Recombinant pichia pastoris capable of efficiently expressing III-type human-like collagen and application of recombinant pichia pastoris

Abstract

The invention relates to recombinant pichia pastoris capable of efficiently expressing III-type human-like collagen and application of the recombinant pichia pastoris, and belongs to the technical field of microorganisms. According to the invention, an III-type human-like collagen expression cassette is constructed in pichia pastoris GS115 and co-expressed with a molecular chaperone, a transcription factor and a translation factor, so that the expression efficiency of the III-type human-like collagen is improved, and the shake-flask fermentation yield reaches 0.93 g / L. And then fermentation conditions, material supplementing conditions, methanol feeding induction conditions and the like are optimized, so that the yield of the III-type human-like collagen in a 5L fermentation tank by the constructed recombinant pichia pastoris reaches 10.3 g / L, the efficient production of target protein is realized, a favorable tool is provided for the application of the III-type human-like collagen in the fields of food, cosmetics, medicines and the like, and the engineering application prospect is broad. Good industrial application prospects are realized.

Description

Technical Field

[0001] The present invention relates to the field of microbial technology, in particular to a recombinant Pichia pastoris capable of efficiently expressing type III human collagen and an application thereof. Background Art

[0002] Collagen is the main structural protein in humans and animals, accounting for 30% of the total animal protein. It is widely distributed in various tissues and organs. Collagen molecules form filamentous and microfibril structures through self-assembly. Its main unit is a triple helix composed of three parallel α chains. An important component of the helical structure is the (Gly-XY) in each chain. n A tripeptide repeat sequence, where Gly represents glycine, X typically represents proline, and Y is typically hydroxyproline. There are at least 29 known different types of collagen. Due to their unique structural properties and physiological functions, collagen has become a popular ingredient in diverse fields, including food, biomaterials, cosmetics, and medicine. Recombinant type III collagen plays a role in chronic wound repair and regeneration, cartilage regeneration, healing of UV-damaged skin, and neointimal healing. Its antioxidant capacity and bioactivity underlie its application in tissue engineering, wound healing, and anti-aging products.

[0003] The market demand for collagen has been growing steadily. Currently, most commercially available collagen products are derived from mammalian tissues and organs, including sheep, pigs and cattle. However, animal-derived collagen may pose risks of immune rejection, pathogen carriage, and potential cultural or religious restrictions. Extraction of collagen from plant and marine species has recently become increasingly popular, but heavy metal contamination, solvent residues and insufficient batch stability still require improvement of these technologies. In order to avoid potential risks and meet growing industrial demand, researchers have developed a variety of expression systems for the production of recombinant collagen III. However, natural collagen is an insoluble fibrous protein with a unique (Gly-XY) n Due to its highly repetitive sequence, it is subject to strict post-translational modification and complex self-assembly during synthesis, and therefore has always faced problems such as difficult expression, low expression level, difficult secretion, and complex purification. As a result, there are currently few types of type III collagen and it is expensive. Therefore, seeking soluble and efficient secretory expression of type III collagen has always been the direction of researchers' efforts. Summary of the Invention

[0004] To address the above technical issues, the present invention provides a Pichia pastoris that efficiently expresses type III human-like collagen and its applications. This invention constructs a type III human-like collagen expression cassette in Pichia pastoris GS115 and co-expresses it with molecular chaperones, transcription factors, and translation factors to enhance the expression efficiency of type III human-like collagen. Subsequently, by optimizing fermentation conditions, feeding conditions, and methanol fed-batch induction conditions, efficient production of the target protein is achieved.

[0005] The first object of the present invention is to provide a recombinant Pichia pastoris expressing type III human collagen, wherein the recombinant Pichia pastoris co-expresses the molecular chaperone HAC1, the transcription factor Prm1, the translation factor Pab1 and type III human collagen, and the nucleotide sequence of the type III human collagen is shown in SEQ ID NO.1.

[0006] Furthermore, the type III human collagen is expressed using AOX1 promoter and DAS2 promoter.

[0007] Furthermore, the expression cassette of human type III collagen also includes MFα signal peptide and AOX1 terminator.

[0008] Furthermore, the nucleotide sequence of the expression cassette of type III human collagen is shown in SEQ ID NO.2.

[0009] Furthermore, the recombinant Pichia pastoris uses Pichia pastoris GS115-hlCOLIII as a chassis strain.

[0010] Furthermore, the expression cassette of the molecular chaperone HAC1 includes a GAP promoter, a molecular chaperone HAC1 gene fragment and an AOX1 terminator.

[0011] Furthermore, the nucleotide sequence of the molecular chaperone HAC1 is shown in SEQ ID NO.3.

[0012] Furthermore, the nucleotide sequence of the expression cassette of the molecular chaperone HAC1 is shown in SEQ ID NO.4.

[0013] Furthermore, the expression cassette of the transcription factor Prm1 includes a DAS2 promoter, a transcription factor Prm1 gene fragment and a CYC1 terminator.

[0014] Furthermore, the nucleotide sequence of the expression cassette of the transcription factor Prm1 is shown in SEQ ID NO.6.

[0015] Furthermore, the expression cassette of the translation factor Pab1 includes an AOX1 promoter, a translation factor Prm1 gene fragment and an AOX1 terminator.

[0016] Furthermore, the nucleotide sequence of the expression cassette of the translation factor Pab1 is shown in SEQ ID NO.8.

[0017] Type III human-like collagen Type III human-like collagen Type III human-like collagen The second object of the present invention is to provide a microbial agent comprising the above-mentioned recombinant Pichia pastoris.

[0018] The third object of the present invention is to provide the use of the above-mentioned recombinant Pichia pastoris or the above-mentioned microbial agent in the production of type III human collagen.

[0019] A fourth object of the present invention is to provide a method for producing type III human collagen, comprising adding the above-mentioned recombinant Pichia pastoris or the above-mentioned microbial agent to a fermentation system, using methanol to induce expression of type III human collagen, and the flow acceleration of the methanol being 4-18 mL / h.

[0020] Furthermore, the fermentation temperature is 23-28°C.

[0021] Furthermore, the dissolved oxygen during the fermentation process is controlled at 0-30%.

[0022] Furthermore, the seed liquid of the recombinant Pichia pastoris is inoculated into the fermentation system at an inoculation rate of 5-10%.

[0023] Furthermore, the fermentation system includes K2SO4 15-20 g / L, MgSO4·7H2O 13-17 g / L, CaSO4 0.8-1 g / L, H3PO4 25-30 mL / L, glycerol 30-60 g / L, and PTM1 3-5%.

[0024] Furthermore, after the fermentation is carried out for 12-24 hours, 30-50% by volume of glycerol is used for feeding.

[0025] Furthermore, glycerol feeding was stopped when the wet weight of the bacteria reached 180-250 g / L, and the time for stopping glycerol feeding was 1-3 hours.

[0026] Beneficial effects of the present invention:

[0027] The present invention constructs a recombinant Pichia pastoris capable of efficiently producing type III human-like collagen. By co-expressing the molecular chaperone HAC1 to activate protein folding, co-expressing the transcription factor Prm1 to enhance transcription, and co-expressing the translation factor Pab1 to form a synergistic effect, the expression of type III human-like collagen is significantly increased. The recombinant Pichia pastoris produces 0.93g / L of type III human-like collagen at the shake flask level. By adjusting and optimizing the fermentation conditions, the recombinant Pichia pastoris produces 10.3g / L of type III human-like collagen at the 5L fermentation tank level, which is significantly increased compared to the starting strain. The recombinant strain is used to produce type III human-like collagen with clear process parameters and easy scale-up production, providing an efficient technical solution for the large-scale preparation of type III collagen. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] In order to make the content of the present invention more clearly understood, the present invention is further described in detail below based on specific embodiments of the present invention in conjunction with the accompanying drawings, wherein:

[0029] Figure 1 This is the effect of co-expressed molecular chaperones on the expression of type III human collagen hLCOLIII in Example 2 of the present invention;

[0030] Figure 2 This is the effect of co-expressed transcription factors on the expression of type III human collagen hLCOLIII in Example 3 of the present invention;

[0031] Figure 3 This is the effect of co-expressed translation factors on the expression of type III human collagen hLCOLIII in Example 4 of the present invention;

[0032] Figure 4 This is a graph showing the yield and wet weight of the recombinant strain in a 5 L tank in Example 5 of the present invention. 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 method for determining the content of type III human collagen hICOLIII involved in the following examples is as follows:

[0035] SDS-PAGE images were captured by a gel imaging system, and the yields were analyzed using ImagJ software.

[0036] The culture medium components involved in the following examples are as follows:

[0037] Seed culture medium: yeast powder 10-20g / L, peptone 20-40g / L, glucose 10-30g / L;

[0038] Shake flask fermentation medium: glycerol 5-15g / L, peptone 15-25g / L, yeast powder 5-15g / L, YNB 10-15g / L, KH2PO4 10-15g / L, K2HPO4 2-5g / L;

[0039] Fermentation tank culture medium: K2SO4 15-20 g / L, MgSO4·7H2O 13-17 g / L, CaSO4 0.8-1 g / L, H3PO4 25-30 mL / L, glycerol 30-60 g / L, PTM1 3-5%.

[0040] Example 1: Recombinant expression of human type III collagen hLCOLIII

[0041] (1) Construction of the pPIC9K-hlCOLIII plasmid: The hLCOLIII fragment (nucleotide sequence shown in SEQ ID NO. 1) was obtained by PCR, and the hLCOLIII expression cassette (nucleotide sequence shown in SEQ ID NO. 2) was constructed. The expression elements included the AOX1 and DAS2 promoters, the MFα signal peptide, the hLCOLIII fragment, and the AOX1 terminator. The plasmid pPIC9K-hlCOLIII was constructed by homologous recombination using the pPIC9K vector as the backbone.

[0042] PCR amplification was performed in a 40 μL reaction system containing 20 μL of Max, 15 μL of ddH2O, 1 μL of template DNA, and 2 μL of each upstream and downstream primers. The reaction conditions were a 3-minute initial denaturation at 95°C followed by 30 cycles of denaturation at 95°C for 30 seconds, annealing at 55°C for 30 seconds, and extension at 72°C for 30 seconds, followed by a final extension at 72°C for 5 minutes. PCR products were identified by electrophoresis and purified by gel extraction.

[0043] (2) Plasmid transformation:

[0044] The circular plasmid was digested with restriction endonucleases, and then the linearized plasmid was recovered using a product purification kit for later use.

[0045] Table 1 Linearization system

[0046]

[0047] Take 1-2 μg of the recovered linearized plasmid and mix it evenly with the Pichia competent culture, let it stand on ice for 5-10 minutes, and insert the linearized plasmid into the Pichia genome through an electroporator. Quickly add 1 mL of pre-cooled 1M sorbitol, culture at 30°C and 220rpm for 2 hours, spread it on an MD plate, and culture it in a 30°C incubator for 2-3 days until a single colony grows to obtain a recombinant strain. The chassis strain is Pichia GS115, and the recombinant strain is named 1# (GS115-hlCOLIII).

[0048] (3) Shake flask fermentation of recombinant bacteria: Recombinant Pichia pastoris 1# was inoculated into 250 mL shake flask fermentation medium at an inoculum size of 3-10% and cultured for 22-28 h. The cells were collected and centrifuged at 4°C, 5000 rpm for 10 min. The supernatant was discarded and the cells were resuspended in fresh 250 mL shake flask fermentation medium. Methanol was added every 22-25 h. Fermentation was carried out at 23-28°C, 220 rpm, and a hLCOLIII yield of 0.70 g / L was obtained.

[0049] Example 2: Co-expression of molecular chaperones improves hCOLIII secretion

[0050] To further improve the secretion efficiency of hLCOLIII in Pichia pastoris, the pGAPZA plasmid was used to co-express the molecular chaperones LHS1, BMH2, AFT1, KEX2, Ssa4, Kar2, Pichia pastoris-derived PpHAC1, Saccharomyces cerevisiae-derived ScHAC1, human HsHAC1, and the hLCOLIII expression cassette constructed in Example 1, respectively. The recombinant plasmids pGAPZA-LHS1, pGAPZA-BMH2, pGAPZA-AFT1, pGAPZA-KEX2, pGAPZA-Ssa4, pGAPZA-Kar2, pGAPZA-PpHAC1, pGAPZA-ScHAC1, and pGAPZA-HsHAC1 were constructed. The recombinant plasmids were transformed into Pichia pastoris #1 (GS115-hlCOLIII) to obtain recombinant strains 1-LHS1, 1-BM H2, 1-AFT1, 1-KEX2, 1-Ssa4, 1-Kar2, 1-PpHAC1, 1-ScHAC1, and 1-HsH AC1. Shake flask fermentation of these recombinant strains was performed according to the method in Example 1.

[0051] The expression elements of the molecular chaperone expression cassette include a GAP promoter, a molecular chaperone gene fragment and an AOX1 terminator.

[0052] Fermentation results such as Figure 1As shown, among the nine chaperone strains, co-expression of the PpHAC1 chaperone showed the best effect, increasing the hLCOLIII yield to 0.81 g / L, a significant increase compared to the starting strain 1#. PpHAC1 activates the expression of a series of genes related to protein folding, quality control, ER-associated degradation, and lipid synthesis. The nucleotide sequence of PpHAC1 is shown in SEQ ID NO. 3, and the sequence of the PpHAC1 expression cassette is shown in SEQ ID NO. 4.

[0053] Example 3: Co-expression of transcription factors to increase the yield of hLCOLIII

[0054] The AOX1 promoter can promote high-level transcription of exogenous genes under methanol culture conditions, but is strongly inhibited under other carbon source conditions. The activity of the AOX1 promoter is positively regulated by transcription factors such as Mit1, Mxr1 and Prm1, which bind to different sites of AOX1. Therefore, the expression cassettes of the transcription factors were connected in series on the basis of the pGAPZA-PpHAC1 obtained in Example 2 to obtain recombinant plasmids pGAPZA-PpHAC1-Mit1, pGAPZA-PpHAC1-Mxr1 and pGAPZA-PpHAC1-Prm1, respectively. The recombinant plasmids were transformed into the recombinant strain Pichia pastoris 1# (GS115-hlCOLIII) to obtain recombinant strains 1-PpHAC1-Mit1, 1-PpHAC1-Mxr1 and 1-PpHAC1-Prm1. The above recombinant strains were subjected to shake flask horizontal fermentation according to the method in Example 1.

[0055] The expression elements of the transcription factor expression cassette include a DAS2 promoter, a transcription factor gene fragment and a CYC1 terminator.

[0056] Fermentation results such as Figure 2 As shown, overexpression of different transcription factors differentially affects hLCOLIII production. The recombinant strain 1-PpHAC1-Prm1, which overexpresses Prm1, produced the highest hLCOLIII production, reaching 0.85 g / L, an increase compared to strain 1-PpHAC1. This may be due to Prm1's ability to activate its own expression and Mit1, thereby inducing robust activation of PAOX1. The nucleotide sequence of Prm1 is shown in SEQ ID NO. 5, and the nucleotide sequence of the Prm1 expression cassette is shown in SEQ ID NO. 6.

[0057] Example 4: Co-expression of translation factors to increase the yield of hLCOLIII

[0058] In yeast cells, translation is a key step in gene expression, responsible for translating the genetic information on mRNA into a specific protein sequence. The translation initiation phase marks the first step in protein synthesis, ensuring that the correct mRNA is recognized by the ribosome and the translation process is initiated. This phase is usually the rate-limiting step of the entire process. The translation initiation factors eIF4E, eIF4A, eIF4G, Pab1, and Rli1 were co-expressed on the basis of pGAPZA-PpHAC1-Prm1 in Example 3 to obtain recombinant plasmids pGAPZA-PpHAC1-Prm1-eIF4E, pGAPZA-PpHAC1-eIF4G-eIF4A, pGAPZA-PpHAC1-Prm1-eIF4G, pGAPZA-PpHAC1-Prm1-Pab1, and pGAPZA-PpHAC1-Prm1-Rli1. The recombinant plasmids were transformed into the recombinant strain Pichia pastoris #1 (GS115-hlCOLIII) to obtain recombinant strains 1-PpHAC1-Prm1-eIF4E, 1-PpHAC1-Prm1-eIF4A, 1-PpHAC1-Prm1-eIF4G, 1-PpHAC1-Prm1-Pab1, and 1-PpHAC1-Prm1-Rli1. These recombinant strains were fermented in shake flasks according to the method in Example 1.

[0059] The expression elements of the translation factor expression cassette include an AOX1 promoter, a translation factor gene fragment and an AOX1 terminator.

[0060] Fermentation results such as Figure 3 As shown, overexpression of Pab1 resulted in a hICOLIII production of 0.93 g / L, a further increase compared to the production of the recombinant strain 1-PpHAC1-Prm1. This may be due to the activation of translation efficiency by Pab1 expression. The nucleotide sequence of Pab1 is shown in SEQ ID NO. 7, and the nucleotide sequence of the Pab1 expression cassette is shown in SEQ ID NO. 8.

[0061] Example 5: High-density fermentation of recombinant strains in 5L tanks

[0062] The strain 1-PpHAC1-Prm1-Pab1 described in Example 4 was fermented at high density. A seed solution of the recombinant Pichia pastoris strain was inoculated into the fermentation medium at an inoculum size of 5-10%. After 12-24 hours of culture, the culture medium was supplemented with 30-50% glycerol by volume until the cell wet weight reached 180-250 g / L. The glycerol feed was then stopped, and the cells were starved for 1-3 hours. At a suitable temperature, the production of the recombinant protein was induced by controlling dissolved oxygen and feeding methanol at a methanol flow rate of 4-18 mL / h.

[0063] Fermentation results such as Figure 4 As shown in the figure, after 140 hours of fermentation, the wet weight of the cells reached a plateau of over 440 g / L, and the production of hCOLIII continued to rise. Protein yield continued to increase with methanol induction. After 180 hours of induction, the production of hCOLIII reached 10.3 g / L.

[0064] 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 recombinant Pichia pastoris expressing human type III collagen, characterized by: The recombinant Pichia pastoris co-expresses molecular chaperone HAC1, transcription factor Prm1, translation factor Pab1 and type III human collagen. The nucleotide sequence of the type III human collagen is shown in SEQ ID NO.

1.

2. The recombinant Pichia pastoris according to claim 1, wherein: The recombinant Pichia pastoris uses Pichia pastoris GS115-hlCOLIII as the chassis strain.

3. The recombinant Pichia pastoris according to claim 1, wherein: The human type III collagen was expressed using the AOX1 promoter and the DAS2 promoter.

4. The recombinant Pichia pastoris according to claim 1, wherein: The nucleotide sequence of the molecular chaperone HAC1 is shown in SEQ ID NO.

3.

5. A microbial agent comprising the recombinant Pichia pastoris according to any one of claims 1 to 4.

6. Use of the recombinant Pichia pastoris according to any one of claims 1 to 4 or the microbial agent according to claim 5 in the production of type III human collagen.

7. A method for producing type III human collagen, characterized in that: The recombinant Pichia pastoris according to any one of claims 1 to 4 or the microbial agent according to claim 5 is added to the fermentation system, and methanol is used to induce the expression of type III human collagen, wherein the flow acceleration of the methanol is 4-18 mL / h.

8. The method according to claim 7, wherein: The fermentation system includes K2SO4 15-20 g / L, MgSO4·7H2O 13-17 g / L, CaSO4 0.8-1 g / L, H3PO4 25-30 mL / L, glycerol 30-60 g / L, and PTM1 3-5%.

9. The method according to claim 7, wherein: After 12-24 hours of fermentation, 30-50% by volume of glycerol was used for feeding.

10. The method according to claim 9, characterized in that: When the wet weight of the bacteria reaches 180-250 g / L, stop feeding with glycerol, and the time for stopping glycerol feeding is 1-3 hours.

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