeng Liu: Writing – review & editing. Jianghua Li: Supervision. Guocheng Du: Supervision, Resources. Long Liu: Conceptualization, Supervision, Funding acquisition, Resources, Writing – review & editing.
Declaration of competing interest
The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.
Acknowledgements
This work was supported by the National Natural Science Foundation of China (32021005, 32270096), and the Fundamental Research Funds for the Central Universities (JUSRP222007, JUSRP622004).
Footnotes
Peer review under responsibility of KeAi Communications Co., Ltd.
Supplementary data to this article can be found online at https://doi.org/10.1016/j.synbio.2023.12.002.
Appendix A. Supplementary data
The following is/are the supplementary data to this article:
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