A recombinant pichia pastoris strain for efficiently secreting human lactoferrin and a construction method and application thereof

Abstract

This invention discloses a recombinant Pichia pastoris strain that efficiently secretes and expresses human lactoferrin, its construction method, and its applications. The genome of the recombinant Pichia pastoris strain integrates a human lactoferrin expression cassette, in which the codon-optimized hLTF coding gene is located at the 3' end of the coding sequence for the Ste13 protease recognition cleavage site of the α-mating factor signal peptide. The construction method includes the following core steps: First, the codon-optimized hLTF coding gene is linked to the EAEA coding sequence of the pPIC9K plasmid, and then electrotransformed into Pichia pastoris strain GS115. A high-expression strain, GS115-pPIC9K-hLTF, is obtained through G418 gradient selection. Next, copy number fine-tuning is performed using the pPICZHαA plasmid, adding one or two copies to the high-expression strain GS115-pPIC9K-hLTF. Finally, the Mxr1 regulatory element is introduced into the strain with two additional copies using the pGAPZαhA plasmid. The resulting optimized strain, GS115-pPIC9K-hLTF-2C-Mxr1, exhibits considerable expression levels in shake flasks and has been successfully scaled up in a 10 L fermenter. This invention achieves efficient secretory expression of human lactoferrin in Pichia pastoris through an integrated strategy of EAEA site configuration optimization, multi-copy gene dosage regulation, and synergistic enhancement of secretory pathway regulatory elements.

Description

Technical Field

[0001] This invention belongs to the field of genetic engineering and microbial fermentation technology, specifically relating to a recombinant Pichia pastoris strain that efficiently secretes and expresses human lactoferrin, its construction method, and its application. Background Technology

[0002] Human lactoferrin (hLTF) is an iron-binding glycoprotein with a molecular weight of approximately 80 kDa. It belongs to the transferrin family and contains 703 amino acid residues. It has a variety of important physiological functions, including broad-spectrum antibacterial, antiviral, antioxidant, immune response regulation, and iron absorption promotion. It has broad application prospects in infant formula, nutritional supplements, cosmetics, and pharmaceuticals.

[0003] Currently, commercially available lactoferrin mainly relies on extraction from bovine milk. However, the background content of lactoferrin in bovine milk is only 0.03-0.49 g / L, resulting in high extraction costs and low yields. Furthermore, bovine lactoferrin is a heterologous protein, posing potential immunogenicity risks to humans. Therefore, the preparation of recombinant human lactoferrin using microbial heterologous expression has become an important alternative route.

[0004] Pichia pastoris is recognized by the US FDA as a GRAS (Generally Recognized As Safe) microorganism. It combines the ease of handling and rapid growth of prokaryotes with the ability of eukaryotic proteins to fold, glycosylate, and secrete for processing and modification, making it a mainstream host for recombinant protein production. The most commonly used signal peptide for secretory expression in Pichia pastoris is the pre-pro peptide of α-factor (MFα) derived from Saccharomyces cerevisiae. After translation, this signal peptide is sequentially cleaved at the KR site by the Kex2 protease and at the EAEA site by the Ste13 protease, releasing the mature exogenous protein.

[0005] Existing techniques for enhancing hLTF expression in Pichia pastoris can be mainly divided into three categories:

[0006] The first category involves signal peptide modification: patents such as CN119776401A, CN115960175A, and CN120737998A mostly employ overall replacement or large-fragment modification of the α-factor signal peptide to construct hybrid signal peptides. Significant modification of the signal peptide can lead to abnormal protease processing sites, the introduction of non-natural amino acid sequences, resulting in incomplete cleavage and decreased protein activity.

[0007] The second category involves the co-expression of molecular chaperones and transporters: protein folding and vesicle transport are improved by overexpressing genes such as KAR2, PDI1, ERO1, and BMH2. This strategy mainly addresses the post-translational folding bottleneck of proteins.

[0008] The third category involves multi-copy integration: obtaining multi-copy Pichia pastoris transformants using the G418 gradient screening is a routine procedure. However, for the larger molecular weight hLTF, gene copy number and protein expression are not simply positively correlated; excessively high copy numbers can overload the host transcription-secretion pathway, increase protein misfolding, and actually decrease yield. Current techniques lack research on the optimal copy number for the human lactoferrin system.

[0009] Furthermore, existing technologies for introducing regulatory elements are mostly based on low-copy or single-copy strains, lacking systematic research on the further introduction of regulatory elements on multi-copy lactoferrin strains.

[0010] Therefore, the present invention aims to overcome the above-mentioned defects of the prior art and provide a complete technical solution that takes into account signal peptide site optimization, fine control of copy number, and transcriptional enhancement of high copy defect strains, so as to achieve efficient secretion of human lactoferrin. Summary of the Invention

[0011] The technical problem solved by this invention is to address the current technical status of low secretion efficiency and insufficient expression level of human lactoferrin in Pichia pastoris. This invention provides a recombinant Pichia pastoris strain that efficiently secretes and expresses human lactoferrin and its construction method. The aim is to achieve efficient secretory expression of human lactoferrin in Pichia pastoris through an integrated strategy of optimizing the configuration of EAEA cleavage sites, screening multi-copy strain systems, and synergistic enhancement of regulatory elements.

[0012] To solve the above-mentioned technical problems, the present invention adopts the following technical solution:

[0013] In a first aspect, the present invention provides a recombinant Pichia pastoris strain that efficiently secretes and expresses human lactoferrin. The genome of the recombinant Pichia pastoris strain integrates a human lactoferrin expression cassette. The human lactoferrin expression cassette includes an operatively linked promoter, an α-mating factor signal peptide coding sequence, and a codon-optimized human lactoferrin coding gene. The codon-optimized human lactoferrin coding gene is located at the 3' end of the Ste13 protease recognition cleavage site coding sequence in the α-mating factor signal peptide coding sequence.

[0014] As a further preferred embodiment of the present invention, the human lactoferrin expression cassette has an additional copy number of 1 to 2 in the genome.

[0015] As a further preferred embodiment of the present invention, the copy number is increased by one to two copies, and the genome of the recombinant Pichia pastoris strain also integrates an Mxr1 regulatory element expression cassette, the Mxr1 regulatory element expression cassette comprising a constitutive GAP promoter and an Mxr1 coding gene, the Mxr1 coding gene being located at the 3' end of the GAP promoter.

[0016] As a further preferred embodiment of the present invention, the nucleotide sequence of the codon-optimized human lactoferrin encoding gene is as shown in SEQ ID NO.2, or is a nucleotide sequence that has at least 90% sequence identity with the sequence shown in SEQ ID NO.2 and encodes a polypeptide with human lactoferrin activity.

[0017] As a further preferred embodiment of the present invention, the amino acid sequence of the Mxr1 encoding gene is as shown in SEQ ID NO.3, or is the encoding sequence of a protein that has at least 80% sequence identity with the sequence shown in SEQ ID NO.3 and has Mxr1 transcriptional regulatory activity.

[0018] As a further preferred embodiment of the present invention, the Pichia pastoris host strain is Pichia pastoris GS115 strain.

[0019] As a further preferred embodiment of the present invention, the promoter is the AOX1 promoter, and the human lactoferrin expression cassette also includes the AOX1 terminator.

[0020] Secondly, the present invention provides a method for constructing a recombinant Pichia pastoris strain that efficiently secretes and expresses human lactoferrin, comprising the following steps:

[0021] (1) Construction of recombinant expression plasmid: The codon-optimized human lactoferrin coding gene was linked to a Pichia pastoris expression plasmid containing the coding sequence of the α-mating factor signal peptide EAEA cleavage site. The codon-optimized human lactoferrin coding gene was located at the 3' end of the coding sequence of the EAEA cleavage site, and the recombinant expression plasmid pPIC9K-EAEA-hLTF was obtained.

[0022] (2) Pichia pastoris transformation and positive screening: The recombinant expression plasmid obtained in step (1) was linearized by single enzyme digestion with SacⅠ, and then electrotransformed into Pichia pastoris GS115 competent cells. The transformants were plated on MD solid medium, and His was obtained by histidine auxotrophic complementation screening. + Positive transformants;

[0023] (3) G418 gradient screening of multi-copy integrated strains: The His⁺ positive transformants obtained in step (2) were spread on YPD solid medium containing different concentrations of G418. By screening with G418 concentration gradient, single colonies growing on high concentration G418 plates were selected to obtain recombinant Pichia pastoris strains that integrate multiple copies of human lactoferrin expression cassettes.

[0024] As a further preferred embodiment of the present invention, in step (3), the G418 screening concentration gradient is 1.0 mg / mL, 3.0 mg / mL and 4.0 mg / mL, and a strain expressing high human lactoferrin is obtained by screening on a 4.0 mg / mL G418 plate and named GS115-pPIC9K-hLTF.

[0025] As a further preferred embodiment of the present invention, the method further includes step (4): linearizing the pPICZHαA-hLTF recombinant plasmid containing a single human lactoferrin expression cassette with SalⅠ enzyme, electroporating the high-expression strain GS115-pPIC9K-hLTF obtained in step (3), and obtaining a recombinant Pichia pastoris strain with an additional copy of the human lactoferrin expression cassette by Zeocin resistance screening, named GS115-pPIC9K-hLTF-1C.

[0026] As a further preferred embodiment of the present invention, the method further includes step (5): linearizing the pPICZHαA-hLTFⅡ recombinant plasmid containing two human lactoferrin expression cassettes with SalⅠ enzyme, electroporating the high-expression strain GS115-pPIC9K-hLTF obtained in step (3), and obtaining a recombinant Pichia pastoris strain with two additional copies of human lactoferrin expression cassettes by Zeocin resistance screening, named GS115-pPIC9K-hLTF-2C.

[0027] As a further preferred embodiment of the present invention, the method further includes step (6): linearizing the pGAPZαhA-Mxr1 recombinant plasmid by digestion with BspHI, electroporating the five-copy strain GS115-pPIC9K-hLTF-2C competent cells obtained in step (5), and screening them with YPD plates containing 250 μg / mL hygromycin to obtain a recombinant Pichia pastoris strain whose genome also integrates the Mxr1 regulatory element expression cassette, named GS115-pPIC9K-hLTF-2C-Mxr1.

[0028] The pPICZHαA-hLTF recombinant plasmid is constructed using pPICZHαA as its backbone. pPICZHαA is a recombinant plasmid obtained by inserting the HIS4 target gene after the PciⅠ restriction site of the pPICZHαA plasmid. The pPICZHαA-hLTFⅡ recombinant plasmid is a recombinant plasmid containing two hLTF expression cassettes constructed by ligating the human lactoferrin expression cassette (AOX1 promoter-α-factor-hLTF-AOX1 terminator) to the BamHⅠ restriction site on the basis of pPICZHαA-hLTF.

[0029] Thirdly, the present invention provides a recombinant expression plasmid containing a human lactoferrin expression cassette, the human lactoferrin expression cassette comprising an operably linked AOX1 promoter, an α-mating factor signal peptide coding sequence, and a codon-optimized human lactoferrin coding gene, wherein the codon-optimized human lactoferrin coding gene is located at the 3' end of the Ste13 protease recognition cleavage site (EAEA) coding sequence in the α-mating factor signal peptide coding sequence.

[0030] As a further preferred embodiment of the present invention, the recombinant expression plasmid is constructed using pPIC9K as a backbone and named pPIC9K-EAEA-hLTF.

[0031] As a further preferred embodiment of the present invention, the recombinant expression plasmid is constructed with pPICZHαA as the backbone and contains a human lactoferrin expression cassette, named pPICZHαA-hLTF; or it is constructed with pPICZHαA as the backbone and contains two human lactoferrin expression cassettes, named pPICZHαA-hLTFⅡ.

[0032] Fourthly, the present invention provides the application of the above-mentioned recombinant Pichia pastoris strain in the production of human lactoferrin.

[0033] As a further preferred embodiment of the present invention, a method for producing human lactoferrin using recombinant Pichia pastoris includes the following steps:

[0034] (1) The above-mentioned recombinant Pichia pastoris strain was inoculated into YPD medium for activation, and after seed culture, it was subjected to methanol-induced fermentation in BMMY induction medium;

[0035] (2) Add methanol every 24 h to a final concentration of 0.5% to 1.0% (v / v) and continue induction for 96 to 120 h;

[0036] (3) Centrifuge and collect the fermentation supernatant to obtain a fermentation broth containing human lactoferrin.

[0037] As a further preferred embodiment of the present invention, the methanol-induced fermentation temperature in step (1) is 28-30°C and the rotation speed is 220-250 rpm.

[0038] As a further preferred embodiment of the present invention, the method is carried out in a fermenter of 10 L or more, and the fermentation process includes a glycerol batch stage, a glycerol feeding stage, and a methanol induction stage; the temperature in the methanol induction stage is maintained at 28°C, the methanol concentration is maintained at 0.5% to 1.0% (v / v), and the specific growth rate is controlled at 0.015 to 0.020 h. -1 The pH was maintained at 5.5.

[0039] The beneficial effects of adopting the above technical solution are as follows:

[0040] 1. Without altering the overall structure of the α-factor signal peptide, this invention significantly enhances the shake-flask secretion of hLTF by placing the codon-optimized hLTF encoding gene after the EAEA coding sequence of the Ste13 protease recognition cleavage site, compared to the control group placed after the KR coding sequence of the Kex2 protease recognition cleavage site. This indicates that the complete dual enzymatic digestion process of Kex2 and Ste13 facilitates the correct cleavage of the hLTF precursor peptide and the efficient secretion of the mature protein.

[0041] 2. This invention accurately obtained the high-expression strain GS115-pPIC9K-hLTF through G418 gradient screening, and its expression level in shake flasks was approximately 11 times that of the single-copy strain. The expression level was further increased after adding one copy to the high-expression strain. However, the expression level decreased when two more copies were added. This invention systematically reveals for the first time that there is an optimal window phenomenon for the integration copy number of human lactoferrin in Pichia pastoris, which has important reference value for guiding the recombinant expression of high molecular weight exogenous proteins.

[0042] 3. Based on the GS115-pPIC9K-hLTF-2C strain, this invention drives the continuous expression of the Mxr1 regulatory element through the constitutive GAP promoter, which effectively alleviates the bottleneck caused by the high copy number of multiple gene expression load on the host secretion pathway. The final expression level is about 58.8% higher than that of the five-copy strain, achieving a dual synergistic effect of gene dosage optimization and secretion pathway enhancement.

[0043] 4. The final optimized strain GS115-pPIC9K-hLTF-2C-Mxr1 obtained in this invention has been successfully scaled up in a 10 L fermenter, demonstrating its good potential for large-scale production. Attached Figure Description

[0044] Figure 1This is an SDS-PAGE image of recombinant hLTF with different α-factor cleavage sites in the supernatant of induced fermentation for 120 h, as shown in Example 1 of this invention. Wherein, M: 10-180 kDa Protein Marker; 1: X33-pPICZαA-EAEA-hLTF-single colony 1; 2: X33-pPICZαA-EAEA-hLTF-single colony 2; 3: X33-pPICZαA-EAEA-hLTF-single colony 3; 4: X33-pPICZαA-EAEA-hLTF-single colony 4; 5: X33-pPICZαA-EAEA-hLTF-single colony 5; 6: X33-pPICZαA-KR-hLTF-single colony 1; 7: X33-pPICZαA-KR-hLTF-single colony 2; 8: X33-pPICZαA-KR-hLTF single colony 3; 9: X33-pPICZαA-KR-hLTF single colony 4; 10: X33-pPICZαA-KR-hLTF single colony 5; 11: Positive control (hLTF purified standard);

[0045] Figure 2 The plasmid map of the recombinant plasmid pPIC9K-EAEA-hLTF;

[0046] Figure 3 The image shows the SDS-PAGE results of different clones of strain GS115 in the supernatant of induced fermentation after 120 h of screening for multiple copies in Example 2 of this invention; where 1: GS115-pPIC9K-EAEA-hLTF-single colony 1; 2: GS115-pPIC9K-EAEA-hLTF-single colony 2; 3: GS115-pPIC9K-EAEA-hLTF-single colony 3; 4: GS115-pPIC9K-EAEA-hLTF-single colony 4; 5: GS115-pPIC9K-EAEA-hLTF-single colony 5; 6: GS115-pPIC9K-EAEA-hLTF-single colony 6; 7: GS115-pPIC9K-EAEA-hLTF-single colony 7; 8: GS115-pPIC9K-EAEA-hLTF-single colony 8; 9: GS115-pPIC9K-EAEA-hLTF-single colony 9; 10: GS115-pPIC9K-EAEA-hLTF-single colony 10; 11: positive control (hLTF purified standard);

[0047] Figure 4 The plasmid map of the recombinant plasmid pPICZHαA-hLTF;

[0048] Figure 5The plasmid map of the recombinant plasmid pPICZHαA-hLTFⅡ;

[0049] Figure 6 The image shows the SDS-PAGE results of the supernatant from the 120 h induced fermentation of the strain GS115-pPIC9K-hLTF-1C, which was added as a single copy in Example 3 of this invention. M: 10-180 kDa Protein Marker; r1: GS115-pPIC9K-hLTF; 2: GS115-pPIC9K-hLTF-1C single colony 1; 3: GS115-pPIC9K-hLTF-1C single colony 2; 4: GS115-pPIC9K-hLTF-1C single colony 3; 5: GS115-pPIC9K-hLTF-1C single colony 4; 6: GS115-pPIC9K-hLTF-1C single colony 5; 7: GS115-pPIC9K-hLTF-1C single colony 6; 8: GS115-pPIC9K-hLTF-1C single colony 7; 9: GS115-pPIC9K-hLTF-1C single colony 8; 10: GS115-pPIC9K-hLTF-1C single colony 9; 11: GS115-pPIC9K-hLTF-1C single colony 10; 12: Positive control (hLTF purified standard);

[0050] Figure 7 This is an SDS-PAGE result of the GS115-pPIC9K-hLTF-2C strain with two copies added in Example 3 of the present invention in the supernatant of induced fermentation for 120 h; where M: 10-180 kDa Protein Marker; 1: GS115-pPIC9K-hLTF; 2: GS115-pPIC9K-hLTF-1C; 3: GS115-pPIC9K-hLTF-2C - single colony 1; 4: GS115-pPIC9K-hLTF-2C - single colony 2; 5: GS115-pPIC9K-hLTF-2C - single colony 3; 6: GS115-pPIC9K-hLTF-2C - single colony 4; 7: GS115-pPIC9K-hLTF-2C - single colony 5; 8: GS115-pPIC9K-hLTF-2C single colony 6; 9: GS115-pPIC9K-hLTF-2C single colony 7; 10: GS115-pPIC9K-hLTF-2C single colony 8; 11: GS115-pPIC9K-hLTF-2C single colony 9; 12: GS115-pPIC9K-hLTF-2C single colony 10; 13: Positive control (hLTF purified standard);

[0051] Figure 8 This is the plasmid map of the recombinant plasmid pGAPZαhA-Mxr1.

[0052] Figure 9 This is an SDS-PAGE result of GS115-pPIC9K-hLTF-2C-Mxr1 in the supernatant of induced fermentation after 120 h following the introduction of the Mxr1 regulatory element into a five-copy strain in Example 4 of this invention; where M: 10-180 kDa Protein Marker; 1: GS115-pPIC9K-hLTF-2C; 2: GS115-pPIC9K-hLTF-2C-Mxr1 single colony 1; 3: GS115-pPIC9K-hLTF-2C-Mxr1 single colony 2; 4: GS115-pPIC9K-hLTF-2C-Mxr1 single colony 3; 5: GS115-pPIC9K-hLTF-2C-Mxr1 single colony 4; 6: GS115-pPIC9K-hLTF-2C-Mxr1 single colony 5; 7: Single colony 6 of GS115-pPIC9K-hLTF-2C-Mxr1; 8: Single colony 7 of GS115-pPIC9K-hLTF-2C-Mxr1; 9: Single colony 8 of GS115-pPIC9K-hLTF-2C-Mxr1; 10: Single colony 9 of GS115-pPIC9K-hLTF-2C-Mxr1; 11: Single colony 10 of GS115-pPIC9K-hLTF-2C-Mxr1; 12: Positive control (purified hLTF standard);

[0053] Figure 10 The image shows the SDS-PAGE results of the fermentation supernatant diluted 10-fold at different methanol induction time periods during the scale-up verification of the final optimized strain GS115-pPIC9K-hLTF-2C-Mxr1 in Example 5 of this invention in a 10 L fermenter; where 1: 48 h; 2: 72 h; 3: 96 h; 4: 120 h; 5: positive control (hLTF purified standard);

[0054] Figure 11 This is a graph showing the change in human lactoferrin expression levels over induction time. Detailed Implementation

[0055] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be described clearly and completely below with reference to specific embodiments. Where specific conditions are not specified in the embodiments, they shall be implemented according to conventional conditions or manufacturer-recommended conditions.

[0056] The core biological materials used in this invention are described below:

[0057] Pichia pastoris strains X-33 and GS115 were both routine laboratory strains. Restriction endonucleases and PCR reagents were commercially available. The preparation of competent Pichia pastoris cells and electroporation procedures were performed in accordance with the Invitrogen Pichia pastoris manual.

[0058] The PCR amplification system and procedure used in this invention are shown in Table 1 and Appendix Table 2.

[0059] Table 1 PCR amplification system

[0060]

[0061] Table 2 PCR amplification program

[0062]

[0063] The serial numbers of this invention are as follows:

[0064] SEQ ID NO.1: Amino acid sequence of human lactoferrin (hLTF);

[0065] SEQ ID NO.2: The codon-optimized hLTF nucleotide sequence;

[0066] SEQ ID NO.3: Amino acid sequence of Mxr1 regulatory element;

[0067] SEQ ID NO.4: The codon-optimized nucleotide sequence of Mxr1;

[0068] SEQ ID NO.5: hLTF amplification primers: F sequence;

[0069] SEQ ID NO.6: hLTF amplification primers: R sequence;

[0070] SEQ ID NO.7: Mxr1 amplification primers: pGAPZαhA-Mxr1-F sequence;

[0071] SEQ ID NO.8: Mxr1 amplification primers: pGAPZαhA-Mxr1-R sequence;

[0072] SEQ ID NO.9: Full nucleotide sequence of the pPICZHαA vector;

[0073] SEQ ID NO.10: Full nucleotide sequence of the pPICZHαA-hLTFⅡ vector;

[0074] SEQ ID NO.11: Full nucleotide sequence of the pGAPZαhA vector;

[0075] Example 1: Effect of EAEA cleavage site configuration on hLTF secretion efficiency

[0076] This embodiment constructs two cleavage site configurations and systematically compares the effects of different α-factor connection positions on hLTF secretion efficiency.

[0077] 1.1 Construction of recombinant expression vectors

[0078] Using pPICZαA plasmid as a backbone, the following two recombinant expression vectors were constructed:

[0079] pPICZαA-KR-hLTF: The hLTF encoding gene with codon optimization as shown in SEQ ID NO.2 is placed after the Kex2 protease recognition cleavage site and before the Ste13 protease recognition cleavage site of the α-factor coding sequence. That is, the N-terminus of the signal peptide is intact but does not contain the EAEA sequence, and hLTF is directly exposed after Kex2 cleavage.

[0080] pPICZαA-EAEA-hLTF: The hLTF encoding gene with codon optimization as shown in SEQ ID NO.2 is placed after the Ste13 protease recognition cleavage site of the α-factor encoding sequence. The signal peptide retains the complete KR and EAEA sequences. hLTF needs to be released after two-step enzymatic digestion by Kex2 and Ste13.

[0081] The codon-optimized hLTF gene, pPICZαA-KR-hLTF, and pPICZαA-EAEA-hLTF shown in SEQ ID NO.2 above were all synthesized by Genewiz Ltd.

[0082] 1.2 Pichia pastoris transformation and screening

[0083] The two recombinant plasmids were extracted separately and linearized by single digestion with restriction endonuclease SacⅠ. After successful linearization was confirmed by agarose gel electrophoresis, they were purified using a Qiagen gel extraction kit, dissolved in 20 μL of sterile water, and stored at -20℃ for later use.

[0084] Pichia pastoris X-33 competent cells were prepared according to the Invitrogen Pichia pastoris expression manual. 20 μg of linearized recombinant plasmid was mixed with 100 μL of competent cells and transferred to a pre-chilled 0.2 cm electroporation cuvette. The cells were incubated on ice for 5 min, and the electroporation instrument parameters were set as follows: voltage 1500 V, capacitance 25 μF, resistance 200 Ω, and pulse duration approximately 2 ms. Electroporation transformation was performed immediately after electroporation. Immediately after electroporation, 1 mL of 1 M sorbitol solution (on ice) was added, and the cells were incubated at 30°C for 1.5 h to recover. The cells were then streaked onto YPDS agar plates containing 100 μg / mL Zeocin and incubated at 30°C for 5 days for initial screening. Single colonies from the initial screening were picked and plated onto YPDS agar plates containing 100 μg / mL Zeocin and incubated at 30°C for 5 days for secondary screening. Single colonies from the secondary screening were picked and plated onto YPDS agar plates and incubated at 30°C for 3 days for activation.

[0085] 1.3 Shake-flask fermentation and expression detection

[0086] (1) The preserved strain was streaked onto YPD solid plates and incubated upside down at 30°C for 72 h to activate it;

[0087] (2) Select large, round single colonies after activation, inoculate them into YPD seed culture medium, and incubate at 30℃ and 250 rpm for 12 h until OD. 600 ≥2, obtain first-grade seed solution;

[0088] (3) The primary seed culture was transferred to BMGY fermentation medium and cultured at 30°C and 250 rpm for 16 h until OD. 600 ≈5, complete the secondary seed propagation;

[0089] (4) Collect the bacterial cells by centrifugation at 4000×g and 4℃ for 5 min, and discard the supernatant; resuspend the bacterial cells in LB medium without sodium chloride pre-cooled at 4℃, centrifuge at 4000×g and 4℃ for 5 min, discard the supernatant, and wash 3 times in the same way.

[0090] (5) The clean bacterial body was suspended in BMMY induction medium and cultured at 28℃ and 250 rpm with shaking. Methanol was added every 24 h to a final concentration of 0.5% (v / v) and induced continuously for 120 h. During this period, the supernatant was collected by centrifugation at 12000 rpm and 4℃ for 10 min, which is the fermentation broth containing the target protein.

[0091] (6) Mix the fermentation supernatant with 5× protein loading buffer in equal proportion, heat in a metal bath at 100℃ for 5 min, cool and centrifuge at 12000 rpm for 5 min, take the supernatant for SDS-PAGE analysis, and use ImageJ software to perform grayscale analysis and quantification of the target band.

[0092] 1.4 Experimental Results

[0093] The SDS-PAGE results are shown in Figure 1. Both recombinant Pichia pastoris strains with modified signal peptide conformations showed a target protein band at approximately 100 kDa, consistent with the theoretical molecular weight of hLTF. Gradient electrophoresis using human lactoferrin standards was performed, and the band grayscale was read using ImageJ software to plot a standard curve for absolute quantification. The hLTF secretion expression level of the pPICZαA-EAEA-hLTF strain was approximately 10.6 mg / L, higher than that of pPICZαA-KR-hLTF. These results indicate that placing the hLTF encoding gene after the intact EAEA cleavage site of the α-factor can utilize the sequential processing mechanism of the Kex2 / Ste13 protease to enhance the secretory expression level of high molecular weight hLTF.

[0094] Example 2: Construction of multi-copy integrating strains and obtaining triple-copy high-expression strains

[0095] In this embodiment, the pPIC9K plasmid platform combined with G418 gradient screening was used to systematically screen for human lactoferrin high-copy integration strains and to identify the expression performance of triple-copy strains.

[0096] 2.1 Construction of recombinant plasmid pPIC9K-EAEA-hLTF

[0097] Using the pPIC9K plasmid carrying AOX1 as a promoter as a backbone, the hLTF coding gene with optimized codons as shown in SEQ ID NO.2 was ligated to the EAEA coding sequence downstream of the α-factor coding sequence to construct the recombinant plasmid pPIC9K-EAEA-hLTF. The plasmid map is shown below. Figure 2 pPIC9K carries the HIS4 gene, which can be integrated into the genome in the GS115 host via homologous recombination, and carries the G418 resistance gene for multicopy selection.

[0098] 2.2 Pichia pastoris electroconversion

[0099] After linearizing the pPIC9K-EAEA-hLTF recombinant plasmid by SacⅠ restriction enzyme, Pichia pastoris GS115 competent cells were prepared according to the method described in Example 1 and then subjected to electroporation.

[0100] 2.3 MD plate screening of His + Positive transformants

[0101] Prepare MD solid culture medium containing 1.34% YNB and 4×10⁴ mg / L. -51% biotin, 2% glucose, and 1.5% agar powder were autoclaved and poured onto plates. The revived bacterial culture was centrifuged at 3000×g for 5 min, most of the supernatant was discarded, and approximately 200 μL of the bacterial cells were resuspended. 100 μL and 200 μL of the resuspended cells were then spread onto MD plates and incubated upside down at 30°C for 3 days. His... + Positive transformants are those in which pPIC9K-EAEA-hLTF has been successfully integrated into the GS115 genome.

[0102] 2.4 Screening of multi-copy integrated strains using G418 concentration gradient

[0103] The copy number of the G418 resistance gene carried by pPIC9K is positively correlated with its integration copy number, and multi-copy transformants can be enriched by increasing the G418 screening concentration.

[0104] (1) Prepare YPD solid culture medium containing 1.0 mg / mL, 3.0 mg / mL and 4.0 mg / mL G418 respectively. The specific operation is to add the G418 stock solution to the YPD culture medium cooled to about 55°C, mix well and pour into plates.

[0105] (2) Pipette 1 mL of sterile water onto an MD plate and gently scrape off all of the His solution with a sterile swab. + Transformant colonies were formed, and bacterial suspensions were measured. 600 According to the Pichia pastoris density conversion formula, based on 1 OD 600 ≈5×10 7 Calculate the total number of cells per mL;

[0106] (3) Dilute the bacterial suspension with sterile water to 1×10⁻⁶. 7 5×10 7 1×10 8 5×10 8 100 μL cells / 100 μL;

[0107] (4) Take 100 μL of bacterial suspension at each gradient cell concentration and spread it evenly on YPD plates of each concentration of G418, and incubate at 30℃ for 5 days.

[0108] (5) Pick tolerant single colonies from 4.0 mg / mL G418 plates.

[0109] 2.5 Experimental Results

[0110] After screening using MD plates and G418 gradient screening, the strain obtained from 4.0 mg / mL G418 plates was named GS115-pPIC9K-hLTF. Expression was induced for 120 h using the shake-flask fermentation method described in Example 1. Figure 3The SDS-PAGE analysis shown was performed using gradient electrophoresis of human lactoferrin standards. ImageJ software was used to read the band grayscale and plot a standard curve for absolute quantification. The hLTF expression level of the high-expression strain reached 118.3 mg / L in the shake flask, which is about 11 times that of the single-copy strain, indicating that the integration of three copies significantly enhanced the secretory expression of hLTF in this expression system.

[0111] Example 3: Construction of strains with one and two copies of the pPICZHαA plasmid-mediated copy number increase.

[0112] In this embodiment, based on the GS115-pPIC9K-hLTF strain, the copy number of the hLTF expression cassette was superimposed using the pPICZHαA series plasmids to construct one-copy and two-copy strains, respectively, and the effect of different copy numbers on the hLTF expression level was compared.

[0113] 3.1 Construction of pPICZHαA series recombinant plasmids

[0114] (1) Construction of pPICZHαA backbone: Using pPICZαA plasmid as backbone, the HIS4 target gene was added after the PciⅠ restriction site as a selection marker for homologous recombination and named pPICZHαA; This plasmid also carries the Zeocin resistance gene, which can be used for double resistance screening with the integrated pPIC9K-EAEA-hLTF (G418 resistance);

[0115] (2) Construction of single-copy pPICZHαA-hLTF: After ligating the codon-optimized hLTF coding gene (SEQ ID NO.2) into the EAEA coding sequence of the pPICZHαA vector, a recombinant plasmid containing a single hLTF expression cassette was constructed and named pPICZHαA-hLTF. The plasmid map is shown in [link to plasmid map]. Figure 4 ;

[0116] (3) Construction of double-copy pPICZHαA-hLTFⅡ: The complete hLTF expression cassette, carrying the AOX1 promoter-α-factor-hLTF-AOX1 terminator, was PCR-amplified from pPICZHαA-hLTF and then ligated to the BamHI restriction site of the pPICZHαA-hLTF vector to construct a recombinant plasmid containing two tandem hLTF expression cassettes, named pPICZHαA-hLTFⅡ. The plasmid map is shown in [link to plasmid map]. Figure 5 The full sequence is shown in SEQ ID NO.10.

[0117] 3.2 Copy Number Stacking Conversion

[0118] (1) Extract the pPICZHαA-hLTF and pPICZHαA-hLTFⅡ recombinant plasmids respectively, linearize them by SalⅠ single enzyme digestion, and then dissolve them in 20 μL of sterile water after gel recovery and purification;

[0119] (2) Prepare competent cells of the high-expression strain GS115-pPIC9K-hLTF according to the method described in Example 1;

[0120] (3) Linearized pPICZHαA-hLTF and pPICZHαA-hLTFⅡ were electrotransformed into GS115-pPIC9K-hLTF competent cells (electrotransformation parameters were the same as in Example 1), spread on YPDS plates containing 100 μg / mL Zeocin, and cultured at 30°C for 5 days for initial screening; single colonies were picked and placed on YPDS plates containing 100 μg / mL Zeocin for secondary screening and confirmation.

[0121] 3.3 Experimental Results

[0122] The transformed individuals were induced to express their expression for 120 h using the shake-flask fermentation method described in Example 1. SDS-PAGE analysis results (…) Figure 6 , Figure 7 ) and ImageJ quantitative display:

[0123] The strain infused with pPICZHαA-hLTF was given an additional copy and named GS115-pPIC9K-hLTF-1C. Its hLTF expression level was further increased compared to GS115-pPIC9K-hLTF, reaching 141.5 mg / L, which is about 1.2 times that of the GS115-pPIC9K-hLTF strain.

[0124] The strain introduced with pPICZHαA-hLTFⅡ, i.e., with two additional copies, was named GS115-pPIC9K-hLTF-2C. Its hLTF expression level was 117.4 mg / L, which was actually lower than that of the GS115-pPIC9K-hLTF-1C strain.

[0125] The above results indicate that increasing the number of hLTF copies by one leads to an upward trend in hLTF expression, while increasing the number of copies by two results in a significant decrease in expression. This suggests that there is an optimal window for hLTF integration copy number in this expression system. Exceeding the optimal window may trigger increased endoplasmic reticulum stress or secretory pathway overload, leading to a decrease in expression.

[0126] Example 4: pGAPZαhA plasmid-mediated introduction of Mxr1 regulatory elements and acquisition of highly efficient expression strains

[0127] 4.1 Construction of pGAPZαhA-Mxr1 recombinant plasmid

[0128] The Mxr1 coding gene with codon optimization as shown in SEQ ID NO.4 was placed at the BstBI restriction site of the pGAPZαhA vector and synthesized by Genscript Biotech Co., Ltd., named pGAPZαhA-Mxr1. The plasmid map is shown below. Figure 8 The pGAPZαhA plasmid is driven by a constitutive GAP promoter to continuously and stably express the target gene and carries a hygromycin resistance gene as an integration selection marker.

[0129] 4.2 Linearization of recombinant plasmids

[0130] The pGAPZαhA-Mxr1 recombinant plasmid was extracted, linearized by single digestion with restriction endonuclease BspHI, and the linearization was confirmed by agarose gel electrophoresis. After purification using a gel extraction kit, the plasmid was dissolved in 20 μL of sterile water and stored at -20℃ for later use.

[0131] 4.3 Preparation of competent cells of GS115-pPIC9K-hLTF-2C strain

[0132] (1) Pick a single colony of five-copy strain GS115-pPIC9K-hLTF-2C, inoculate it into 5 mL of YPD liquid medium, and culture overnight at 30℃ and 230 rpm with shaking;

[0133] (2) Transfer the above culture to 50 mL of YPD liquid medium and incubate at 30°C and 230 rpm with shaking until OD. 600 ≈1.5;

[0134] (3) Transfer the bacterial culture to a 50 mL sterile centrifuge tube, centrifuge at 4℃ and 5000×g for 5 min, and discard the supernatant;

[0135] (4) Resuspend the bacterial cells in 30 mL of pre-cooled sterile water, centrifuge at 4°C and 5000×g for 5 min, discard the supernatant, and repeat once;

[0136] (5) Resuspend in 10 mL of pre-cooled 1 M sorbitol solution, centrifuge at 4°C and 5000×g for 5 min, discard the supernatant, and repeat once;

[0137] (6) Resuspend the bacterial cells in 500 μL of pre-cooled 1 M sorbitol solution, dispense into 100 μL / tube, and store at -80℃ for later use.

[0138] 4. Electroconversion and Hygromycin Resistance Screening

[0139] (1) Take 100 μL of GS115-pPIC9K-hLTF-2C competent cells, add 10 μg of linearized pGAPZαhA-Mxr1 recombinant plasmid, mix gently, transfer to a pre-cooled 0.2 cm electroporation cuvette, and incubate on ice for 5 min;

[0140] (2) Set the electric shock parameters: voltage 1500 V, capacitor 25 μF, resistor 200 Ω, pulse time about 2 ms, and perform electric shock;

[0141] (3) Immediately after the electric shock, add 1 mL of 1 M sorbitol in an ice bath, mix gently, transfer to a sterile centrifuge tube, and incubate at 30°C for 1 hour to recover.

[0142] (4) Take 200 μL of the revived bacterial solution, spread it on a YPD plate containing 250 μg / mL hygromycin, and incubate it upside down at 30℃ for 4 days until clear single colonies appear;

[0143] (5) Pick single colonies and screen them again on YPD plates containing 250 μg / mL hygromycin.

[0144] 5. Experimental Results

[0145] The transformed individuals were induced to express their expression for 120 h using the shake-flask fermentation method described in Example 1. SDS-PAGE analysis results were obtained. Figure 9 ImageJ quantitative analysis showed that after introducing the regulatory element Mxr1 into the five-copy strain, the hLTF expression level of the resulting strain GS115-pPIC9K-hLTF-2C-Mxr1 reached 186.4 mg / L, which was the highest level among all the strains tested in this invention. This was an increase of approximately 58.8% compared to the five-copy strain and approximately 31.7% compared to the GS115-pPIC9K-hLTF-2C strain (141.5 mg / L).

[0146] The results showed that the introduction of the Mxr1 regulatory element effectively alleviated the bottleneck effect of high-copy exogenous gene expression on the host secretion pathway, achieving a dual synergistic effect of "multi-copy gene dosage optimization + secretion pathway transcription enhancement".

[0147] Example 5: Scale-up validation of the final optimized strain in a 10 L fermenter

[0148] In this embodiment, the final optimized strain GS115-pPIC9K-hLTF-2C-Mxr1 was scaled up in a 10 L fermenter to evaluate its suitability for large-scale production.

[0149] 5.1 Seed liquid preparation

[0150] A single colony of GS115-pPIC9K-hLTF-2C-Mxr1 was picked and inoculated into 5 mL of YPD liquid medium. The culture was incubated at 30°C and 250 rpm for 12 h to obtain the primary seed culture. The primary seed culture was then transferred to a 1 L shake flask containing 400 mL of BMGY medium and incubated at 30°C and 250 rpm for 14 h until OD (Organic Difference) was reached. 600 ≥4.0, to obtain secondary seed liquid.

[0151] 5.2 Sterilization and Inoculation of Fermentation Tanks

[0152] 7.6 L of FM21 medium (KH2PO4, 7 g / L; CaSO4, 0.2 g / L; K2SO4, 3 g / L; MgSO4-7H2O, 1.5 g / L; glycerol 40 g / L) was added to a 10 L fermenter and autoclaved at 121 °C for 20 min. Then, 400 mL of secondary seed culture was pumped into the fermenter and high-density fermentation was started at 30 °C.

[0153] 5.3 High-density fermentation process

[0154] The fermentation process is divided into three stages:

[0155] Glycerol batch stage: Initial growth was carried out using glycerol in FM21 medium as a carbon source. Dissolved oxygen (DO) was maintained above 20% by controlling the stirring speed at 200-800 rpm and the aeration rate at 2-20 vvm. The pH was maintained at around 5.5 throughout the process by adding ammonia.

[0156] Glycerin replenishment phase: When DO rebounds to above 80%, add a 50% glycerin solution containing 12 mL / L LPTM1 trace element solution at a rate of 2.42 mL / min, and continue replenishing until OD reaches 80%. 600 Once the glycerol level reaches 220, stop adding glycerol and starve the animal for 1 hour.

[0157] Methanol induction stage: The temperature was lowered to 28℃, and methanol feeding induction was initiated. The methanol concentration was maintained at 0.5%–1.0% (v / v) using offline methanol monitoring. The methanol solution contained 12 mL / L PTM1 trace element solution. Feeding was controlled according to the specific growth rate, which was maintained at 0.015–0.020 h. -1 When the specific growth rate is less than 0.015 h -1 At that time, increase the feeding rate by 0.5 ml / h·L.

[0158] 5.4 Sample Testing

[0159] Fermentation broth samples were collected at different time points during methanol induction, and the supernatant was collected by centrifugation. The supernatant was diluted 10 times and mixed with 5× protein loading buffer. The mixture was heated in a metal bath at 100℃ for 5 min, cooled, centrifuged, and the supernatant was collected for SDS-PAGE analysis.

[0160] 5.5 Experimental Results

[0161] The results are as follows Figure 10 and Figure 11 As shown, the target protein band continued to increase with the extension of induction time. According to ImageJ quantitative analysis and combined with the standard curve, the human lactoferrin yield reached 1.7 g / L after 120 h of methanol induction.

[0162] Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art can still modify the technical solutions described in the foregoing embodiments or make equivalent substitutions for some of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. A recombinant Pichia pastoris strain that efficiently secretes and expresses human lactoferrin, characterized in that, The genome of the recombinant Pichia pastoris strain integrates a human lactoferrin expression cassette, which includes an operatively linked promoter, an α-mating factor signal peptide coding sequence, and a codon-optimized human lactoferrin coding gene located at the 3' end of the Ste13 protease recognition cleavage site coding sequence in the α-mating factor signal peptide coding sequence.

2. The recombinant Pichia pastoris strain according to claim 1, characterized in that, The human lactoferrin expression cassette is integrated into the genome by 1 to 2 copies.

3. The recombinant Pichia pastoris strain according to claim 1, characterized in that, The copy number is increased by one or two copies, and the genome of the recombinant Pichia pastoris strain also integrates an Mxr1 regulatory element expression cassette, which includes a constitutive GAP promoter and an Mxr1 coding gene, wherein the Mxr1 coding gene is located at the 3' end of the GAP promoter.

4. The recombinant Pichia pastoris strain according to claim 1, characterized in that, The codon-optimized human lactoferrin encoding gene nucleotide sequence is shown in SEQ ID NO.2, or is a nucleotide sequence that has at least 90% sequence identity with the sequence shown in SEQ ID NO.2 and encodes a polypeptide with human lactoferrin activity.

5. The recombinant Pichia pastoris strain according to claim 3, characterized in that, The amino acid sequence of the Mxr1 encoding gene is shown in SEQ ID NO.3, or is the encoding sequence of a protein that has at least 80% sequence identity with the sequence shown in SEQ ID NO.3 and has Mxr1 transcriptional regulatory activity.

6. A method for constructing a recombinant Pichia pastoris strain that efficiently secretes and expresses human lactoferrin as described in any one of claims 1-5, characterized in that, Includes the following steps: (1) Construction of recombinant expression plasmid: The codon-optimized human lactoferrin coding gene was linked to a Pichia pastoris expression plasmid containing the coding sequence of the α-mating factor signal peptide EAEA cleavage site. The codon-optimized human lactoferrin coding gene was located at the 3' end of the coding sequence of the EAEA cleavage site, and the recombinant expression plasmid pPIC9K-EAEA-hLTF was obtained. (2) Pichia pastoris transformation and positive screening: The recombinant expression plasmid obtained in step (1) was linearized by single enzyme digestion with SacⅠ, and then electrotransformed into Pichia pastoris GS115 competent cells. The transformants were plated on MD solid medium, and His⁺ positive transformants were obtained by histidine auxotrophic complementation screening. (3) G418 gradient screening of multi-copy integrated strains: The His⁺ positive transformants obtained in step (2) were spread on YPD solid medium containing different concentrations of G418. By screening with G418 concentration gradient, single colonies growing on high concentration G418 plates were selected to obtain recombinant Pichia pastoris strains that integrate multiple copies of human lactoferrin expression cassettes. As a further preferred embodiment of the present invention, in step (3), the G418 screening concentration gradient is 1.0 mg / mL, 3.0 mg / mL and 4.0 mg / mL. A strain with high expression of human lactoferrin was obtained by screening on a 4.0 mg / mL G418 plate and named GS115-pPIC9K-hLTF.

7. The method for constructing the recombinant Pichia pastoris strain according to claim 6, characterized in that, The method further includes one or more of the following steps: Step (4): After linearizing the pPICZHαA-hLTF recombinant plasmid containing a single human lactoferrin expression cassette with SalⅠ, the high-expression strain GS115-pPIC9K-hLTF obtained in step (3) was electroporated. The recombinant Pichia pastoris strain with an additional copy of the human lactoferrin expression cassette was obtained by Zeocin resistance screening and named GS115-pPIC9K-hLTF-1C. Step (5): After linearizing the pPICZHαA-hLTFⅡ recombinant plasmid containing two human lactoferrin expression cassettes with SalⅠ, the high-expression strain GS115-pPIC9K-hLTF obtained in step (3) was electroporated. The recombinant Pichia pastoris strain integrating two additional copies of human lactoferrin expression cassettes was obtained by Zeocin resistance screening and named GS115-pPIC9K-hLTF-2C. Step (6): After linearizing the pGAPZαhA-Mxr1 recombinant plasmid with BspHI, electroporate the GS115-pPIC9K-hLTF-2C competent cells obtained in step (5), and screen them with YPD plates containing 250 μg / mL hygromycin to obtain a recombinant Pichia pastoris strain whose genome also integrates the Mxr1 regulatory element expression cassette, named GS115-pPIC9K-hLTF-2C-Mxr1.

8. A recombinant expression plasmid, characterized in that, The recombinant expression plasmid contains a human lactoferrin expression cassette, which includes an operatively linked AOX1 promoter, an α-mating factor signal peptide coding sequence, and a codon-optimized human lactoferrin coding gene located at the 3' end of the Ste13 protease recognition cleavage site coding sequence in the α-mating factor signal peptide coding sequence.

9. The recombinant expression plasmid according to claim 8, characterized in that, Choose from any of the following: The recombinant expression plasmid was constructed using pPIC9K as the backbone and named pPIC9K-EAEA-hLTF; The recombinant expression plasmid is constructed with pPICZHαA as the backbone and contains a human lactoferrin expression cassette, named pPICZHαA-hLTF; or it is constructed with pPICZHαA as the backbone and contains two human lactoferrin expression cassettes, named pPICZHαA-hLTFⅡ.

10. The use of the recombinant Pichia pastoris strain according to any one of claims 1-5 in the production of human lactoferrin.

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