We integrated four distinct promoters—AOX1, CAT1, FDH1, and AOX713—into the pPIC9K backbone plasmid to drive the expression and secretion of the antimicrobial peptide NZ2114. These promoters function in conjunction with other regulatory elements present in the vector, including signal peptides, to enable coordinated transcriptional control and efficient protein secretion. In this study, we constructed a series of recombinant plasmids based on the pPIC9K-AOX1-⍺Factor-NZ2114 framework, in which the AOX1 promoter was systematically replaced with FDH1, CAT1, or AOX713, resulting in three derivative constructs. Together with the original AOX1-containing construct, this yielded a total of four promoter variants for comparative evaluation.

Fig. 1 Schematic diagram of plasmid design in Snapgene

As shown in Figure 2, during the plasmid construction process, we first amplified the synthetic target gene fragment by PCR and subsequently purified it to obtain the desired DNA sequence. Using homologous recombination, we integrated promoter-containing fragments into the pPIC9K backbone plasmid and transformed the recombinant constructs into Escherichia coli DH5α.

Fig .2 DNA electrophoresis of PCR product of promoter fragment and plasmid backbone fragment.

Fig. 3 Transformation of Escherichia coli competent cells with promoter plasmids and verification of colony monoclonal clones

Colony PCR analysis confirmed successful plasmid assembly, with amplification of the expected bands observed in multiple clones (Figure 3). Positive colonies were then selected for large-scale culture and plasmid extraction to prepare for downstream applications.

Fig. 4 Sanger sequencing was used to identify the constructed plasmid DNA

Following identification of positive DH5α clones and plasmid extraction, Sanger sequencing was performed to verify the correct assembly of the recombinant plasmids. As shown in Figure 4, the sequencing results confirmed accurate integration of the target fragments, thereby validating the successful construction of the expression vectors.

The recombinant plasmids were successfully transformed into Pichia pastoris GS115, and following antibiotic selection, engineered yeast strains harboring distinct promoter constructs were obtained. These transformants were subjected to large-scale cultivation in BMMY medium with methanol induction to drive the expression and secretion of Plectasin NZ2114 into the culture supernatant. The presence of the target protein in the supernatants was subsequently analyzed by SDS-PAGE, and as shown in Figure 5, a band corresponding to the expected molecular weight of Plectasin NZ2114 was clearly detected in all engineered strains, confirming successful expression and extracellular accumulation.

Fig. 5 Expression of plectasin NZ2114 driven by different promoter and verification by Tris-Tricine PAGE Electrophoresis

The culture supernatants from various yeast fermentation batches were evaluated for antimicrobial activity against Escherichia coli DH5α using the Oxford cup diffusion assay. E. coli DH5α, a widely used laboratory strain, served as the indicator organism. All supernatants derived from strains harboring different promoters exhibited detectable inhibitory effects. Notably, the strain driven by the AOX713 promoter produced the smallest inhibition zone, indicating relatively lower activity, whereas the supernatants from strains under the control of AOX1, FDH1, and CAT1 promoters displayed strong and comparable antibacterial efficacy, as evidenced by similarly sized inhibition zones.

Fig. 6 The inhibition zone experiment was used to detect the fermentation products of different promoter/signal peptide strain strains.

Protein electrophoresis and antimicrobial zone assays demonstrated that all tested promoter constructs successfully drove the expression of the NZ2114 antimicrobial peptide in Pichia pastoris, resulting in detectable antibacterial activity. Notably, strains harboring the AOX1, FDH1, and CAT1 promoters exhibited higher levels of protein expression and stronger inhibitory effects compared to others. Among these, the AOX1-driven strains displayed superior consistency across biological replicates, indicating enhanced expression stability. Furthermore, considering the broad applicability and widespread adoption of the AOX1 promoter—particularly in combination with the pPIC9K vector system—it has become a standard choice for recombinant protein expression in Pichia pastoris. Based on its strong performance, reproducibility, and established utility, AOX1 was selected as a key regulatory element for constructing the high-yield Plectasin NZ2114-producing yeast strain.

As shown in the Fig .8, First, based on the construction of the expression plasmid with the promoter, we chose pPIC9K-AOX1-NZ2114 as the backbone plasmid for constructing different signal peptide expression plasmids. We obtained gene fragments of the 0030, SP4, and SP14 signal peptide sequences through PCR amplification.

Fig. 8 Gel electrophoresis of agarose nucleic acid for identifying signal peptide sequence fragments and plasmid backbone fragments

The amplified target fragments were purified and subsequently ligated into the linearized vector via homologous recombination, followed by transformation into Escherichia coli DH5α competent cells. Transformants were selected by culturing on solid agar plates, and positive colonies were screened by colony PCR. As shown in Figure 9, the presence of PCR amplicons of the expected size confirmed the successful construction of the recombinant plasmids containing different signal peptides. Positive clones were then expanded in liquid culture, and plasmid DNA was isolated for further use.

Fig. 9 Transformation of signal peptide plasmids into Escherichia coli competent cells and colony identification

Following the isolation of plasmid DNA from constructs containing different signal peptides, Sanger sequencing was performed. As shown in Figure 10, the sequencing results confirmed that the obtained sequences precisely matched the intended design, with strong and clean signals across all reads and no evidence of nucleotide mismatches or mutations.

Fig. 10 Sanger sequencing for identifying the plasmid containing the signal peptide and its construction.

Following successful construction of the signal peptide-containing plasmid DNA, the plasmids were linearized and transformed into chemically competent GS115 cells. After screening on selective plates and molecular identification, stable Pichia pastoris transformants harboring different signal peptide constructs were successfully obtained. These engineered yeast strains were then subjected to large-scale cultivation and induced for recombinant antimicrobial peptide expression with methanol. Protein electrophoresis analysis revealed distinct expression levels among the strains, as shown in Figure 11. The ⍺-factor-mediated construct demonstrated significantly higher secretion and expression of the NZ2114 antimicrobial peptide compared to those directed by SP4, SP14, and 0030 signal peptides.

Fig. 11 Expression of Plectasin NZ2114 driven by different signal peptide and Tris-Tricine PAGE Electrophoresis verification

To evaluate the functional efficacy of the secreted antimicrobial peptides, inhibition zone assays were performed using culture supernatants from Pichia pastoris strains expressing NZ2114 under the control of different signal peptides. As shown in Figure 12, the supernatant from the ⍺-factor-expressing strain produced a significantly larger inhibition zone compared to those of the SP4, SP14, and 0030 signal peptide strains. In contrast, the strain carrying the 0030 signal peptide exhibited almost no detectable antibacterial activity.

Fig. 12 The inhibition zone experiment was used to detect the fermentation products of different promoter/signal peptide strain strains.

In this engineering cycle, we constructed and functionally evaluated engineered yeast strains expressing Plectasin NZ2114 using different signal peptides. Among the tested variants, the ⍺-factor signal peptide exhibited significantly enhanced secretion efficiency. Based on these results, ⍺-factor was selected as a key regulatory element for subsequent development of a high-yield Plectasin NZ2114-producing yeast strain.

Building upon the results from the first two engineering cycles, small-scale fed-batch fermentations were carried out for the engineered yeast strains harboring the selected promoter and signal peptide combination. Induced expression was performed under methanol induction, and two independent batches of fermentation supernatants were collected from both the control strain (empty pPIC9K Pichia pastoris) and the recombinant strain (pPIC9K-AOX1-⍺-factor-NZ2114 Pichia pastoris). The antimicrobial activity in the harvested supernatants was comprehensively evaluated to validate the functional efficacy of the target product.

Two engineered Pichia pastoris strains were selected for fermentation: the control strain harboring the empty pPIC9K vector and the recombinant strain expressing NZ2114. Both strains were initially inoculated into 3 mL YPD medium in test tubes and incubated with shaking. A 1% (v/v) volume of the resulting seed culture was transferred to 30 mL BMGY medium for overnight growth until the OD600 reached approximately 4. Cells were then harvested and resuspended in 50 mL BMMY induction medium. Methanol was added every 24 hours to a final concentration of 1% (v/v) to maintain induction of the AOX1 promoter. After 120 hours of induced expression, cultures were centrifuged, and the supernatants were collected and stored at –80°C for subsequent protein activity assays.

Escherichia coli DH5α and Bacillus subtilis were selected as model bacterial strains for the subsequent evaluation of antimicrobial peptide activity. The assessment included growth curve analysis, cell viability assays, and scanning electron microscopy (SEM) examination to comprehensively characterize the biological effects of the peptide.

The growth curve assay

1% (v/v) seed cultures of Escherichia coli and Bacillus subtilis were inoculated as the initial inoculum and mixed with varying proportions of yeast culture supernatants. The mixtures were incubated in a shaking incubator, and the OD600 was measured at predetermined time intervals to monitor bacterial growth.

As shown in Figures 13A and 13B, the E. coli control group (treated with empty vector supernatant) exhibited normal growth, reaching an OD600 of approximately 1. In contrast, the NZ2114-containing supernatant significantly suppressed bacterial growth in a dose-dependent manner: 10% and 25% supplementation resulted in partial inhibition, while 50% and 100% supplementation completely abolished the growth of E. coli DH5α. Similarly, as depicted in Figures 13C and 13D, B. subtilis in the control group grew robustly, whereas all NZ2114 treatment groups—except the 0% (no supernatant) control—completely inhibited bacterial growth, indicating potent and broad-spectrum antimicrobial activity of the expressed peptide.

Fig. 13 Determination of the inhibitory growth curve of the culture supernatant of engineered yeast strains: 2h, 4h, 6h, 24h, 30h

Cell viability assay

Luminescence signals were quantified using a microplate reader, and the results are presented in Figure 14. Compared to the control group treated with supernatant from the empty vector-carrying GS115 yeast strain, the supernatant from the NZ2114-expressing strain significantly reduced the viability of both Escherichia coli and Bacillus subtilis at 50% concentration. Notably, the inhibitory effect on Bacillus subtilis was more pronounced, achieving nearly 80% reduction in cell viability.

Fig.14 Luminescence detection of control and NZ2114 treated DH5 alpha and B.subtilis in a 50% ratio of fermentation culture medium.

SEM analysis of bacterial morphological changes indicative of AMP killing

To further evaluate the membrane-disrupting activity and bactericidal efficacy of the antimicrobial peptide NZ2114, Bacillus subtilis was selected as a structurally relevant model organism for SEM analysis, owing to its shared cellular features with drug-resistant Staphylococcus aureus.

Fig. 15 SEM electron microscope image of Bacillus subtilis morphology at a 1-micron scale, control group (A, B, C), NZ2114 treatment group (D, E, F)

As shown in Fig. 15, SEM observations at a 1-micron scale were performed across multiple fields of view. In the control group (Fig. 15 A, B, C), Bacillus subtilis cells and spores exhibited smooth and intact surface morphologies. In contrast, NZ2114-treated Bacillus subtilis (Fig. 15 D, E, F) displayed clear morphological alterations, including cell surface wrinkling, collapse, and irregularity. These structural changes are consistent with the known mechanism of antimicrobial peptides, which exert their bactericidal effects by disrupting the bacterial membrane. The observed damage provides direct visual evidence that Plectasin NZ2114 compromises membrane integrity in Bacillus subtilis.

Furthermore, high-resolution SEM imaging at a 200-nanometer scale (Fig. 16) revealed more pronounced ultrastructural damage in the treatment group. Compared to the uniformly smooth and intact surfaces of control bacteria, NZ2114-treated cells not only exhibited severe wrinkling but also developed crack-like fissures on their surfaces. These findings further substantiate the membrane-disrupting mode of action of NZ2114 and confirm its potent bactericidal effect against Bacillus subtilis.

Fig. 16 SEM electron microscope image of Bacillus subtilis morphology at a 200-nanometer scale, control group (A, B), NZ2114 treatment group (D, E)

The results from multiple analytical methods consistently demonstrate that the engineered Pichia pastoris strain developed for high-yield production of Plectasin NZ2114 effectively expresses and secretes the functional antimicrobial peptide into the culture supernatant, exhibiting strong antibacterial activity. These findings establish a solid foundation for future industrial-scale applications. Future efforts should focus on optimizing large-scale fermentation conditions, refining purification protocols, and evaluating product stability to advance the commercialization of this innovative antimicrobial peptide in broader therapeutic and biotechnological contexts.