Method for improving yield of antibacterial peptide expressed by pichia pastoris

Abstract

The application discloses a method for improving the yield of an antibacterial peptide expressed by Pichia pastoris, and comprises the following steps: integrating a coding gene of an exogenous protein selected from the following group into the genome of a Pichia pastoris host bacterium for expressing the antibacterial peptide: glutathione S-transferase (GST), protein disulfide isomerase (PDI), 6-phosphofructo-2-kinase (PFK2), bZIP transcription factor (HAC1) and aspartyl aminopeptidase (APE2); and screening a positive clone with improved antibacterial peptide expression level compared with the Pichia pastoris host bacterium. The recombinant bacterium constructed by the method is used for producing the antibacterial peptide cerulenin NZ2114, the expression amount of the NZ2114 is significantly improved, and the method has a good popularization and application prospect.

Description

Technical Field

[0001] This invention belongs to the fields of protein engineering and fermentation engineering technology, and specifically relates to a method for increasing the yield of antimicrobial peptides, especially plectasin such as NZ2114, expressed in Pichia pastoris. Background Technology

[0002] Antimicrobial peptides (AMPs) are one of the most promising new types of antimicrobial drugs. AMPs typically consist of fewer than 100 positively charged hydrophobic amino acid residues, forming a short, bioactive peptide sequence with strong thermal stability. Antimicrobial peptides are attracting increasing attention in the food, pharmaceutical, feed, and agricultural sectors, and have broad application prospects.

[0003] Mycotoxins NZ2114, an antimicrobial peptide, is a fungal defensin derivative. Compared to traditional fungal defensins, it has three amino acid mutations (D9N, M13L, Q14R). Composed of 40 amino acids, it can directly bind to lipid II, a precursor of bacterial cell walls, inhibiting its binding to peptidoglycan, an important component of Gram-positive bacteria. This interferes with cell wall biosynthesis, thus exhibiting good inhibitory effects against Gram-positive bacteria and being insensitive to drug resistance. Furthermore, mycotoxin NZ2114 exhibits inhibitory activity against methicillin-resistant Staphylococcus aureus (MRSA), a strain that exhibits refractory resistance. Drug sensitivity testing showed that NZ2114's inhibitory activity against Staphylococcus aureus is stronger than that of fungal defensins. NZ2114 demonstrates high inhibitory efficacy against Staphylococcus aureus both in vitro (bovine mammary epithelial cells) and in vivo (mouse mastitis model). This suggests that mycelium, which has biological functional activity, can be used as an antibiotic alternative.

[0004] Studies on the preparation methods of the antimicrobial peptide mycoplasmycin have shown that the separation of natural products results in low yield, complex processes, and high costs; artificial chemical synthesis methods are more suitable. Higher synthesis costs and the inability to mass-produce mycotoxins have become limiting factors for their widespread application. Therefore, utilizing genetic engineering and microbial fermentation expression to address the supply shortage and enable large-scale production of mycotoxins has been a research hotspot. For example, Wang Jianhua et al. In 2014, mycomycin NZ2114 was first introduced and successfully expressed in *Pichia pastoris*, with total secreted protein and purified protein reaching 2390 mg / L and 583 mg / L, respectively. Compared with traditional antibiotics, NZ2114 showed stronger activity (0.028–0.9 μmol / L) than ampicillin (1.35–172.50 μmol / L) and vancomycin (0.71–5.67 μmol / L), and also exhibited a post-antibiotic effect (PAE). Since its initial isolation, mycomycin's heterologous expression system has achieved multiple technological breakthroughs: from the construction of the *Pichia pastoris* system, to the application of fusion tag strategies, and the continuous optimization of high-density fermentation processes, the expression level of mycomycin NZ2114 has been gradually increased to the gram scale. However, significant room for yield improvement remains in areas such as systematic screening of expression elements and targeted gene modification of secretion pathways. Future synergistic use of multiple technologies is expected to further unlock the industrial production potential of mycelium. Summary of the Invention

[0005] We conducted a series of explorations on the production of mycelium-derived antimicrobial peptide NZ2114 using Pichia pastoris fermentation. First, we systematically screened four different Pichia pastoris host strains—X-33, GS115, SMD1168, and KM71H—to determine the optimal host, Pichia pastoris X-33 (Invitrogen). Second, we screened expression elements, including promoters, terminators, and signal peptides, that were optimally adapted to the antimicrobial peptide. Based on this, we optimized the copy number of the exogenous gene to obtain a high-copy recombinant strain. Subsequently, through comparative transcriptomics analysis, we compared the differences between the high-copy strain and the non-target gene strain to explore the regulatory mechanism of antimicrobial peptide secretion via cellular metabolic pathways. Based on the transcriptomics results, we promoted the synthesis of mycelium-derived antimicrobial peptides by overexpressing genes from key metabolic pathways. The Pichia pastoris strain culture technology established through high-density fermentation enabled the high-density fermentation of the antimicrobial peptide NZ2114 in a medium-sized fermenter, laying the foundation for large-scale production. Specifically, this invention includes the following technical solutions.

[0006] The first aspect of the present invention provides a method for improving the expression of antimicrobial peptides in Pichia pastoris, characterized by comprising the following steps:

[0007] The genes encoding exogenous methanol metabolism pathway proteins selected from the following groups were integrated into the genome of Pichia pastoris host strain expressing antimicrobial peptides: glutathione S-transferase (GST), protein disulfide isomerase (PDI), phosphofructo-2-kinase (PFK2), bZIP transcription factor HAC1 (ATF / CREB1 homolog), aspartate aminopeptidase (APE2), and the GST+PDI combination.

[0008] Positive clones with higher expression levels of antimicrobial peptides than the Pichia pastoris host strain were screened out, and the engineered Pichia pastoris strain overexpressing the exogenous protein was constructed to produce the antimicrobial peptides through fermentation.

[0009] The aforementioned exogenous methanol metabolism pathway proteins can serve as cofactors for increasing the production of antimicrobial peptides expressed in Pichia pastoris.

[0010] Preferably, the exogenous protein is a methanol metabolism pathway protein derived from Komagataella phaffii GS115: wherein

[0011] Glutathione S-transferase (GST) is gene number PAS_chr1-4_0226, and its amino acid sequence is shown in SEQ ID NO: 3, NCBI accession number XP_002490341.1 (2023). The nucleotide sequence of the CDS region of its encoding gene is shown in SEQ ID NO: 4, NCBI accession number XM_002490296.1 (2023).

[0012] The protein disulfide isomerase, PDI, is identified by gene number PAS_chr4_0844, with its amino acid sequence shown in SEQ ID NO: 5, NCBI accession number XP_002494292.1 (2023). The nucleotide sequence encoding the CDS region of the gene is shown in SEQ ID NO: 6, NCBI accession number XM_002494247.1 (2023).

[0013] 6-Phosphotrexate-2-kinase, or PFK2, is gene number PAS_chr2-1_0870, and its amino acid sequence is shown in SEQ ID NO:7, NCBI accession number XP_002491545.1 (2023). The nucleotide sequence encoding the CDS region of the gene is shown in SEQ ID NO:8, NCBI accession number XM_002491500.1 (2023).

[0014] The bZIP transcription factor HAC1 is gene number PAS_chr1-1_0381, and its amino acid sequence is shown in SEQ ID NO: 9, NCBI accession number XP_002490039.1 (2023). The nucleotide sequence of the CDS region encoding the gene is shown in SEQ ID NO: 10, NCBI accession number XM_002489994.1 (2023).

[0015] Aspartate aminopeptidase APE2 is gene number PAS_chr4_0913, amino acid sequence as shown in SEQ ID NO: 11, NCBI accession number XP_002493442.1 version in 2023; nucleotide sequence of the CDS region encoding the gene is shown in SEQ ID NO: 12, NCBI accession number XM_002493397.1 version in 2023.

[0016] More preferably, the exogenous protein is a glutathione S-transferase, or GST, derived from Komagataella phaffii GS115, with the amino acid sequence shown in SEQ ID NO: 3.

[0017] In one embodiment, the aforementioned antimicrobial peptide is plectasin.

[0018] Preferably, the above-mentioned antimicrobial peptide is mycelium-based mycelium NZ2114, and the amino acid sequence of mycelium-based mycelium NZ2114 is shown in SEQ ID NO: 1:

[0019] GFGCNGPWNEDDLRCHNHCKSIKGYKGGYCAKGGFVCKCY (SEQ ID NO: 1).

[0020] Optionally, the nucleotide sequence of the gene encoding the mycelium NZ2114 is shown in SEQ ID NO: 2:

[0021] GGTTTTGGTTGTAACGGTCCATGGAACGAAGATGATTTGAGATGTCATAACCATTGTAAGTCTATTAAGGGTTACAAGGGTGGTTACTGTGCTAAGGGTGGTTTTGTTTGTAAGTGTTAC (SEQ ID NO: 2).

[0022] In one embodiment, the genome of the Pichia pastoris host strain contains a promoter located upstream of the antimicrobial peptide encoding gene and / or a terminator located downstream of the antimicrobial peptide encoding gene, wherein the promoter is the AOX1 promoter with a nucleotide sequence as shown in SEQ ID NO: 13, and the terminator is the AOX1 terminator with a nucleotide sequence as shown in SEQ ID NO: 14.

[0023] The AOX1 promoter, the antimicrobial peptide encoding gene, and the AOX1 terminator constitute the antimicrobial peptide gene expression cassette / expression frame.

[0024] Furthermore, the genome of the Pichia pastoris host strain also contains a signal peptide, which is an α-factor signal peptide, preferably derived from Saccharomyces cerevisiae (amino acid sequence as shown in SEQ ID NO: 15, nucleotide sequence of the encoding gene as shown in SEQ ID NO: 16).

[0025] Signal peptides, as key components guiding protein secretion, play an important regulatory role in the secretion efficiency of target proteins. The genes encoding signal peptides are located upstream of the genes encoding antimicrobial peptides such as mycotoxins NZ2114.

[0026] Preferably, the genome of the Pichia pastoris host includes two or more copies, more preferably four or more copies, six or more copies, eight or more copies, and more preferably ten copies of an antimicrobial peptide gene or an antimicrobial peptide gene expression cassette / expression frame.

[0027] More preferably, the genome of the Pichia pastoris host strain includes ten copies of an antimicrobial peptide gene.

[0028] Optionally, the Pichia pastoris host strains mentioned above are selected from the following group: Pichia pastoris X-33 (Invitrogen), Pichia pastoris KM71H (Mut S Type, Invitrogen), Pichia pastoris GS115 (Mut + ΔHis type, Invitrogen), Pichia pastoris SMD1168 (Mut + ,ΔPep4,ΔHis type, Invitrogen.

[0029] In one embodiment, the gene encoding the exogenous protein is integrated into the genome of the Pichia pastoris host strain in the following manner:

[0030] A. The gene encoding the exogenous protein was cloned into the genome using gene editing technology;

[0031] B. Construct a recombinant plasmid for overexpressing the foreign protein in Pichia pastoris, and transform the recombinant plasmid containing the gene encoding the foreign protein into Pichia pastoris to obtain positive transformants.

[0032] The transformation of the above recombinant plasmids can be achieved using traditional electrochemical transformation, chemical transformation, or thermal shock methods.

[0033] The gene editing technologies mentioned above can be selected from the following group: homologous double crossover, Red homologous recombination, TALEN system, CRISPR-Cas9 system, CRISPR-Cpf1 system, CRISPR-Cas12a system, MuGENT (multiplex genome editing by natural transformation), and MUCICAT (multi-copy chromosomal integration by CRISPR-associated transposase, a bacterial chromosome multicopy integration technology based on CRISPR-associated transposases).

[0034] For example, the coding gene of the foreign protein can be integrated into the genome of the Pichia pastoris host strain using the CRISPR-Cas9 system.

[0035] A second aspect of the present invention provides an engineered Pichia pastoris strain expressing mycoplasmycin NZ2114, a positive clone of which is constructed by the method described above, and whose genome integrates a gene encoding a methanol metabolism pathway protein selected from the following group: Komagataella phaffiiGS115.

[0036] Glutathione S-transferase, or GST, gene number PAS_chr1-4_0226, has an amino acid sequence as shown in SEQ ID NO: 3, NCBI accession number XP_002490341.1 (2023); the nucleotide sequence of its encoding gene's CDS region is shown in SEQ ID NO: 4, NCBI accession number XM_002490296.1 (2023).

[0037] Protein disulfide isomerase (PDI), gene number PAS_chr4_0844, amino acid sequence as shown in SEQ ID NO: 5, NCBI accession number XP_002494292.1 (2023); nucleotide sequence of the CDS region encoding the gene as shown in SEQ ID NO: 6, NCBI accession number XM_002494247.1 (2023).

[0038] 6-Phosphotrug-2-kinase, or PFK2, gene number PAS_chr2-1_0870, amino acid sequence is shown in SEQ ID NO:7, NCBI accession number XP_002491545.1 (2023); nucleotide sequence of the CDS region encoding the gene is shown in SEQ ID NO:8, NCBI accession number XM_002491500.1 (2023).

[0039] bZIP transcription factor HAC1 (bZIP transcription factor (ATF / CREB1 homolog), gene number PAS_chr1-1_0381, amino acid sequence as shown in SEQ ID NO: 9, 2023 NCBI accession number XP_002490039.1; nucleotide sequence of the encoding gene's CDS region as shown in SEQ ID NO: 10, 2023 NCBI accession number XM_002489994.1)

[0040] Aspartate aminopeptidase, or APE2, gene number PAS_chr4_0913, has an amino acid sequence as shown in SEQ ID NO: 11, NCBI accession number XP_002493442.1 (2023); the nucleotide sequence of the CDS region encoding the gene is shown in SEQ ID NO: 12, NCBI accession number XM_002493397.1 (2023).

[0041] GST+PDI combination.

[0042] A third aspect of the invention provides the use of the above-described engineered Pichia pastoris strain in the production of mycelium-based cytosine NZ2114.

[0043] In one embodiment, mycelium-based mycelium NZ2114 is obtained by fermentation of the above-mentioned engineered Pichia pastoris.

[0044] Our research shows that by using Pichia pastoris X-33, a strain producing mycelium NZ2114, as the chassis cell, and through systematic metabolic engineering and fermentation process optimization, we successfully constructed a high-performance recombinant Pichia pastoris strain X-33-NZ2114-H16-GST, and established a corresponding high-density fermentation process. Experimental results show that in a 5 L fermenter, after 108 hours of induction culture, the mycelium yield reached 2.3 g / L, significantly higher than previously reported levels, demonstrating the application potential of this technology in large-scale production. This achievement not only overcomes the technical bottleneck of heterologous expression of antimicrobial peptides but also provides a reliable and efficient preparation scheme for subsequent industrial applications. Attached Figure Description

[0045] Figure 1 The image shows a gel electrophoresis diagram of the NZ2114 gene fragment containing XhoI and XbaI restriction sites obtained by PCR amplification using primer self-annealing products as templates.

[0046] Figure 2 The image shows a gel electrophoresis diagram of the pPICZαA plasmid fragment after double digestion with restriction endonucleases Xho1 and Xba1.

[0047] Figure 3 The image shows a gel electrophoresis diagram of colony PCR verification of the pPICZαA-NZ2114 recombinant plasmid constructed in this invention.

[0048] Figure 4 The comparison of sequencing results for the pPICZαA-NZ2114 plasmid is shown.

[0049] Figure 5 The image shows a gel electrophoresis photograph of the recombinant yeast colony PCR validation. The PCR used the primer pair 5'AOX1 / 3'AOX1.

[0050] Figure 6 The diagram shows the inhibition zones of the fermentation supernatant of four different chassis-based recombinant Pichia pastoris strains constructed according to the present invention.

[0051] Figure 7 The supernatant protein gel images of recombinant Pichia pastoris strains fermented on four different chassis are shown.

[0052] Figure 8 Gel electrophoresis images showing the digestion verification of recombinant plasmids containing different promoters are displayed.

[0053] Figure 9 Gel electrophoresis images of Pichia pastoris engineered strains containing different promoters are shown.

[0054] Figure 10 The diagram shows the inhibition zones in the fermentation supernatant of Pichia pastoris engineered strains containing different promoters.

[0055] Figure 11 The fermentation supernatant protein gel diagram of Pichia pastoris engineered strains containing different promoters is shown.

[0056] Figure 12 The effects of different promoters on the relative expression levels of antimicrobial peptides in engineered Pichia pastoris strains were shown. Lowercase letters a, b, c, ab, and bc indicate significant differences at the p ≤ 0.05 level, as determined by Duncan's multiple range test.

[0057] Figure 13 Gel electrophoresis images showing the results of enzyme digestion verification of recombinant plasmids containing different signal peptides are displayed.

[0058] Figure 14 Gel electrophoresis images show PCR verification of engineered Pichia pastoris strains containing different natural signal peptides.

[0059] Figure 15 Gel electrophoresis images showing PCR verification of engineered Pichia pastoris strains containing different hybridization signal peptides are displayed.

[0060] Figure 16 The diagram shows the inhibition zones in the fermentation supernatant of engineered Pichia pastoris strains containing different natural signal peptides.

[0061] Figure 17 The image shows a gel image of fermentation supernatant from engineered Pichia pastoris strains containing different natural signal peptides.

[0062] Figure 18 The diagram shows the inhibition zones in the fermentation supernatant of engineered Pichia pastoris strains containing different hybridization signal peptides.

[0063] Figure 19 The fermentation supernatant protein gel image shows Pichia pastoris engineered strains containing different hybridization signal peptides.

[0064] Figure 20 The effects of different signal peptides on the relative expression levels of antimicrobial peptides in engineered Pichia pastoris were shown. Lowercase letters a, b, and c indicate significant differences at the p ≤ 0.05 level, as determined by Duncan's multiple range test.

[0065] Figure 21 Gel electrophoresis images showing the digestion verification of recombinant plasmids containing different terminators are displayed.

[0066] Figure 22 Gel electrophoresis images of Pichia pastoris engineered strains containing different terminators are shown.

[0067] Figure 23 The diagram shows the inhibition zones in the fermentation supernatant of Pichia pastoris engineered strains containing different terminators.

[0068] Figure 24 The fermentation supernatant protein gel diagram of Pichia pastoris engineered strains containing different terminators is shown.

[0069] Figure 25 The study showed the effect of different terminators on the relative expression levels of antimicrobial peptides in engineered Pichia pastoris. Lowercase letters 'a' and 'b' indicate significant differences at the p ≤ 0.05 level, as determined by Duncan's multiple range test.

[0070] Figure 26 The standard curve of primer amplification efficiency for the target gene is shown.

[0071] Figure 27 The standard curve of amplification efficiency of the internal reference gene primers is shown.

[0072] Figure 28 The diagram shows the inhibition zone of the fermentation supernatant from a high-copy Pichia pastoris engineered strain.

[0073] Figure 29 The image shows a gel image of the fermentation supernatant from a high-copy Pichia pastoris engineered strain.

[0074] Figure 30 The graph shows the relationship between relative expression levels and copy numbers of different transformants of high-copy strains.

[0075] Figure 31 The fermentation supernatant protein gel image of the strain overexpressing the target gene is shown.

[0076] Figure 32 The relative expression levels of antimicrobial peptides in strains overexpressing the target gene are shown. CK represents high-copy strains that did not overexpress the peptide; lowercase letters a, b, c, ab, and bc indicate significant differences at the p ≤ 0.05 level, as determined by Duncan's multiple range test.

[0077] Figure 33 The image shows a gel electrophoresis photograph verifying the loss of plasmids during passage of the engineered strain.

[0078] Figure 34 The fermentation supernatant protein gel image of the target gene combination overexpression strain is shown.

[0079] Figure 35 The relative expression levels of antimicrobial peptides in strains overexpressing the target gene combination are shown. CK represents high-copy strains that did not overexpress the peptides; lowercase letters a, b, c, d, e, ab, bc, cd, and de indicate significant differences at the p ≤ 0.05 level according to Duncan's multiple range test.

[0080] Figure 36 Images showing the antibacterial effect of high-density fermentation supernatant of recombinant yeast strain X-33-NZ2114-H16-GST at different times are displayed.

[0081] Figure 37 The image shows protein gel images of the supernatant from the high-density fermentation of recombinant yeast strain X-33-NZ2114-H16-GST at different times.

[0082] Figure 38 The changes in cell wet weight and yield of recombinant yeast strain X-33-NZ2114-H16-GST over time in a fermenter are shown. Detailed Implementation

[0083] To overcome the shortcomings of existing technologies, such as low yield, instability in high-density fermentation, and difficulty in large-scale fermentation production when expressing mycelium-based antimicrobial peptides in Pichia pastoris, we attempted to construct recombinant strains with significantly improved mycelium-based antimicrobial peptide production capacity. The main research contents of this invention include the following aspects:

[0084] (1) Select different types of Pichia pastoris strains to construct recombinant strains expressing mycomycin antimicrobial peptides, and screen out the dominant host strains from them;

[0085] (2) Screening for expression elements with the best compatibility with antimicrobial peptides to enhance the expression level of mycomycin antimicrobial peptides;

[0086] (3) On this basis, increase the copy number of expression cassettes, analyze the relationship between copy number and expression level, and further improve the yield of mycomycin antimicrobial peptide;

[0087] (4) By comparing the transcriptome data of high-copy strains and control strains, the genes of key metabolic pathways were overexpressed, thereby promoting the synthesis of mycelium antimicrobial peptides;

[0088] (5) Verify the high-density fermentation process of the optimal strain.

[0089] In this article, the terms “(antimicrobial peptide expression level / yield / capacity) increase”, “enhancement” or “enhancement” used above refer to an increase of at least 10% or more compared to the reference level, such as at least 20% or more, 30% or more, at least 50% or more, at least 80% or more, at least about 1 time, at least about 2 times, at least about 3 times, at least about 4 times or at least about 5 times.

[0090] In this document, for the sake of simplicity, the name of a protein, such as glutathione S-transferase (GST), and its encoding gene (DNA) name, GST, are sometimes used interchangeably. Those skilled in the art should understand that they represent different types of substances in different descriptive contexts. Their meanings are readily understood by those skilled in the art based on the context. For example, when describing the function or category of glutathione S-transferase, GST refers to a protein; when described as an encoding gene, it refers to the gene encoding that protein.

[0091] For ease of description, mycotocin NZ2114 will sometimes be referred to as NZ2114 or 114 in this article. They have the same meaning and can be used interchangeably.

[0092] As used herein, "exogenous" or "heterogeneous" refers to the relationship between two or more nucleic acid or protein sequences from different bacterial / microbial species, or the relationship between proteins (or nucleic acids) from different sources and host cells. For example, if the combination of nucleic acid and host cell is not normally naturally occurring, then the nucleic acid is exogenous to that host cell. A particular sequence is "exogenous" to the cell or organism into which it is inserted.

[0093] Starting with four different mycelium-producing strains of NZ2114, including Pichia pastoris X-33, GS115, SMD1168 and KM71H, and through a series of studies and optimized combinations of multiple biological elements, we successfully constructed a high-performance recombinant Pichia pastoris strain X-33-NZ2114-H16-GST and established a corresponding high-density fermentation process.

[0094] Compared with the prior art, the beneficial effects of the present invention are mainly reflected in the following aspects:

[0095] 1. Chassis strain screening and optimization: Pichia pastoris host strains with clear genetic background and stable expression were successfully screened, laying the foundation for efficient exogenous protein expression.

[0096] 2. Expression element optimization: By screening and combining key expression elements such as strong promoters and signal peptides, the secretory expression efficiency of target proteins is effectively improved.

[0097] 3. Increased copy number: By adopting a high copy number screening strategy, the copy number of the target gene was significantly increased, thereby greatly improving the expression level of antimicrobial peptides.

[0098] 4. Enhancement through cofactor engineering: By screening and overexpressing genes related to molecular chaperones, transcriptional regulation, protein degradation, and reactive oxygen species scavenging, the endoplasmic reticulum stress, folding load, and oxidative damage caused by high expression were reduced, thereby enhancing the expression stability and protein synthesis capacity of the strain.

[0099] 5. Fermentation process scale-up: A high-density fermentation process was established and optimized, achieving simultaneous improvement in cell density and total product under large-scale conditions, demonstrating good potential for production application.

[0100] The technical solution of the present invention will be further described in detail below with reference to specific embodiments. It should be understood that the following embodiments are only used to illustrate the present invention and are not intended to limit the scope of protection of the present invention.

[0101] Example

[0102] The examples involve the addition amount, content and concentration of various substances, and unless otherwise specified, the percentage content refers to the mass percentage content.

[0103] In the embodiments described herein, unless otherwise specified, the temperature generally refers to room temperature (15-30°C).

[0104] Materials and methods

[0105] The molecular biology experiments in the examples included plasmid construction, enzyme digestion, ligation, competent cell preparation, transformation, culture medium preparation, etc., mainly referring to "Molecular Cloning: A Laboratory Manual" (4th Edition), edited by M.R. Green and J. Sambrook (USA), translated by He Fuchu, Science Press, Beijing, 2017. Specific experimental conditions can be determined through simple experiments if necessary.

[0106] PCR amplification experiments should be performed according to the reaction conditions provided by the plasmid or DNA template supplier or the kit instructions. Adjustments can be made through simple experiments if necessary.

[0107] Enzymes and reagents used in the examples: KOD-Plus-Neo enzyme and KOD-FX enzyme were purchased from Toyobo (Shanghai) Biotechnology Co., Ltd.; restriction endonucleases SacI, SalⅠ, XhoI, and XbaI were purchased from Thermo Fisher Scientific; Solution Ⅰ ligase and Lysis buffer were purchased from Takara. Kits: Plasmid extraction kit and gel extraction kit were purchased from Axygen; Pichia pastoris competent cell preparation and transformation kit was purchased from Beijing Coollab Technology Co., Ltd.; Tricine-SDS-PAGE kit was purchased from Wuhan Sewell Biotechnology Co., Ltd. Antibiotics: Bleomycin (zeocin) was purchased from Solarbio; ampicillin (Amp) was purchased from Sangon Biotech (Shanghai) Co., Ltd.; YNB was purchased from Beijing Coollab Technology Co., Ltd.; biotin was purchased from Sangon Biotech (Shanghai) Co., Ltd.; glucose was purchased from Sangon Biotech (Shanghai) Co., Ltd.; tryptone and yeast extract were purchased from OXOID; agarose and agar powder were purchased from YEASEN. Methanol, glycerol, and other inorganic chemical reagents were all purchased from domestic analytical grade reagents.

[0108] The following are the culture medium formulations:

[0109] LB medium: 10 g / L tryptone, 5 g / L yeast extract, 10 g / L NaCl; solid LB medium is supplemented with 20 g / L agarose.

[0110] Low-salt LB medium: 10 g / L tryptone, 5 g / L yeast extract, 5 g / L NaCl; solid low-salt LB medium is prepared with 20 g / L agar powder.

[0111] YPD medium: 20 g / L tryptone, 10 g / L yeast extract, 20 g / L glucose; solid YPD medium is prepared with 20 g / L agar powder.

[0112] MD medium: amino acid-free yeast ammonia source (YNB) 13.4 g / L, D-biotin 0.0004 g / L, glucose 20 g / L, agar powder 20 g / L.

[0113] BMGY medium (L): 10 g yeast extract, 20 g tryptone, 500 mL glycerol, 100 mL 13.4% amino acid-free yeast ammonia source (YNB), 2 mL 0.02% biotin, 1 mol / L phosphate buffer, pH=6.0, 100 mL.

[0114] BMMY medium (L): 10 g yeast extract, 20 g tryptone, 500 mL methanol, 100 mL 13.4% amino acid-free yeast ammonia source (YNB), 2 mL 0.02% biotin, 1 mol / L phosphate buffer, pH=6.0, 100 mL.

[0115] BMDY medium (L): 10 g yeast extract, 20 g tryptone, 20 g glucose, 100 mL 13.4% amino acid-free yeast ammonia source (YNB), 2 mL 0.02% biotin, 1 mol / L phosphate buffer, pH=6.0, 100 mL.

[0116] BMEY medium (L): 10 g yeast extract, 20 g tryptone, 500 mL anhydrous ethanol, 100 mL 13.4% amino acid-free yeast ammonia source (YNB), 2 mL 0.02% biotin, 1 mol / L phosphate buffer, pH=6.0, 100 mL.

[0117] Basic salt medium (L): 10 mL phosphate, 1.72 g ammonium sulfate, 10 g magnesium sulfate, 6 g potassium hydroxide, 40 g glycerol, 0.5 g sodium chloride, 0.5 g defoamer.

[0118] Trace element PTM1 (L): 6 g copper sulfate, 3 g manganese sulfate, 65 g ferrous sulfate, 20 g zinc chloride, 0.4 g potassium iodide, 0.1 g sodium molybdate, 0.1 g boric acid, 0.25 g cobalt chloride, 0.1 g D-biotin, 5 mL concentrated sulfuric acid.

[0119] For instructions on using LB medium, low-salt LB, YPD, and other media, please refer to the Invitrogen Pichia pastoris manual.

[0120] Table 1 lists the main strains and plasmids used in this invention.

[0121] Table 1. Tested bacterial strains and plasmids

[0122] Materials and plasmids characteristic Source Explanation pPICZαA Zeocine Henan University of Technology pPIC9K Amp,Kan Henan University of Technology E.coLi DH5a Purchased from Shanghai Bioengineering Co., Ltd. Pichia pastoris X-33 wild type This laboratory preserves Pichia pastoris GS115 <![CDATA[Mut + ,ΔHis]]> This laboratory preserves Pichia pastoris SMD1168 <![CDATA[Mut + ,ΔPep4,ΔHis]]> This laboratory preserves Pichia pastoris KM71H <![CDATA[Mut S ]]> Purchased from Invitrogen pPICZαA-NZ2114 Recombinant vector This invention constructs X-33-NZ2114 Recombinant yeast This invention constructs GS115-NZ2114 Recombinant yeast This invention constructs SMD1168-NZ2114 Recombinant yeast This invention constructs KM71H-NZ2114 Recombinant yeast This invention constructs X-33-NZ2114-H16 Recombinant yeast This invention constructs X-33-NZ2114-H16-GST Recombinant yeast This invention constructs Staphylococcus aureus This laboratory preserves

[0123] The PCR amplification primers for some gene fragments in the examples are listed in Table 2.

[0124] Table 2. Some of the PCR amplification primers used in this example

[0125] Primers (Sequence 5'-3') 5'AOX1 GACTGGTTCCAATTGACAAGC 3'AOX1 GGCAAATGGCATTCTGACAT NZ2114-f1 GTTTTGGTTGTAACGGTCCATGGAACGAAGATGATTTGAGATG NZ2114-r1 ACTTACAAACAAAACCACCCTTAGCACAGTAACCACCCTTG NZ2114-f2 TCTCTCGAGAAGAGAGGTTTTGGTTGTAACGGTCCATGG NZ2114-r2 TGTTCTAGATTATTAGTAACACTTACAAACAAAACCACCC pPIC9K-F1 CGAGAATTCATGGATCCGAATTAATTCGCCTTAGAC pPIC9K-R1 TTCGGATCCATGAATTCTCGAATAATAACTGTTATT AOX1-EcoR1-F1 CGGAATTCGATCTAACATCCAAAGACG NZ2114-BamH1-R1 GCGGATCCTTATTAGTAACACTTACAAAC 9kNZ2114ver-f1 CCTTTCGTCTTCAAGAATTA GAP-EcoR1-F1 CGGAATTCTTTTTGTAGAAATGTCTTGG CAT1-EcoR1-F1 CGGAATTCTTTAATTGTAAGTCTTGAC FDH1-EcoR1-F1 CGGAATTCGAAGGTGGAAATGGCAGAAG GCW14HR-F1 CAGTTATTTCGATTTTGTTGTTGAGTGAAGCG NZ2114HR-R1 GGCGAATTAATTCTTATTAGTAACACTTACAAAC NZ2114HR-F1 GTGTTACTAAGAATTAATTCGCCTTAGACATG GCW14HR-R1 CTCAACAACAAATTCGAATAATAACTGTTATT ALD4HR-F1 CAGTTATTTCGAACGAGCCATTTTTCGATCCG ALD4HR-R1 CGAAAAATGGCTCGTTCGAATAATAACTGTTATT FLD1HR-F1 GTTATTATTCGAGCATGCAGGAATCTCTGGCAC FLD1HR-R1 GGAAATCTCATTGTGAATATCAAGAATTGTATG 9K-FLD1HR-F1 CTTGATATTCAATGAGATTTCCTTCAATTT 9K-FLD1HR-R1 GATTCCTGCATGCTCGAATAATAACTGTTATT AOX2-F1 ATTATTCGAGAATTCGCTTAAAGGACTCCATTTCC AOX2-R1 GGAAATCTCATCCATGGTTCTCAGTTGATTTG 9BOX2-F1 CAACTGAGAACCATGGATGAGATTTCCTTCAATTTTTAC 9BOX2-R1 GGAGTCCTTTAAGCGAATTCTCGAATAATAACTG PGK1-F1 TTATTCGAGAATTCAGTTGGGTATTCAAATAGTTG PGK1-R1 GGAAATCTCATTTTCGTAATCAATTGGGCTATG 9KPGK1-F1 TTGATTACGAAAATGATTTCCTTCAATTTTTAC 9KPGK1-R1 GAATACCCAACTGAATTCTCGAATAATAACTG mut-F1 GAACACTGAAAATAACAGTTATTTCGAGAATTC mut-R1 CAAACTTAGTTCATCTTGGATGAGATCACGCTTTTG mut-F2 GCGTGATCTCATCCAAGATGAACTAAGTTTGGTTCG mut-R2 GTTATTTTTCAGTGTTCCCGATCTGCGTCTATTTC AOX-S34-F1 TCCTGGCCCCCCTGGCGAGGTTCATGCCGAATGCAACAAGCTCCGC AOX-S34-R1 CGCCAGGGGGGCCAGGATAGACAGGCGTCATGGTGTTAGTAGCCTA Ver-AOX921-F1 AGGTTCATGCCGAATGCA 0074-F1 ATTCGAGAATTCTGAATTCCTGGCTGCCCAC 0074-R1 GCTTTAGTTAAAAAAATAGGTCATTCAATCTCG cPDAS-F1 GACCTATTTTTTTAACTAAAGCAGGATGCCTGA cPDAS-R1 GGAAATCTCATTTTGTTCGATTATTCTCCAG 9KDAS-F1 CGAACAAAATGAGATTTCCTTCAATTT 9K0074-R1 CAGGAATTCAGAATTCTCGAATAATAACTG 9k-LguⅠ-F1 GAGAAGAGCATGCTCTTCAGGTTTTGGTTGTAACGGTCC 9k-LguⅠ-R1 CTGAAGAGCATGCTCTTCTCGTTTCGAATAATTAGTTGT 9KHR-F1 GAAAACCTCCAGCAAAAGGCCAGGAACCG 9KHR-R1 CCTTTTGCTGGAGGTTTTCACCGTCATCAC Green-ori-R1 CCTCTGACTTGAGCGTCGA α-MF pre-F1 CAGCTCTTCAACGATGAGATTTCCTTCAATTT α-MF pre-R1 GTGCTCTTCTACCAGCTAATGCGGAGGATGCTG α-amylase-F1 CAGCTCTTCAACGATGGTCGCTTGGTGGTCTT α-amylase-R1 GTGCTCTTCTACCAGCCAAAGCAGGTGCAGCG Serum albumin-F1 CAGCTCTTCAACGATGAAGTGGGTTACCTTTA Serum albumin-R1 GTGCTCTTCTACCAGAGTAAGCAGAAGAGAAAAG Glucoamylase-F1 CAGCTCTTCAACGATGTCTTTTAGATCCTTGT Glucoamylase-R1 GTGCTCTTCTACCAGCCAAACCAGAACAAACC Lysozyme-F1 CAGCTCTTCAACGATGCTGGGTAAGAACGACC Lysozyme-R1 GTGCTCTTCTACCACCTTGACAGATACCCAAC Killer protein-F1 CAGCTCTTCAACGATGACTAAGCCAACCCAAG Killer protein-R1 GTGCTCTTCTACCAGCTACGACTAGATGTAGT Inulinase-F1 CAGCTCTTCAACGATGAAGTTAGCATACTCCT Inulinase-R1 GTGCTCTTCTACCAGCACTGACTCCTGCCAAT Invertase-F1 CAGCTCTTCAACGATGCTTTTGCAAGCTTTCC Invertase-R1 GTGCTCTTCTACCTGCAGATATTTTGGCTGCA OST-F1 CAGCTCTTCAACGATGAGGCAGGTTTGGTTCTC OST-R1 GACTGGAGCAGCAGAAGACACGTTGAAAA SP-OST-F1 CTTCTGCTGCTCCAGTCAACACTACAAC SP-LguⅠ-R1 GTGCTCTTCTACCAGCTTCAGCCTCTCTTTTC INU-F1 CAGCTCTTCAACGATGAAGTTAGCATACTCCC INU-R1 GACTGGAGCTCTCTTGTAATTGATAACTG SP-INU-F1 CAAGAGAGCTCCAGTCAACACTACAAC SUC-F1 CAGCTCTTCAACGATGCTTTTGCAAGCTTTCC SUC-R1 GACTGGAGCTGCAGATATTTTGGCTGCAA SP-SUC-F1 ATATCTGCAGCTCCAGTCAACACTACAAC SCW10-F1 CAGCTCTTCAACGATGCAGGTCAAGTCCATTG SCW10-R1 GACTGGAGCACGCTTGTCATGTTGGTGGT SP-SCW10-F1 GACAAGCGTGCTCCAGTCAACACTACAAC UTH1-F1 CAGCTCTTCAACGATGAAATCCCAACTGATCTTC UTH1-R1 TGACTGGAGCTGAAGCAACCAATGAAGCC SP-UTH1-F1 GTTGCTTCAGCTCCAGTCAACACTACAAC MEL1-F1 CAGCTCTTCAACGATGAGAGCTTTCTTGTTTC MEL1-R1 GACTGGAGCAGTCTCGTTCACCCCAAAAAAC SP-MEL1-F1 GAACGAGACTGCTCCAGTCAACACTACAAC PHO11-F1 CAGCTCTTCAACGATGTTGAAGTCAGCCGTTTA PHO11-R1 GACTGGAGCTGCATTAACCAAAGAAGCG SP-PHO11-F1 GTTAATGCAGCTCCAGTCAACACTACAAC 0030-F1 CAGCTCTTCAACGATGAAGTTCGCAATTTCAAC 0030-R1 CTGGAGCAGCAAAAAACAGCGGCCAGCCTG SP-0030-F1 GTTTTTGCTGCTCCAGTCAACACTACAAC SP4α-factor-F1 CAGCTCTTCAACGATGAAGCTGATCTCCGTGGG SP-α-factor-R1 GTGCTCTTCTACCTCTCTTCTCGAGAGAGACAC SP14α-factor-F1 CAGCTCTTCAACGATGTTAAACAAGCTGTTCAT term-free-f1 AGTTCGTTTGTGCAAGCTT term-free-r1 TTAGAATCTAGCAAGACCG tAOX2-f1 GTCTTGCTAGATTCTAATTTATGTTGTATCTATGAA tAOX2-r1 GCTTGCACAAACGAACTTTAGACTACTCTGAATCCG tDAS1-f1 GTCTTGCTAGATTCTAAACGGGAAGTCTTTACAGTT tDAS1-r1 GCTTGCACAAACGAACTCCAAATTGTAATCATCAGTG tGAP-f1 GTCTTGCTAGATTCTAAATCGATTTGTATGTGAAAT tGAP-r1 GCTTGCACAAACGAACTAGTTTTTCATGTTCAATTA tFDH1-f1 GTCTTGCTAGATTCTAATTGAATGTATTTAATTTG tFDH1-r1 GCTTGCACAAACGAACTTGAACGATGTACAATCTGAG tARG4-f1 GTCTTGCTAGATTCTAAAGGTTTTATACTGAGTTTG tARG4-r1 GCTTGCACAAACGAACTATACATAGGAGATCTAATAC III-15-intver-f1 CAAAATGAACCCAGCCCCATTG II-6-intver-f1 AGTGAACTTTGCAGACAGCC ARG4-f1 TCCATTGACTCCCGTTTTGAG ARG4-r2 TCCTCCGGTGGCAGTTCTT X-33-qPCR-F GGTTTTGGTTGTAACGGTCCAT X-33-qPCR-R GGTTTTGGTTGTAACGGTCCAT

[0126] In Table 2, the suffix "F" in primer names indicates forward direction; "R" indicates reverse direction.

[0127] Example 1: Design and PCR amplification of the target gene NZ2114

[0128] 1.1 The expression sequence of the antimicrobial peptide NZ2114 gene was optimized based on the codon preference of Pichia pastoris.

[0129] Based on the amino acid sequence of the mycotoxin antimicrobial peptide NZ2114 (SEQ ID NO: 1), a gene encoding the antimicrobial peptide NZ2114 was designed. The DNA sequence (NZ2114) after codon optimization is shown in SEQ ID NO: 2.

[0130] GFGCNGPWNEDDLRCHNHCKSIKGYKGGYCAKGGFVCKCY (SEQ ID NO: 1);

[0131] GGTTTTGGTTGTAACGGTCCATGGAACGAAGATGATTTGAGATGTCATAACCATTGTAAGTCTATTAAGGGTTACAAGGGTGGTTACTGTGCTAAGGGTGGTTTTGTTTGTAAGTGTTAC (SEQ ID NO: 2).

[0132] Sequence characteristics: 126 bp; Type: nucleic acid; Strand type: double strand; Topology: linear; GC%: 38%; Molecular type: double-stranded DNA.

[0133] 1.2 Target gene design

[0134] To effectively terminate the translation and expression of NZ2114, two consecutive TAA stop codons were inserted at the C-terminus of the NZ2114 coding sequence. To achieve native secretory expression of NZ2114 in Pichia pastoris, the signal peptide cleavage site kex2 was inserted at the N-terminus of NZ2114. To achieve clonal expression of the NZ2114 gene on a plasmid, Xho1 and Xba1 endonuclease sites were designed at both ends of the NZ2114 gene. The expression cassette gene design is as follows:

[0135] CTCGAGAAGAGAGGTTTTGGTTGTAACGGTCCATGGAACGAAGATGATTTGAGATGTCATAACCATTGTAAGTCTATTAAGGGTTACAAGGGTGGTTACTGTGCTAAGGGTGGTTTTGTTTGTAAGTGTTACTAATAA.

[0136] 1.3 PCR amplification of the NZ2114 gene

[0137] Synthesize complementary primer pairs NZ2114-syn-f1 / NZ2114-syn-r1:

[0138] Forward NZ2114-syn-f1:

[0139] GAACGAAGATGATTTGAGATGTCATAACCATTGTAAGTCTATTAAGGGTTACAAGGGTGGTTACTGTGCTAA,

[0140] Reverse NZ2114-syn-r1:

[0141] TTAGCACAGTAACCACCCTTGTAACCCTTAATAGACTTACAATGGTTATGACATCTCAAATCATCTTCGTTC.

[0142] The primers were annealed together to synthesize double-stranded fragments. First, the double-stranded sequence was formed by annealing. The reaction system was as follows: ddH2O: 35 μL, T4 Ligase buffer: 5 μL, two primers: 5 μL each (20 μM), for a total of 50 μL.

[0143] The reaction conditions were as follows: 95 ℃ for 5 min, decreasing the ℃ by 5-10 ℃ per minute, and then 16 ℃ for 10 min. The annealed product was diluted 10 times to obtain a fragment with a size of 72 bp.

[0144] Using the above fragment as a template, amplification was performed using primer pair NZ2114-f1 / NZ2114-r1, resulting in a size of 114 bp. After DNA agarose gel identification, 1 μL was used as a template and amplified using primer pair NZ2114-f2 / NZ2114-r2, resulting in a size of 150 bp.

[0145] The reaction system is as follows: KODFX Neo plus enzyme: 1 μL, KOD Neo plus buffer: 25 μL, ddH2O: 11 μL, dNTP: 10 μL, primer F / R: 1 μL, template: 1 μL.

[0146] The reaction conditions were as follows: Initial denaturation: 94 ℃, 2 min. Denaturation: 98 ℃, 10 s. Annealing: 55 ℃, 30 s. Extension: 68 ℃, 15 s. Termination extension: 68 ℃, 10 min. Number of cycles: 30.

[0147] The PCR product was purified and recovered using a gel extraction kit (purchased from UE Everbright) to obtain a high-purity NZ2114 gene fragment, which was stored at -20 ℃ for later use. Figure 1 The gel electrophoresis results show the PCR amplification of the NZ2114 target gene.

[0148] Example 2: Construction of Recombinant Expression Vector

[0149] 2.1 Double-digested fragments and vectors

[0150] The purified NZ2114 gene fragment obtained in step 1.3 was digested with Xho1 and Xba1, and the fragment was recovered by gel electrophoresis, with a size of 150 bp. The vector pPICZαA was digested with Xho1 and Xba1, and the fragment was recovered by gel electrophoresis, with a size of 3506 bp. Figure 2 .

[0151] The double enzyme digestion system is as follows: Xho1: 2 μL, Xba1: 2 μL, buffer: 5 μL, DNA: 10 μL, ddH2O: 31 μL. Total: 50 μL. Digestion conditions: 37 ℃ water bath for 30 min.

[0152] After gel recovery of the enzyme digestion products, the vector and the NZ2114 gene were ligated using T4 ligase. The system is as follows:

[0153] T4 ligase: 4.5 μL, pPICZαA: 0.5 μL, NZ2114: 3.5 μL. Total volume: 9 μL. Ligation conditions: 16℃, 1.5 h.

[0154] 2.2 Construction of Recombinant Vectors

[0155] The ligation system was added to competent *E. coli* DH5α cells, incubated on ice for 30 min, then heat-shocked in a 42 °C water bath for 90 s, followed by 2–3 min on ice. 800 μL of LB liquid medium was then added, mixed well, and incubated at 37 °C on a shaker for 1 h. After centrifugation at 3000 rpm for 3 min, 800 μL of supernatant was removed. The remaining bacterial culture was gently pipetted and spread onto LB solid medium containing 50 μg / mL low-salt culture, and incubated at 37 °C for 12–16 h.

[0156] 2.3 Identification of Recombinant Plasmids

[0157] Single colonies were randomly selected from the plate for PCR verification using primer pair NZ2114-f1 / 3'AOX1. DNA agar gel analysis showed a band size of 299 bp, consistent with the target band. Figure 3 As shown, transformants containing the target band were sequenced using 3'AOX1 primers. The sequencing results were then compared with the target gene. Figure 4 As shown, the results are correct and match the plasmid purchased according to the design specifications. It is named pPICZαA-NZ2114.

[0158] The colony PCR reaction system is as follows: Taq mix 10 μL, F / R: 0.5 / 0.5 μL, plasmid: 1 μL, ddH2O: 8 μL. Total: 20 μL.

[0159] The PCR reaction conditions were as follows: initial denaturation: 94 ℃, 2 min; denaturation: 94 ℃, 30 s; annealing: 55 ℃, 30 s; extension: 72 ℃, 10 s; final extension: 72 ℃, 10 min; 30 cycles.

[0160] Example 3: Construction of a recombinant Pichia pastoris strain containing the NZ2114 gene

[0161] 3.1 Linearization of the recombinant vector pPICZαA-NZ2114

[0162] The recombinant vector pPICZαA-NZ2114 obtained above was linearized using the Sac1 endonuclease and transformed into four types of Pichia pastoris competent cells: X-33, GS115, SMD1168, and KM71H.

[0163] The linearization system is as follows: SacⅠ: 5 μL, recombinant vector: 20 μL, buffer: 10 μL, ddH2O: 25 μL, total: 50 μL.

[0164] 3.2 Preparation of Pichia pastoris competent cells

[0165] The preparation and transformation of Pichia pastoris competent cells were carried out in accordance with the instructions of the Pichia pastoris competent cell preparation and transformation kit from Beijing Coolplay Technology Co., Ltd.

[0166] (1) Glyceryl bacteria were activated by streaking on YPD solid medium to obtain single colonies of Pichia pastoris. Fresh single colonies were inoculated into a medium containing 3 mL of YPD liquid medium and cultured at 30 ℃ and 240 rpm for 12-16 h.

[0167] (2) Inoculate the culture at a rate of 1% (v / v) into a 250 mL shake flask containing 50 mL of YPD liquid medium and incubate at 30 °C on a shaker for about 5 h until OD. 600 The value is between 0.6 and 0.8;

[0168] (3) Dispense 50 mL of culture into sterile centrifuge tubes at 10 mL per tube, centrifuge at 3000 rpm / min for 3 min to collect cells, discard the supernatant, add 5 mL of B1 solution to each tube to resuspend, centrifuge again to collect cells, add 200 μL of B1 solution to each tube to suspend and transfer to 1.5 mL sterile centrifuge tubes to obtain competent cells, which can be stored at -80℃ after slow freezing for later use.

[0169] 3.3 Chemical Transformation

[0170] The Pichia pastoris competent cells preparation and transformation kit (purchased from Beijing Cooler Technology Co., Ltd.) were used.

[0171] (1) After linearizing the recombinant plasmid to be transformed with restriction endonuclease, take 3~5 μg and add it together with 10 μL Carrier into the prepared competent cells. After mixing, place it in a 30 ℃ water bath to thaw, then add 1.4 mL B2 transformation solution and incubate in a 30 ℃ water bath for 1 hour, gently inverting and mixing every 10 minutes.

[0172] (2) After incubation, centrifuge at 3000 rpm for 3 minutes to collect cells, discard the supernatant, and add 1 mL of B3 solution to wash the precipitate.

[0173] (3) Centrifuge again at 3000 rpm for 3 minutes, discard the supernatant, and resuspend the cells in 200 μL of B3 solution. Finally, spread the bacterial culture evenly on the corresponding resistant or defective plate and incubate in a constant temperature incubator at 30 ℃.

[0174] 3.4 Colony PCR Validation of Recombinant Pichia pastoris Strain

[0175] Place 30 μL of Lysis Buffer into a 1.5 mL EP tube. Randomly pick 5 transformants from the selection plate with a sterile toothpick and place them in a centrifuge tube. Lyse at 80 °C for 15 min to obtain the colony PCR template. Use primer pair 5'AOX1 / 3'AOX1. Since 5'AOX1 and 3'AOX1 binding sites also exist in the Pichia pastoris chromosome, the DNA agarose gel should show two bands, 2200 bp and 641 bp, respectively. Figure 5 As shown, the size is consistent with the target band.

[0176] Example 4: Screening of Pichia pastoris host strains

[0177] 4.1 Induced Fermentation

[0178] Three positive transformants each of Pichia pastoris X-33, GS115, SMD1168, and KM71H were selected and inoculated into 3 mL of YPD liquid medium (containing 100 μg / m zeocin) and cultured at 30 ℃ and 240 rpm for 16–24 h. They were then transferred to 30 mL of BMGY medium and cultured overnight. After centrifugation to collect the cells, they were inoculated into 50 mL of BMMY medium and cultured on a shaker at 30 ℃ and 240 rpm for methanol induction. Methanol was added every 24 h to a final concentration of 1% (v / v). During the 120 h of expression, 500 μL of bacterial culture samples were collected every 24 h, centrifuged, and the supernatant was stored at -80 ℃ for protein expression assays to screen for the strain with the highest protein expression and the optimal harvest time of the bacterial culture.

[0179] 4.2 Antibacterial activity detection of recombinant Pichia pastoris fermentation broth

[0180] To evaluate the antibacterial activity of the fermentation product of recombinant Pichia pastoris, the agar diffusion method was used, with Staphylococcus aureus as the indicator bacterium for antibacterial effect analysis. The specific steps are as follows: First, 10 μL of Staphylococcus aureus from the preserved strain was inoculated into LB liquid medium and cultured at 37℃ and 220 rpm for 3–4 h with shaking until OD... 600The target concentration was 1.0. The melted LB solid medium was cooled to approximately 45°C, and the bacterial culture was added at an inoculum rate of 0.4% (v / v). After gentle mixing, the mixture was poured into plates to prepare LB solid plates uniformly containing the indicator bacteria. After the plates solidified, uniformly perforated circular wells of consistent diameter were made on the agar surface using a sterile punch. 70 μL of the supernatant from the fermentation endpoint was added to each well. The supernatant from the fermentation of a strain without the target gene served as a blank control. Positive control wells were also prepared, with 10 μL of Lamb antibiotic solution (1 mg / mL) added. The plates were incubated overnight at 37°C. The antimicrobial activity of the antimicrobial peptide was evaluated by observing and measuring the diameter of the inhibition zone. Results are as follows: Figure 6 As shown.

[0181] 4.3 Detection of protein expression levels in recombinant Pichia pastoris fermentation broth

[0182] To analyze the expression levels of antimicrobial peptides in the fermentation supernatant of recombinant Pichia pastoris, Tricine-SDS-PAGE was used for protein separation and detection. The specific steps were as follows: 30 µL of fermentation supernatant was taken, and an appropriate amount of 5× loading buffer was added to dilute to a final concentration of 1×. The mixture was then heated in a boiling water bath for 10 min to denature, followed by centrifugation at 12000 rpm for 5 min. The supernatant was collected as the electrophoresis sample. For sample loading, 10 µL of the sample and 3 µL of low molecular weight protein marker were added to the gel wells. The electrophoresis conditions were set to an initial voltage of 100 V. After the sample entered the separating gel, the voltage was increased to 150 V, and electrophoresis continued until the bromophenol blue front migrated to the bottom of the gel. After electrophoresis, the gel was placed in staining solution and stained on a shaker at room temperature for 5 min. The stained gel was then transferred to destaining solution and destained on a shaker until the background was clear and the bands were distinct. Electrophoresis images were acquired using a fully automated gel imaging system. The presence of the target protein was confirmed based on the position of the protein marker bands, and the relative expression level of the antimicrobial peptide was assessed. like Figure 7 As shown, considering the antibacterial effect, X-33 was selected as the chassis cell expressing mycorrhizal antimicrobial peptides.

[0183] Tricine-SDS-PAGE gels were prepared according to the instructions of the accompanying kit. Staining solution: 50% methanol, 10% glacial acetic acid, 0.2% Coomassie Brilliant Blue G-250, the remainder being deionized water. Destaining solution: 5% anhydrous ethanol, 10% glacial acetic acid, the remainder being deionized water.

[0184] Example 5: Screening of expression elements from recombinant Pichia pastoris

[0185] 5.1 Promoter selection

[0186] 5.1.1 Construction of plasmids with different promoters

[0187] The NZ2114 gene was cloned into the pPIC9K plasmid, and the recombinant plasmid pPIC9K-AOX1-NZ2114 was successfully constructed. Using this plasmid as a template, the original AOX1 promoter was replaced with a series of different promoters, thereby constructing various recombinant expression plasmids, including: pPIC9K-AOX2-NZ2114, pPIC9K-FDH1-NZ2114, pPIC9K-FLD1-NZ2114, pPIC9K-CAT1-NZ2114, pPIC9K-AOX713-NZ2114, pPIC9K-AOX995-NZ2114, pPIC9K-AOX921-NZ2114, pPIC9K-0074DAS-NZ2114, pPIC9K-GAP-NZ2114, pPIC9K-ALD4-NZ2114, pPIC9K-PGK1-NZ2114, and pPIC9K-GCW14-NZ2114. The plasmid validation results are correct. Figure 8 As shown.

[0188] 5.1.2 Construction of Pichia pastoris engineered strains with different promoters

[0189] 5.1.2.1 Linearization of recombinant plasmids with different promoters

[0190] The successfully constructed recombinant plasmid-containing glycerol bacteria were inoculated into 4 mL of LB-Amp liquid medium and cultured overnight at 37 ℃ and 180 rpm. The plasmids were then extracted using a plasmid extraction kit. The plasmids were linearized using SpeedyCut SalI rapid digestion enzyme, as shown in the table below. The digestion reaction was carried out at 37 ℃ for 2 h, and the fragments were cleaned and recovered. Multiple preparations were performed, ensuring a yield of 3-5 μg of plasmid fragment.

[0191] Table 3. Enzyme digestion reaction system

[0192] reaction system Reaction volume (μL) DNA (plasmid or fragment) 25 SpeedyCut SalI 5 10×FastDigest buffer 5 <![CDATA[dH2O]]> 15 Total volume 50

[0193] 5.1.2.2 Pichia pastoris transformation

[0194] Linearized vectors with different promoters were transformed into the GS115 strain, and positive transformants were screened using MD medium. The preparation and transformation of competent cells were carried out in the same manner as in steps 3.2 and 3.3.

[0195] 5.1.2.3 PCR identification of Pichia pastoris colonies

[0196] The yeast colony PCR method is the same as in step 3.4. Colony PCR verification was performed on recombinant transformants with different promoters using specific primers, and the results are as follows. Figure 9 As shown, the verification is correct, and the names are as shown in the table below:

[0197] Table 4. Constitutive and Inducible Pichia pastoris Engineered Strains

[0198] strain name Startup subtype Substrate carbon source GS115-pPIC9K-AOX1-NZ2114 Induced methanol GS115-pPIC9K-AOX2-NZ2114 Induced methanol GS115-pPIC9K-FDH1-NZ2114 Induced methanol GS115-pPIC9K-FLD1-NZ2114 Induced methanol GS115-pPIC9K-CAT1-NZ2114 Induced methanol GS115-pPIC9K-AOX713-NZ2114 Induced methanol GS115-pPIC9K-AOX995-NZ2114 Induced methanol GS115-pPIC9K-AOX921-NZ2114 Induced methanol GS115-pPIC9K-0074DAS-NZ2114 Induced ethanol GS115-pPIC9K-GAP-NZ2114 Constitutive glucose GS115-pPIC9K-ALD4-NZ2114 Constitutive glucose GS115-pPIC9K-PGK1-NZ2114 Constitutive glucose GS115-pPIC9K-GCW14-NZ2114 Constituent glucose

[0199] 5.1.3 Fermentation culture of recombinant strains and screening of high-expression strains under different carbon sources

[0200] 1) Fermentation culture method of strains using methanol as carbon source

[0201] Initial colony screening: Three PCR-positive transformants were selected and inoculated into 3 mL YPD medium tubes for culture. Recombinant protein induction: 1% (v / v) initial seed culture was inoculated into 30 mL BMGY medium and cultured overnight until OD500. 600 The concentration was approximately 10. After centrifugation to remove the supernatant, the mixture was resuspended in 50 mL of BMMY fermentation medium. Methanol was added every 24 h to a final concentration of 1% (v / v). After fermentation for 120 h, the supernatant was collected by centrifugation and stored at -20 ℃ for protein expression assay to screen for strains with the highest protein expression levels.

[0202] 2) Fermentation culture method of strains using ethanol as carbon source

[0203] Initial colony screening: Three PCR-positive transformants were selected and inoculated into 3 mL YPD medium tubes for culture. Recombinant protein induction: 1% (v / v) initial seed culture was inoculated into 30 mL BMGY medium and cultured overnight until OD500. 600 The concentration was approximately 10. After centrifugation to remove the supernatant, the mixture was resuspended in 50 mL of BMEY fermentation medium. Anhydrous ethanol was added every 24 h to a final concentration of 1% (v / v). After fermentation for 120 h, the supernatant was collected by centrifugation and stored at -20 ℃ for protein expression assay to screen for strains with the highest protein expression levels.

[0204] 3) Fermentation culture method using glucose as a carbon source

[0205] Initial colony screening: Three PCR-positive transformants were selected and inoculated into 3 mL YPD medium tubes for culture. Recombinant protein induction: 1% (v / v) initial seed culture was inoculated into 30 mL BMGY medium and cultured overnight until OD500. 600 The concentration was approximately 10. After centrifugation to remove the supernatant, the mixture was resuspended in 50 mL of BMDY fermentation medium. Glucose was added every 24 h until the final concentration was 1% (v / v). After fermentation for 120 h, the supernatant was collected by centrifugation and stored at -20 ℃ for protein expression assay to screen for strains with the highest protein expression levels.

[0206] 5.1.4 Detection of induced expression level of recombinant strains

[0207] The antibacterial activity and protein expression level of the fermentation broth were detected according to the methods described in steps 4.2 and 4.3, respectively, and the results are as follows: Figure 10 and Figure 11 As shown. Further grayscale analysis of the protein bands was performed using ImageJ software. Figure 12 The results showed that the engineered strain using AOX1 as the promoter exhibited the highest expression level. Therefore, the AOX1 promoter was selected in this invention for expressing mycoplasmycin antimicrobial peptides.

[0208] 5.2 Screening of signal peptides

[0209] 5.2.1 Construction of different signal peptide plasmids

[0210] Using pPIC9K-AOX1-NZ2114 plasmid as a template, the full-length α-factor signal peptide in the vector was replaced with natural signal peptides from different sources, successfully constructing a series of recombinant plasmids containing natural signal peptides, including pPIC9K-α-mating-NZ2114, pPIC9K-α-amylase-NZ2114, pPIC9K-Glucoamylase-NZ2114, pPIC9K-Serum-NZ2114, pPIC9K-Killer-NZ2114, pPIC9K-Lysozyme-NZ2114, pPIC9K-Inulinase-NZ2114, and pPIC9K-Invertase-NZ2114. Furthermore, by using fusion modification technology, the pro region of the α-factor signal peptide was chimeric with different natural signal peptide sequences to obtain a series of hybrid signal peptides. Then, using pPIC9K-AOX1-NZ2114 plasmid as a template, the α-factor signal peptide in the vector was replaced with the above-mentioned hybrid signal peptide sequences, thereby constructing a series of recombinant plasmids of hybrid signal peptides, specifically pPIC9K-0030-NZ2114, pPIC9K-OST-NZ2114, pPIC9K-SUC2-NZ2114, pPIC9K-INU1-NZ2114, pPIC9K-UTH1-NZ2114, pPIC9K-PHO11-NZ2114, pPIC9K-SCW10-NZ2114, pPIC9K-MEL1-NZ2114, pPIC9K-SP4-NZ2114, and pPIC9K-SP14-NZ2114. plasmid validation results are as follows Figure 13 As shown.

[0211] 5.2.2 Construction of Pichia pastoris engineered strains with different signal peptides

[0212] Linearization, transformation, and Pichia pastoris colony PCR identification of different signal peptide recombinant plasmids were performed in the same steps as described in 5.1.2.1, 3.2, 3.3, and 3.4. The colony PCR results are as follows: Figure 14 and Figure 15 As shown.

[0213] 5.2.3 Induced Fermentation

[0214] The fermentation methods for different signal peptide Pichia pastoris engineered strains are the same as those in step 5.1.3 for the fermentation culture method of strains using methanol as a carbon source.

[0215] 5.2.4 Detection of induced expression level of recombinant strains

[0216] The methods for detecting the antibacterial activity and protein expression level of the fermentation broth were the same as those in steps 4.2 and 4.3, respectively. The experimental results are as follows: Figures 16 to 19 As shown. Further grayscale analysis of the protein bands was performed using ImageJ software, and the results are as follows. Figure 20 As shown, the Pichia pastoris engineered strain using the α-factor signal peptide exhibited the highest expression level.

[0217] 5.3 Terminator Screening

[0218] 5.3.1 Construction of different terminator plasmids

[0219] Using pPIC9K-AOX1-NZ2114 plasmid as a template, the original AOX1 terminator was replaced with a series of different terminators, thereby constructing various recombinant expression plasmids, including: pPIC9K-tDAS1-NZ2114, pPIC9K-tFDH1-NZ2114, pPIC9K-tAOX2-NZ2114, pPIC9K-tGAP-NZ2114, and pPIC9K-tARG4-NZ2114. The plasmid validation results are correct, as shown below. Figure 21 As shown.

[0220] 5.3.2 Construction of Pichia pastoris engineered strains with different terminators

[0221] Linearization, transformation, and Pichia pastoris colony PCR identification of recombinant plasmids with different terminators were performed in the same steps 5.1.2.1, 3.2, 3.3, and 3.4. The colony PCR results were correct. Figure 22 As shown.

[0222] 5.3.3 Induced Fermentation

[0223] The fermentation methods for Pichia pastoris engineered strains with different terminators are the same as those in step 5.1.3 for the fermentation culture method of strains using methanol as the carbon source.

[0224] 5.3.4 Detection of induced expression level of recombinant strains

[0225] The detection of antibacterial activity in the fermentation broth and the detection of protein expression levels in the fermentation broth were performed in steps 4.2 and 4.3, respectively. The results are as follows: Figure 23 and Figure 24 As shown. Further grayscale analysis of the protein bands was performed using ImageJ software. Figure 25 This indicates that the engineered strain with AOX1 as the terminator had the highest expression level.

[0226] Example 6: High-copy screening of engineered strains for optimal chassis and components

[0227] Introducing multiple copies of a foreign gene is an effective strategy to enhance the expression level of heterologous proteins. Generally, an increase in the copy number of a foreign gene is positively correlated with the expression level of the heterologous protein. This invention, based on the Pichia pastoris X-33 chassis, constructed an engineered strain expressing mycorrhizalmycin using the AOX1 promoter, α-factor signal peptide, and AOX1 terminator, and utilized Aw et al. The developed Liquid PTVA method was used for high-copy screening. The main process of this screening method is as follows: the strain is allowed to grow adaptively in a liquid medium with an increasing resistance concentration gradient, and finally plated on a high-resistance plate. After cultivation, high-copy transformants are obtained.

[0228] 6.1 Copy number determination of each transformant

[0229] Gene copy number determination in different transformants was performed using real-time quantitative PCR. SYBR Green I dye, a highly specific double-stranded DNA-binding dye, was used to detect the accumulation of PCR products during PCR cycles. Target gene-specific primers X-33-qPCR-F / X-33-qPCR-R and internal control gene primers ARG4-f1 / ARG4-r2 were designed. The qPCR system was as follows: SYBR Green I: 10 μL, ddH2O: 8 μL, F / R: 0.5 μL, DNA: 1 μL. Reaction conditions: pre-denaturation 95 ℃, 2 min; denaturation 95 ℃, 2 min; annealing: 60 ℃, 1 min, 40 cycles. Melting curve: 95 ℃, 15 s; 60 ℃, 15 s; 20 min; 95 ℃, 15 s. The same transformant was used as the target gene and diluted to 10 μL. -1 10 -2 10 -3 10 -4 The amplification efficiency of two primer pairs was determined, and the cycle threshold (Ct) was measured on the ordinate. A standard curve was plotted with the negative logarithm of the template content on the x-axis. R 2 All were above 0.99, as shown in the results. Figure 26 , 27 As shown, the k value is obtained through the standard curve, and then according to the formula: The amplification efficiency of the internal reference gene was 109.92%, and the amplification efficiency of the target gene was 106.31%.

[0230] Genomic DNA from different monoclonal transformants was used as templates for real-time quantitative PCR analysis. The gene copy number of each transformant was calculated using the obtained Ct values. The results are shown in the table below.

[0231] Table 5. Copy number of different transformants

[0232] Transformer -∆ct <![CDATA[Copy number 2 -（-∆ct） > X-33-NZ2114-H1 -0.432 1.349 X-33-NZ2114-H2 -2.025 4.07 X-33-NZ2114-H3 -2.985 7.92 X-33-NZ2114-H4 -2.515 5.72 X-33-NZ2114-H5 -3.365 10.30 X-33-NZ2114-H6 -2.802 6.96 X-33-NZ2114-H7 -3.090 8.51 X-33-NZ2114-H8 -2.170 4.50 X-33-NZ2114-H9 -3.490 11.24 X-33-NZ2114-H10 -1.707 3.27 X-33-NZ2114-H11 -2.392 5.24 X-33-NZ2114-H12 -1.830 3.56 X-33-NZ2114-H13 -1.073 2.10 X-33-NZ2114-H14 -0.055 1.04 X-33-NZ2114-H15 -1.830 3.56 X-33-NZ2114-H16 -3.370 10.34 X-33-NZ2114-H17 -1.805 3.49 X-33-NZ2114-H18 -1.592 3.01 X-33-NZ2114-H19 -2.825 7.09 X-33-NZ2114-H20 -2.037 4.11 X-33-NZ2114-H21 -2.695 6.48 X-33-NZ2114-H23 -2.728 6.63 X-33-NZ2114-H24 -1.395 2.63 X-33-NZ2114-H25 -2.705 6.52 X-33-NZ2114 (before copying) -0.335 1.26

[0233] By combining the copy number results with the inhibition zone size and analyzing the protein abundance in the gel sponge image, it was found that ( Figure 28 , 29 30): The production and antibacterial activity of antimicrobial peptides showed a trend of first increasing and then decreasing with the increase of gene copy number. When the copy number was below 10, the two were significantly positively correlated; however, after the copy number exceeded the inflection point of 10, the expression level dropped significantly, suggesting that the host has a tolerance limit to the expression of high-copy exogenous genes.

[0234] Example 7: Transcriptome Analysis of High-Copy Strains

[0235] To investigate the differences in gene transcription between high-copy strains and the original strain, the high-copy strain X-33-H16 with the highest expression level of antimicrobial peptides was used as the experimental group, and the original strain X-33-pPICZαA was used as the control group. The three groups were cultured in parallel and the culture method was the same as in 4.1. The samples were then sent for transcriptome analysis.

[0236] 7.1 Analysis of Transcription Results

[0237] The expression of antimicrobial peptides has a significant impact on biological processes such as cell growth, development, and metabolism. Therefore, a large number of differentially expressed genes (DEGs) were identified in both the control and experimental groups. A total of 158 genes were identified as differentially expressed genes, of which 102 were significantly upregulated and 56 were significantly downregulated. In the experimental group, the genes PAS_chr1-4_0226 (encoding glutathione S-transferase, GST) in the methanol oxidation pathway and PAS_chr2-1_0870 (phosphofructosyl-2-kinase, PFK2) related to the glycolysis pathway were significantly upregulated. The GST gene can enhance the methanol catabolism pathway, neutralize oxidative stress products, maintain intracellular redox balance, and ensure efficient carbon source metabolism. The upregulation of the PFK2 gene directly accelerates the metabolic flux from glucose to pyruvate, rapidly generating ATP through anaerobic metabolism, ensuring energy supply during the large-scale synthesis of antimicrobial peptides, reflecting the host's adaptive adjustment of energy metabolism to meet the high demand for heterologous protein expression.

[0238] Furthermore, the abundant synthesis of antimicrobial peptides in high-copy strains induces endoplasmic reticulum (ER) protein folding stress, leading to a significant upregulation of the expression of unfolded protein response (UPR)-related genes in the experimental group. Specifically, the expression of the UPR core transcription factor HAC1 (encoding gene PAS_chr1-1_0381) was increased (log2FC=1.3); the expression of the key ER molecular chaperone BIP (encoding gene PAS_chr2-1_0140) also increased synchronously (log2FC=1.2). The upregulation of HAC1 activates the expression of downstream folding helper genes, while BIP promotes correct folding by binding to unfolded peptide chains. The synergistic effect of these two mechanisms alleviates ER protein overload stress and is a core regulatory mechanism by which the host responds to ER stress induced by high antimicrobial peptide expression. Although UPR assists protein folding by upregulating BIP (molecular chaperone) and HAC1 (transcription factor), some incorrectly folded antimicrobial peptide precursors or miscellaneous proteins still need to be degraded by ER-related degradation (ERAD). For example, the aspartic aminopeptidase (APE2) gene significantly upregulates PAS_chr4_0913 (log2FC=14.00). The degradation products (short peptides) need to be further broken down into free amino acids by aminopeptidase to achieve amino acid recycling and reuse.

[0239] In contrast to the majority of upregulated genes involved in methanol metabolism, protein production, and degradation, the expression of genes related to oxidative phosphorylation of the electron transport chain and ATP synthesis was generally downregulated, constituting a core characteristic of energy metabolism remodeling. Specifically, the expression level of the cytochrome c oxidase encoding gene PAS_chr2-2_0265 was significantly reduced (log2FC = -2.1); the F-type H⁺-ATPase subunit gene PAS_chr2-1_0751, involved in mitochondrial ATP synthesis, also showed a downregulated trend (log2FC = -1.1). The inhibition of these gene expression directly led to a decrease in mitochondrial oxidative phosphorylation efficiency, presumably an adaptive regulatory strategy by the host to reduce the accumulation of reactive oxygen species (ROS) during methanol metabolism, thus mitigating the interference of oxidative stress on cellular physiological functions. In the experimental group, ribosome structural genes related to host growth generally showed downregulated expression, reflecting a tendency for resource allocation to favor heterologous protein synthesis. For example, the expression level of the gene encoding the small ribosomal subunit protein PAS_chr2-2_0257 was significantly reduced (log2FC = -2.0); other genes related to ribosome assembly and function (such as PAS_chr1-4_0504 and PAS_chr1-4_0412) also showed varying degrees of downregulation. These results indicate that the expression of high-copy-value antimicrobial peptides causes the host to preferentially allocate transcriptional and translational resources to the synthesis of foreign proteins, thereby inhibiting its own ribosome biogenesis and cell proliferation, forming a metabolic allocation pattern of "preferential allocation of foreign protein synthesis".

[0240] Based on transcriptome sequencing results, it was found that excessive copy number can cause significant transcriptional and translational burden on cells, specifically manifested as ATP deficiency in methanol metabolism, endoplasmic reticulum stress caused by protein misfolding and accumulation during transcription and translation, and also indirectly leading to insufficient degradation efficiency of the endoplasmic reticulum-associated degradation pathway (ERAD).

[0241] 7.2 Validation of target gene function based on transcriptome results

[0242] To investigate the enhancing effect of key gene overexpression on heterologous protein secretion, this invention selected candidate target genes such as GST, PFK2, APE2, BIP, PDI, and HAC1 based on transcriptome analysis. Using Pichia pastoris genome-directed integration technology, engineered strains expressing these genes were constructed. The effects of each gene on the secretory capacity of the recombinant strains were then systematically evaluated by comparing the antimicrobial peptide content in the fermentation broth.

[0243] 7.2.1 Integration of Cas9 plasmids

[0244] The pGAP-Hscas9-SV40-Nours plasmid was linearized using the StUI restriction endonuclease. The linearized product was transformed into competent cells of the high-copy-rate Pichia pastoris strain X-33-NZ2114-H16. Transformed cells were screened on YPD plates containing 200 μg / mL nourseothricin; positive transformants were named X-33-NZ2114-H16-Cas9. The preparation and transformation methods for competent cells are described in steps 3.2 and 3.3.

[0245] 7.2.2 Construction of Donor Fragments

[0246] Using pPIC9K as a template, the replicon ori region and the Kan resistance expression frame region were amplified, and the two parts were overlapped to obtain a plasmid vector. Using the GS115 genome as a template, the upstream and downstream homologous arms of the III-15 site, the GAP promoter, and the AOX1 terminator were amplified, and Gibson assembly was performed with the above plasmid vector to obtain the III-15 site backbone plasmid named pGAP-Ⅲ-15.

[0247] Using the GS115 genome as a template, the GST, PFK2, APE2, BIP, PDI, and HAC1 genes were amplified (intron regions removed). These genes were then inserted between the promoter and terminator of the pGAP-Ⅲ-15 plasmid. After verification, the plasmids were named as follows: pGAP-Ⅲ-15-GST, pGAP-Ⅲ-15-PFK2, pGAP-Ⅲ-15-APE2, pGAP-Ⅲ-15-PDI, pGAP-Ⅲ-15-BIP, and pGAP-Ⅲ-15-HAC1.

[0248] Using the plasmids described above as templates, donor fragments containing different genes can be obtained by amplifying the fragments using the 5' primer of the upstream homologous arm and the 3' primer of the downstream homologous arm.

[0249] 7.2.3 Gene Integration and Validation

[0250] The competent cells of strain X-33-NZ2114-H16-Cas9 were prepared for use. The donor fragment and the III-15 gRNA plasmid were added to the competent cells for transformation to complete gene integration. Colony PCR was performed using III-15-intver-f and downstream primers from different gene regions for validation. The correctly validated positive transformants were named X-33-NZ2114-H16-GST, X-33-NZ2114-H16-PFK2, X-33-NZ2114-H16-APE2, X-33-NZ2114-H16-BIP, X-33-NZ2114-H16-PDI, and X-33-NZ2114-H16-HAC1.

[0251] 7.2.4 Induced fermentation and content determination

[0252] Clones were selected from the verified positive transformants for methanol-induced fermentation. Protein gel electrophoresis bands in the fermentation supernatant were compared (e.g.,...). Figure 31 , 32 As shown in the figure, except for the molecular chaperone BIP, all the other cofactors tested helped to increase the expression level of antimicrobial peptides.

[0253] 7.2.5 Validation of Gene Synergistic Effects

[0254] Three target genes that help enhance the expression of antimicrobial peptides were selected and combined to obtain the combinations GST+APE2, GST+PDI, and PDI+APE2. The next step of gene integration requires discarding the gRNA plasmid at sites III-15 before integrating genes at different sites. This was verified by passage on plates without gRNA resistance (HYG) for 15 generations, followed by validation on plates containing HYG resistance. The absence of transformants confirms successful plasmid loss.

[0255] To verify the synergistic effect between genes, this invention selected three target genes (GST, APE2, and PDI) that can individually enhance the expression of antimicrobial peptides, constructing three dual-gene combinations: GST+APE2, GST+PDI, and PDI+APE2. To facilitate the subsequent integration of these combinations at different genomic loci, the original III-15 site gRNA plasmid (containing HYG resistance) in the host bacteria needed to be removed first. Therefore, strains X-33-NZ2114-H16-GST and X-33-NZ2114-H16-PDI were passaged 15 times consecutively on YPD plates without hygromycin (HYG) to induce plasmid loss. Subsequently, the 15th generation colonies were streaked onto HYG-containing plates for verification. The results showed that no clonal growth was observed in the experimental groups. Figure 33 This confirms that the gRNA plasmid has been successfully lost and can be integrated at the next site.

[0256] To obtain the gene donor fragment targeting site II-6, the upstream and downstream homologous arm regions of site II-6 were amplified using the GS115 genome as a template. These amplified regions then replaced the upstream and downstream homologous arms of site III-15 in the plasmids pGAP-Ⅲ-15-APE2 and pGAP-Ⅲ-15-PDI, respectively, thus constructing plasmids pGAP-Ⅱ-6-APE2 and pGAP-Ⅱ-6-PDI. Subsequently, using these two plasmids as templates, PCR amplification was performed using the 5' primer of the upstream homologous arm and the 3' primer of the downstream homologous arm at site II-6 to obtain the target donor fragment.

[0257] The plasmid-removed X-33-NZ2114-H16-GST and X-33-NZ2114-H16-PDI strains were prepared as competent cells for later use. Using the X-33-NZ2114-H16-GST competent cells as recipients, the PDI donor fragment at site II-6 and the II-6 gRNA plasmid were co-transformed. Genotypes were verified by colony PCR (using primer II-6-intver-f and the downstream primer inside the PDI gene), yielding the correctly genotyped GST+PDI combined strain. Similarly, the APE2 donor fragment at site II-6 and the II-6 gRNA plasmid were co-transformed, and after PCR verification (using primer II-6-intver-f and the downstream primer inside the APE2 gene), the GST+APE2 combined strain was obtained. Using X-33-NZ2114-H16-PDI competent cells as recipients, the APE2 donor fragment at site II-6 and the II-6 gRNA plasmid were co-transformed. The genotype was verified by colony PCR (using primer II-6-intver-f and the downstream primer inside the APE2 gene) to obtain the PDI+APE2 combined strain with the correct genotype.

[0258] After methanol-induced fermentation of the validated positive transformants, the protein gel electrophoresis bands of the fermentation supernatant were compared. Figure 34 , Figure 35 The study found that among the dual-gene overexpression combinations, only the GST+PDI combination showed a slight increase in antimicrobial peptide production compared to the original strain, while the other dual-gene combinations (GST+APE2 and PDI+APE2) actually led to a decrease in expression levels. Furthermore, compared to strains overexpressing a single gene, all dual-gene combinations showed significantly lower yields. Based on these results, the strain overexpressing the single gene GST achieved the highest yield and was therefore selected as the superior strain for further research.

[0259] Example 8: High-density fermentation with engineered bacteria

[0260] Preparation of seed culture: Single colonies of yeast transformants were picked from YPD plates and inoculated into 3 mL of YPD liquid medium containing 100 μg / mL Zeocin. The culture was incubated at 30℃ and 240 rpm with shaking for 16–18 h to obtain primary seed culture. Subsequently, the culture was transferred to YPG liquid medium at a 1% (v / v) inoculation rate and incubated at 30℃ and 250 rpm with shaking for another 16–18 h until OD (Organic Oxygen Demand) was reached. 600 When the value reaches 5-8, a secondary seed solution is obtained.

[0261] Glycerol growth stage: The initial pH of the basal salt medium was adjusted to 5.0 using ammonia, and the culture temperature was set to 30 °C. A 10% inoculum of the secondary seed culture was inoculated into a fermenter containing 1 L of basal salt medium, and 2 mL / L PTM1 solution was added. During the culture process, an aeration rate of 1 vvm was maintained, and the initial stirring speed was set to 300 rpm. A dissolved oxygen and stirring speed linkage control mode was used (the stirring speed could only increase) to stabilize the dissolved oxygen at 20%–30%. Six hours after inoculation, the linkage was disengaged, and the stirring speed was fixed at 800 rpm.

[0262] Glycerin Feeding Phase: When the dissolved oxygen level suddenly rises above 60% after a slow decline (DO Spike), it indicates that the initial glycerin has been largely depleted. Begin feeding a 50% glycerin solution containing 12 mL / L PTM1 at a flow rate of 12–20 mL / L / h. Increase the rotation speed to 1000 rpm, maintaining dissolved oxygen between 30% and 40%, and continue feeding for 6–8 hours.

[0263] Methanol induction phase: When the cell wet weight reaches approximately 200 g / L, stop adding glycerol, maintain a rotation speed of 1000 rpm and aeration of 1 vvm to raise dissolved oxygen to above 60% and maintain this level for 5 minutes. Then, begin adding methanol containing 12 mL / L PTM1. The initial flow rate is 1–3 mL / L / h, and the adaptation period is 2–3 hours. If dissolved oxygen cannot be maintained above 20% during this period, stop adding the methanol and resume when the dissolved oxygen peak reappears. Generally, the cells are fully adapted to methanol metabolism after 2–4 hours. At this point, gradually increase the flow rate to maintain dissolved oxygen at no less than 20%, and simultaneously adjust the pH to 5.5. Continue induction for 120 hours, then terminate fermentation.

[0264] During the methanol induction phase, 10 mL of fermentation sample was taken every 12 hours to determine the cell wet weight, antimicrobial peptide antibacterial activity, and expression level. Results are as follows: Figure 36 As shown, the antibacterial activity of the recombinant yeast strain X-33-NZ2114-H16-GST gradually increased with prolonged induction time during high-density fermentation. The corresponding changes in protein expression levels are shown in [Figure showing changes in protein expression levels]. Figure 37 , 38 This indicates that the expression level of the antimicrobial peptide also increases continuously with the extension of induction time. After 120 hours of fed induction, the yield of mycelium antimicrobial peptide reached 2.3 g / L at 108 h of induction.

[0265] In summary, this invention successfully constructed a high-performance recombinant Pichia pastoris strain X-33-NZ2114-H16-GST through systematic metabolic engineering and fermentation process optimization, and established a corresponding high-density fermentation process. Ultimately, in a 5 L fermenter, after 108 hours of induction culture, the mycelial mycin yield reached 2.3 g / L, significantly higher than previously reported levels, demonstrating the strong potential of this technology system in large-scale production.

[0266] The above embodiments are only used to illustrate the technical solutions of the present invention. Without departing from the spirit of the present invention, those skilled in the art can make various modifications or alterations to the present invention on this basis. Equivalent forms of various variations or modifications should also fall within the scope of the present invention.

[0267] It should be noted that the listing and discussion of previously disclosed documents in this specification should not be construed as an admission that such documents are prior art or common general knowledge.

[0268] References

[0269] [1] R.E.W. Hancock, H.-G. Sahl, Antimicrobial and host-defensepeptides as new anti-infective therapeutic strategies, Nature Biotechnology,24 (2006) 1551-1557.

[0270] [2] H.v. Jenssen, P. Hamill, R.E.W. Hancock, Peptide AntimicrobialAgents, Clinical Microbiology Reviews, 19 (2006) 491-511.

[0271] [3] E.D. Murphey, G. Fang, E.R. Sherwood, Pretreatment with the Gram-positive bacterial cell wall molecule peptidoglycan improves bacterialclearance and decreases inflammation and mortality in mice challenged withStaphylococcus aureus, Critical Care Medicine, 36 (2008) 3067-3073.

[0272] [4] Y. Zhang, D. Teng, R. Mao, X. Wang, D. Xi, X. Hu, J. Wang, Highexpression of a plectasin-derived peptide NZ2114 in Pichia pastoris and itspharmacodynamics, postantibiotic and synergy against Staphylococcus aureus,Appl. Microbiol. Biotechnol., 98 (2013) 681-694.

[0273] [5] D. Andes, W. Craig, L.A. Nielsen, H.H. Kristensen, In VivoPharmacodynamic Characterization of a Novel Plectasin Antibiotic, NZ2114, ina Murine Infection Model, Antimicrob. Agents Chemother., 53 (2009) 3003-3009.

[0274] [6] K.S. Brinch, P.M. Tulkens, F. Van Bambeke, N. Frimodt-Moller, N.Hoiby, H.H. Kristensen, Intracellular activity of the peptide antibioticNZ2114: studies with Staphylococcus aureus and human THP-1 monocytes, andcomparison with daptomycin and vancomycin, Journal of AntimicrobialChemotherapy, 65 (2010) 1720-1724.

[0275] [7] Y.Q. Xiong, W.A. Hady, A. Deslandes, A. Rey, L. Fraisse, H.H.Kristensen, M.R. Yeaman, A.S. Bayer, Efficacy of NZ2114, a Novel Plectasin-Derived Cationic Antimicrobial Peptide Antibiotic, in ExperimentalEndocarditis Due to Methicillin-Resistant Staphylococcus aureus , Antimicrob.Agents Chemother., 55 (2011) 5325-5330.

[0276] [8] R. Aw, K.M. Polizzi, Liquid PTVA: a faster and cheaperalternative for generating multi-copy clones in Pichia pastoris, MicrobialCell Factories, 15 (2016).

[0277] [9] S. Abad, K. Kitz, A. Hörmann, U. Schreiner, F.S. Hartner, A.Glieder, Real‐time PCR‐based determination of gene copy numbers in Pichiapastoris, Biotechnology Journal, 5 (2010) 413-420.

Claims

1. A method for improving the expression of antimicrobial peptides in Pichia pastoris, characterized in that, Includes the following steps: The coding genes of the following histones were integrated into the genome of Pichia pastoris host strain expressing antimicrobial peptides: glutathione S-transferase (GST), protein disulfide isomerase (PDI), 6-phosphofructo-2-kinase (PFK2), bZIP transcription factor HAC1, aspartate aminopeptidase (APE2), and the GST+PDI combination. Positive clones with higher expression levels of antimicrobial peptides than the Pichia pastoris host strain were screened out, and the engineered Pichia pastoris strains overexpressing the protein were constructed to produce the antimicrobial peptides through fermentation.

2. The method according to claim 1, characterized in that, The exogenous protein is derived from Komagataella phaffiiGS115: where Glutathione S-transferase (GST) is gene number PAS_chr1-4_0226, and its amino acid sequence is shown in SEQ ID NO: 3, NCBI accession number XP_002490341.1 (2023). The nucleotide sequence of the CDS region of its encoding gene is shown in SEQ ID NO: 4, NCBI accession number XM_002490296.1 (2023). The protein disulfide isomerase, PDI, is identified by gene number PAS_chr4_0844, with its amino acid sequence shown in SEQ ID NO: 5, NCBI accession number XP_002494292.1 (2023). The nucleotide sequence encoding the CDS region of the gene is shown in SEQ ID NO: 6, NCBI accession number XM_002494247.1 (2023). 6-Phosphotrug-2-kinase, or PFK2, is gene number PAS_chr2-1_0870, and its amino acid sequence is shown in SEQ ID NO: 7, NCBI accession number XP_002491545.1 (2023). The nucleotide sequence encoding the CDS region of the gene is shown in SEQ ID NO: 8, NCBI accession number XM_002491500.1 (2023). The bZIP transcription factor HAC1 is gene number PAS_chr1-1_0381, and its amino acid sequence is shown in SEQ ID NO: 9, NCBI accession number XP_002490039.1 (2023). The nucleotide sequence of the CDS region encoding the gene is shown in SEQ ID NO: 10, NCBI accession number XM_002489994.1 (2023). Aspartate aminopeptidase APE2 is gene number PAS_chr4_0913, amino acid sequence as shown in SEQ ID NO: 11, NCBI accession number XP_002493442.1 version in 2023; nucleotide sequence of the CDS region encoding the gene is shown in SEQ ID NO: 12, NCBI accession number XM_002493397.1 version in 2023.

3. The method according to claim 1, characterized in that, The antimicrobial peptide is mycelium, preferably mycelium NZ2114.

4. The method according to claim 1, characterized in that, The amino acid sequence of the mycelium-based mycelium NZ2114 is shown in SEQ ID NO: 1, and the nucleotide sequence of the encoding gene is shown in SEQ ID NO:

2.

5. The method according to claim 1, characterized in that, The genome of the Pichia pastoris host strain contains a promoter upstream of the antimicrobial peptide coding gene and / or a terminator downstream of the antimicrobial peptide coding gene, wherein the promoter is the AOX1 promoter with the nucleotide sequence shown in SEQ ID NO: 13, and the terminator is the AOX1 terminator with the nucleotide sequence shown in SEQ ID NO: 14; and / or The genome of the Pichia pastoris host strain also contains a signal peptide, which is an α-factor signal peptide, preferably derived from Saccharomyces cerevisiae, with an amino acid sequence as shown in SEQ ID NO: 15 and a nucleotide sequence encoding the gene as shown in SEQ ID NO:

16.

6. The method according to claim 1, characterized in that, The genome of the Pichia pastoris host includes two or more copies, preferably four or more, six or more, eight or more, and more preferably ten copies of an antimicrobial peptide gene or an antimicrobial peptide gene expression cassette / expression frame.

7. The method according to claim 1, characterized in that, The Pichia pastoris host strain was selected from the following group: Pichia pastoris X-33, Pichia pastoris KM71H (Mut S Pichia pastoris GS115 (Mut) + ΔHis type), Pichia pastoris SMD1168 (Mut + ,ΔPep4,ΔHis type).

8. The method according to claim 1, characterized in that, The gene encoding the protein is integrated into the genome of the Pichia pastoris host strain in the following manner: A. The protein-coding gene is cloned into the genome using gene editing technology; B. Construct a recombinant plasmid for overexpressing the protein in Pichia pastoris, and transform the recombinant plasmid containing the protein-encoding gene into Pichia pastoris to obtain positive transformants.

9. A Pichia pastoris engineered strain expressing mycomycin NZ2114, wherein a positive clone is obtained by constructing according to any one of claims 4-8, characterized in that, Its genome integrates genes encoding the following histones: Glutathione S-transferase, or GST, gene number PAS_chr1-4_0226, has an amino acid sequence as shown in SEQ ID NO: 3, NCBI accession number XP_002490341.1 (2023); the nucleotide sequence of its encoding gene's CDS region is shown in SEQ ID NO: 4, NCBI accession number XM_002490296.1 (2023). The protein disulfide isomerase, PDI, gene number PAS_chr4_0844, has an amino acid sequence as shown in SEQ ID NO: 5, NCBI accession number XP_002494292.1 (2023); the nucleotide sequence of the CDS region encoding the gene is shown in SEQ ID NO: 6, NCBI accession number XM_002494247.1 (2023). 6-Phosphotrug-2-kinase, or PFK2, gene number PAS_chr2-1_0870, amino acid sequence is shown in SEQ ID NO: 7, NCBI accession number XP_002491545.1 (2023); nucleotide sequence of the CDS region encoding the gene is shown in SEQ ID NO: 8, NCBI accession number XM_002491500.1 (2023). The bZIP transcription factor HAC1, gene number PAS_chr1-1_0381, has an amino acid sequence as shown in SEQ ID NO: 9, NCBI accession number XP_002490039.1 (2023); the nucleotide sequence of the CDS region encoding the gene is shown in SEQ ID NO: 10, NCBI accession number XM_002489994.1 (2023). Aspartate aminopeptidase, or APE2, gene number PAS_chr4_0913, has an amino acid sequence as shown in SEQ ID NO: 11, NCBI accession number XP_002493442.1 (2023); the nucleotide sequence of the CDS region encoding the gene is shown in SEQ ID NO: 12, NCBI accession number XM_002493397.1 (2023). GST+PDI combination.

10. The use of the engineered Pichia pastoris strain according to claim 9 in the production of mycelium-based mycelium NZ2114.

Images