Lactoferrin-producing pichia pastoris with enhanced vesicle transport, and method for promoting exocytosis

Abstract

Provided are a lactoferrin-producing Pichia pastoris with enhanced vesicle transport, and a method for promoting exocytosis, which belong to the technical field of biology. It is found that the accumulation of lactoferrin in cells without efflux limits the yield of extracellular lactoferrin. In addition, a large amount of lactoferrin being accumulated in cells also affects the growth of yeast cells. Therefore, it is hoped that a method capable of effectively promoting the secretory expression of lactoferrin is found. It is found by means of screening that after overexpression of BMH2, Sec12 or Sly1, the content of lactoferrin in an extracellular supernatant is significantly increased, and the content of lactoferrin in a supernatant after precipitation and crushing is effectively reduced. By means of the method, the vesicle transport of lactoferrin in Pichia pastoris can be enhanced, so that lactoferrin can be quickly secreted out of cells, an enhancement in the efficient expression of lactoferrin is realized, and the current problem of lactoferrin accumulating in cells without secretion is solved.

Description

A lactoferrin-producing Pichia yeast with enhanced vesicle transport and a method for promoting extracellular secretion Technical Field

[0001] The invention relates to a lactoferrin-producing Pichia yeast capable of strengthening vesicle transport and a method for promoting extracellular secretion, belonging to the field of biotechnology. Background Art

[0002] Lactoferrin (LF), also known as lactotransferrin, is an iron-binding protein with a molecular weight of approximately 80 kDa and a key globulin in breast milk. Studies have shown that LF is not only present in breast milk but is also widely distributed in various secretions, including tears, saliva, and bile. Its highest concentration is in breast milk, followed by human milk, where it reaches a concentration of 1.0-3.0 mg / mL, ten times that found in cow's milk. LF not only transports iron ions but also exhibits biological activities such as antibacterial and anti-inflammatory properties and immune response regulation.

[0003] In addition to its antibacterial activity, LF also possesses significant bactericidal activity. On the one hand, the high alkaline charge density on LF's surface facilitates subspecific binding to certain bacterial biomolecules, disrupting cell membrane function. On the other hand, LF can specifically bind to bacterial cell membranes, increasing membrane permeability and even disrupting the lipid bilayer, thereby exerting its bactericidal effect. Further studies have revealed that the bactericidal activity of LF is primarily located in the N-lobe. Unlike the iron-binding region, the N-terminus of LF relies on its own positive charge to generate electrostatic attraction with negatively charged lipopolysaccharides and teichoic acids on the bacterial cell membrane, ultimately damaging the cell membrane. Therefore, LF is considered a novel antibacterial and anticancer agent. Currently, LF has been added to numerous commercial products, including infant formula, thermotherapy beverages, fermented milk, cosmetics, and toothpaste. LF's diverse health-promoting properties and its widespread real-life applications have sparked growing research interest.

[0004] Currently, the main way to obtain lactoferrin is to separate and extract it from cow's milk. However, cow's milk only contains 0.03-0.49g / L lactoferrin. The extraction process is not only expensive, but the consumption of heterologous proteins by humans may also have certain negative effects and produce antigenic reactions. In order to obtain large amounts of lactoferrin while avoiding its side effects, researchers have attempted to use genetic engineering technology to mass-produce human lactoferrin. Among them, the host cells for producing lactoferrin mainly include Escherichia coli, yeast, mammalian cells and plant cells. The Escherichia coli expression system lacks a glycosylation modification mechanism, resulting in the inability to produce biologically active lactoferrin. Although mammalian cells and plant cells can undergo appropriate glycosylation modification, large-scale production is difficult to achieve due to the long cell growth cycle and complex culture.

[0005] Current research has identified elements suitable for lactoferrin expression through screening for promoters and signal peptides. Furthermore, by overexpressing molecular chaperones involved in protein synthesis in yeast, target protein expression is enhanced. This, in turn, strengthens transcription factor expression at the transcriptome level, leading to an upregulation of overall yeast protein levels and enhanced target protein expression. When the inventors disrupted Pichia pastoris and subjected the supernatant to SDS-PAGE, Western blotting, and ELISA analysis along with the extracellular supernatant to assay lactoferrin production, they found a ratio of 1:2 between extracellular and intracellular lactoferrin production, indicating that a significant amount of lactoferrin is not being effectively secreted from the cells, limiting production. To further increase lactoferrin production, the problem of lactoferrin being trapped within the cells and unable to be excreted promptly must be addressed, thereby enabling the smooth secretion of exogenous proteins.

[0006] Summary of the Invention

[0007] To address the above issues, the present invention fused and expressed a hybrid signal peptide ost-a with lactoferrin, and introduced the vesicle transport genes BMH2, Sec12, or Sly1 into a starting Pichia pastoris strain. This method significantly increased the lactoferrin content in the extracellular supernatant and effectively reduced the lactoferrin content in the supernatant of the precipitated fragments, demonstrating that the transport of lactoferrin from the cell to the extracellular space was effectively promoted. This method increases the expression of related genes within the cell, strengthens the intracellular membrane transport system, and improves the transport efficiency of lactoferrin within the yeast, thereby promoting the extracellular expression of lactoferrin.

[0008] The first object of the present invention is to provide a lactoferrin-producing Pichia pastoris with enhanced vesicle transport pathway, wherein the Pichia pastoris heterologously expresses a lactoferrin gene and a vesicle transport gene BMH2, Sec12 or Sly1.

[0009] Furthermore, the lactoferrin gene includes a human lactoferrin gene, and the amino acid sequence of human lactoferrin is shown in SEQ ID NO.4.

[0010] Furthermore, the expression of the lactoferrin gene is regulated by a signal peptide having a nucleotide sequence as shown in SEQ ID NO.2.

[0011] Furthermore, the lactoferrin gene is expressed by an inducible promoter.

[0012] Furthermore, the inducible promoter includes but is not limited to promoter P aox1 wait.

[0013] Furthermore, the inducible promoter P aox1 The nucleotide sequence is shown in SEQ ID NO.3.

[0014] Furthermore, the GenBank number of the vesicle transport gene BMH2 is FR839629.1; the GenBank number of the vesicle transport gene Sec12 is FN392322.1; and the GenBank number of the vesicle transport gene Sly1 is FR839631.1.

[0015] Furthermore, the vesicle transport gene BMH2 is expressed from a constitutive promoter.

[0016] Furthermore, the constitutive promoter includes but is not limited to promoter P GAP wait.

[0017] Furthermore, the promoter P GAP The nucleotide sequence is shown in SEQ ID NO.1.

[0018] Furthermore, Pichia pastoris X-33 was used as the starting strain.

[0019] A second object of the present invention is to provide a method for constructing the lactoferrin-producing Pichia pastoris, comprising the following steps:

[0020] S1, amplify the vesicle transport gene BMH2 and connect it to the expression vector pGAP-HygR to obtain the recombinant plasmid pGAP-BMH2;

[0021] S2. Amplify the lactoferrin gene, fusion express the signal peptide shown in SEQ ID NO.2, and connect the fusion gene fragment to the expression vector pPICZα to obtain the recombinant plasmid pPICZα-ost-a-LF;

[0022] S3. Transform the recombinant plasmid pGAP-BMH2 and the recombinant plasmid pPICZα-ost-a-LF into the Pichia pastoris host bacteria to construct the lactoferrin-producing Pichia pastoris.

[0023] The third object of the present invention is to provide the use of the above-mentioned Pichia pastoris in the preparation of products containing lactoferrin or its derivatives (such as antibacterial agents) in the fields of biology, pharmacy, food or chemical industry.

[0024] The fourth object of the present invention is to provide a method for producing lactoferrin, comprising the following steps: fermenting and producing lactoferrin using the above-mentioned Pichia pastoris.

[0025] Furthermore, the fermentation system contains inducers, including but not limited to methanol.

[0026] Furthermore, the fermentation conditions are 28-35° C. and 180-260 rpm.

[0027] The fifth object of the present invention is to provide a method for enhancing the secretory expression of lactoferrin in Pichia pastoris, comprising the following steps: introducing the vesicle transport gene BMH2, Sec12 or Sly1 into the Pichia pastoris host bacteria.

[0028] Furthermore, P GAP The promoter expresses the vesicle transport genes BMH2, Sec12 or Sly1.

[0029] Beneficial effects of the present invention:

[0030] The present invention uses a high-copy lactoferrin expression cassette of Pichia pastoris ost-a-hLF as a starting strain, screens for different vesicle transport-related genes for enhancing protein secretion and expression in Pichia pastoris strains, and identifies vesicle transport genes that can enhance lactoferrin production by verifying their effects on lactoferrin expression. This results in a Pichia pastoris strain that efficiently synthesizes lactoferrin. The present invention not only achieves the goal of increasing lactoferrin expression, but also provides a method for increasing protein secretion and expression. Testing with a lactoferrin ELISA kit revealed that the extracellular lactoferrin production of the BMH2 strain reached 721 mg / L. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] Figure 1 shows the SDS-PAGE results of the fermentation broth of the recombinant strain. A: Fermentation broth supernatant; B: Precipitation supernatant; M: 10-180 Protein Marker.

[0032] Figure 2 shows the lactoferrin yield in the supernatant of the fermentation broth of the recombinant strain. DETAILED DESCRIPTION

[0033] The present invention will be further described below with reference to the accompanying drawings and specific embodiments so that those skilled in the art can better understand the present invention and implement it. However, the embodiments are not intended to limit the present invention.

[0034] The materials involved in the following examples are as follows:

[0035] (1) Sequence information:

[0036] BMH2, GenBank: FR839629.1; Sar1, GenBank: FR839629.1; Sec1, GenBank: FR839631.1; Sec4, GenBank: FR839630.1; Sec12, GenBank: FN392322.1; Sly1, GenBank: FR839631.1; Sso1, GenBank: FR839628.1.

[0037] Promoter P GAP, SEQ ID NO.1; ost-a signal peptide, SEQ ID NO.2; promoter P aox1 , SEQ ID NO.3; hLF, SEQ ID NO.4.

[0038] (2) Culture medium

[0039] Liquid YPD medium: 1% yeast extract, 2% peptone, 2% glucose, steam sterilize at 115℃ for 20 min and store at room temperature.

[0040] BMGY medium: 5 g yeast extract, 10 g tryptone, add 350 mL of distilled water, steam sterilize at 121°C for 20 min, then add 50 mL of 13.4% YNB, 50 mL of 10% glycerol, 50 mL of 1 M pH 6.0 potassium phosphate buffer, and 1 mL of 0.02% biotin.

[0041] BMMY medium: 5 g yeast extract, 10 g tryptone, add 350 mL of distilled water, steam sterilize at 121°C for 20 min, then add 50 mL of 13.4% YNB, 50 mL of 10% glycerol, 50 mL of 1 M pH 6.0 potassium phosphate buffer, and 1 mL of 0.02% biotin.

[0042] Example 1 Amplification of vesicle transport genes and construction of PGAP expression plasmid

[0043] Using the Pichia pastoris genome as a template, the corresponding vesicle transport genes were amplified using the primer sequences listed in Table 1. The PCR products were digested and transformed into competent E. coli cells. Plates containing hygromycin were then plated to obtain the corresponding transformants. The transformants were cultured overnight, plasmids were extracted, and the correct transformants were identified by sequencing.

[0044] Table 1 Primer sequences for amplifying molecular chaperones

[0045] Example 2: Linearization of the recombinant plasmid and electrotransformation into Pichia pastoris

[0046] (1) The construction of the starting strain ost-a-hLF is described in Chinese patent CN202210946281.4. The specific steps are as follows: the ost-a signal peptide shown in SEQ ID NO.2 and the hLF coding gene are connected to the vector pPICZα to obtain the recombinant plasmid pPICZα-ost-a-hLF, and the recombinant plasmid is electroporated into Pichia pastoris X-33 to obtain ost-a-hLF expressing lactoferrin.

[0047] (2) Using the plasmid line sequenced correctly in Example 1 as a template, amplify using linearized primers to obtain a linearized fragment, and purify the linearized fragment using a kit. Take 80 μL of competent cells ost-a-hLF and 10 μL of linearized recombinant plasmid DNA, mix them evenly, and transfer them to a 0.2 cm electroporation cup that has been ice-bathed at -20°C, and ice-bath the electroporation cup containing the mixed solution for 5 minutes. Adjust the parameters of the electroporation instrument, set it to the Pichia yeast setting, voltage 1.5 kV, capacitance 25 microfarads, resistance 200 ohms, and time of about 5 milliseconds. After the electric shock, quickly add 1 ml of ice-cold 1 M sorbitol solution to the transformation cup, gently pipette to mix, and aspirate the mixture and transfer it to a centrifuge tube. Incubate at 30°C for 1-2 hours, and centrifuge at 3000 rpm for 5 minutes. Discard 800 μl of supernatant, blow the remaining bacteria evenly, spread them on YPDH (containing 500 μg / mL hygromycin) plates, and culture them in a 30°C incubator for 2-4 days until single colonies grow. Verify the correctness of the colonies by colony PCR.

[0048] Table 2 Linearized primer sequences

[0049] Example 3 Recombinant Pichia pastoris induced expression of lactoferrin

[0050] Transfer the correct colony to 3 mL of YPD medium and incubate overnight at 30°C, 220 rpm. The next day, transfer 1 mL of the culture to a 250 mL Erlenmeyer flask containing 25 mL of BMGY medium and incubate at 30°C, 220 rpm, for 24 hours. Harvest the cells by centrifugation at 3000 rpm for 5 minutes, wash three times with sterile water to remove residual glycerol, then resuspend in 25 mL of BMMY medium and transfer to a 250 mL Erlenmeyer flask. Incubate at 30°C, 220 rpm, for 5 days, supplementing with 1% methanol every 24 hours to ensure continuous induction.

[0051] The fermentation broth was centrifuged to obtain the fermentation supernatant, and lactoferrin synthesis was analyzed by SDS-PAGE. The results showed that the lactoferrin content in the fermentation supernatant of the strain overexpressing the BMH2 gene was significantly increased (Figure 1A), and the lactoferrin content in the supernatant of cell disruption was also significantly reduced, indicating a decrease in intracellular lactoferrin content (Figure 1B). Lactoferrin production was measured using an ELISA kit, and the results showed that the lactoferrin production in the fermentation supernatant of the strain overexpressing the BMH2 gene reached 721 mg / L (Figure 2).

[0052] Obviously, the above embodiments are merely examples for clarity of explanation and are not intended to limit the implementation methods. Those skilled in the art will appreciate that other variations or modifications can be made based on the above description. It is not necessary and impossible to enumerate all implementation methods here. Obvious variations or modifications arising therefrom remain within the scope of protection of the present invention.

Claims

1. A lactoferrin-producing Pichia pastoris with enhanced vesicle transport pathway, characterized in that: The Pichia pastoris heterologously expresses the lactoferrin gene and the vesicle transport gene BMH2, Sec12 or Sly1.

2. The Pichia pastoris according to claim 1, characterized in that: The lactoferrin gene is expressed under the control of a signal peptide whose nucleotide sequence is shown in SEQ ID NO.

2.

3. The Pichia pastoris according to claim 1, characterized in that: The lactoferrin gene is expressed by an inducible promoter.

4. The Pichia pastoris according to claim 3, characterized in that: The inducible promoter includes promoter P aox1 .

5. The Pichia pastoris according to claim 1, characterized in that: The lactoferrin gene includes the human lactoferrin gene hLF.

6. The Pichia pastoris according to claim 1, characterized in that: The vesicle transport gene BMH2 is regulated by the promoter P GAP Start expression.

7. The Pichia pastoris according to claim 1, characterized in that: The GenBank number of the vesicle transport gene BMH2 is FR839629.1; the GenBank number of the vesicle transport gene Sec12 is FN392322.1; and the GenBank number of the vesicle transport gene Sly1 is FR839631.

1.

8. The Pichia pastoris according to claim 1, characterized in that: Pichia pastoris X-33 was used as the starting strain.

9. The method for constructing Pichia pastoris according to any one of claims 1 to 8, characterized in that: The following steps are involved: S1, amplify the vesicle transport gene BMH2, connect it to the expression vector pGAP-HygR, and obtain the recombinant plasmid pGAP-BMH2; S2, amplify lactoferrin gene, fuse and express the signal peptide shown in SEQ ID NO.2, The gene fragment was connected to the expression vector pPICZα to obtain the recombinant plasmid pPICZα-ost-a-LF; S3. Transform the recombinant plasmid pGAP-BMH2 and the recombinant plasmid pPICZα-ost-a-LF into the Pichia pastoris host bacteria to construct the lactoferrin-producing Pichia pastoris.

10. Use of the Pichia pastoris according to any one of claims 1 to 8 in the preparation of products containing lactoferrin or its derivatives in the biological, pharmaceutical, food or chemical fields.

11. The use according to claim 10, characterized in that: The product includes a bacteriostatic agent.

12. A method for producing lactoferrin, characterized in that: The method comprises the step of fermenting the Pichia yeast according to any one of claims 1 to 8.

13. The method according to claim 12, characterized in that: The fermentation conditions are 28-35°C and 180-260rpm.

14. A method for enhancing the secretory expression of lactoferrin in Pichia pastoris, characterized in that: The following steps are involved: The vesicle transport gene BMH2, Sec12 or Sly1 was introduced into the Pichia pastoris host bacteria.

15. The method according to claim 14, characterized in that: P GAP The promoter expresses the vesicle transport genes BMH2, Sec12 or Sly1.

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