Method for enhancing yeast expression recombinant mussel fusion protein based on multi-engineering strategy and application thereof

Abstract

The invention discloses a method for enhancing yeast expression recombinant mussel fusion protein based on multi-engineering strategies and application of the method, recombinant saccharomycetes for over-expressing molecular chaperone, signal peptide, aminoacyl tRNA synthetase, endoplasmic reticulum expansion related factors and recombinant mussel fusion protein are constructed, and the recombinant mussel fusion protein is obtained by combining regulation and control on the fermentation process of the recombinant saccharomycetes. The recombinant mussel fusion protein has the advantages of enhancing the secretion efficiency of the recombinant mussel fusion protein, enhancing the correct folding of the recombinant mussel fusion protein, improving the transfer efficiency of related amino acid raw materials for synthesizing the recombinant mussel fusion protein, improving the synthesis efficiency of the recombinant mussel fusion protein, and further remarkably improving the yield and quality of the recombinant mussel fusion protein. The recombinant yeast disclosed by the invention is fermented in a fermentation culture medium containing related amino acid raw materials for synthesizing the recombinant mussel fusion protein, and the yield of the recombinant mussel fusion protein is up to 698mg / L.

Description

Technical Field

[0001] The present invention belongs to the technical field of genetic engineering, and in particular relates to a method for enhancing yeast expression of recombinant mussel fusion protein based on a multi-engineering strategy and an application thereof. Background Art

[0002] With the continuous development of biotechnology, recombinant proteins play an important role in many fields, including medicine, food, and materials. In recent years, mussel fusion proteins have attracted increasing attention due to their unique biological functions and excellent material properties. These fusion proteins, which often involve multiple functional small peptides (such as Mfp-1 and Mfp-3), have shown broad potential in various bioengineering applications. Yeast, as a major eukaryotic expression system, is widely used in the production of recombinant proteins, especially due to its advantages in genome modification and expression regulation, providing an excellent environment for protein expression.

[0003] However, due to the complex structure and specific expression requirements of mussel fusion proteins, traditional yeast expression systems still face numerous challenges in expressing recombinant proteins, especially those with complex structures, such as low yields, improper protein folding, and inefficient secretion. Achieving efficient and stable expression in yeast has become a key factor in driving development in this field. Therefore, achieving efficient and stable expression of mussel fusion proteins in yeast is of great significance. Summary of the Invention

[0004] The technical problem to be solved by the present invention is to address the problems of low yield, incorrect protein folding and low secretion efficiency of recombinant mussel fusion proteins in the existing technology, and provide a method for enhancing yeast expression of recombinant mussel fusion proteins based on a multi-engineering strategy. By integrating multiple strategies such as optimizing the design of expression plasmids, adjusting the genome construction of yeast strains, and improving culture medium components and fermentation conditions, the expression efficiency, biological activity and yield of recombinant mussel fusion proteins are improved.

[0005] In order to achieve the above object, the technical solution provided by the present invention is as follows:

[0006] A recombinant yeast for enhanced expression of a recombinant mussel fusion protein based on a multi-engineering strategy. The recombinant yeast overexpresses a molecular chaperone, a signal peptide, an aminoacyl-tRNA synthetase, an endoplasmic reticulum expansion-related factor and the recombinant mussel fusion protein.

[0007] The genome of the recombinant yeast contains at least one copy of the gene encoding the recombinant mussel fusion protein. Preferably, the genome of the recombinant yeast contains three copies of the gene encoding the recombinant mussel fusion protein.

[0008] Wherein, the molecular chaperone is any one of CPR5, HAC1, PDI1, PDI, ERO1 and BiP, preferably PDI1; the NCBI accession numbers of the molecular chaperones CPR5, HAC1, PDI, ERO1 and BiP are XM_002489874.1, XM_002489994.1, AJ302014.1, XM_002489600.1 and XM_002490982.1, respectively, and the nucleotide sequence of the gene encoding the molecular chaperone PDI1 is shown in SEQ ID No. 1; the signal peptide is any one of α-MF, αCC, Ost1, αopt and NCW2, and the nucleotide sequences of the genes encoding the molecular chaperones are shown in SEQ ID No. No.2 to 6; the signal peptide is preferably Ost1; the aminoacyl tRNA synthetase is any one or more of lysyl tRNA synthetase, tyrosyl tRNA synthetase and prolyl tRNA synthetase, and the nucleotide sequences of the genes encoding them are shown as SEQ ID No.7 to 9, respectively; the aminoacyl tRNA synthetase is preferably lysyl tRNA synthetase, tyrosyl tRNA synthetase and prolyl tRNA synthetase; the gene encoding the endoplasmic reticulum expansion-related factor is INO4, and its nucleotide sequence is shown as SEQ ID No.10; the recombinant mussel fusion protein is any one of Mfp-1, Mfp-3, Mfp-5, Mfp-6, Mfp-151 and Mfp-1561, and their amino acid sequences are shown as SEQ ID No.11 to 16, respectively, and their nucleotide sequences are shown as SEQ ID No.17 to 22, respectively; the recombinant mussel fusion protein is preferably Mfp-1561.

[0009] The present invention also discloses a method for constructing the recombinant yeast, comprising the following steps:

[0010] (1) linking a signal peptide encoding gene, a recombinant mussel fusion protein encoding gene fragment, a molecular chaperone encoding gene, an aminoacyl-tRNA synthetase encoding gene fragment and an endoplasmic reticulum expansion-related factor encoding gene to obtain a multi-gene fragment; preferably, using overlap extension PCR technology to link the signal peptide encoding gene with a single copy or multiple copies of the recombinant mussel fusion protein encoding gene to obtain a signal peptide encoding gene-recombinant mussel fusion protein encoding gene fragment SP-Mfp, using overlap extension PCR technology to link the SP-Mfp gene fragment with the molecular chaperone gene fragment to obtain the SP-Mfp-Chaperone gene fragment, using overlap extension PCR technology to link the SP-Mfp-Chaperone gene fragment with the aminoacyl-tRNA synthetase encoding gene fragment to obtain the SP-Mfp-Chaperone-aaRS gene fragment, using overlap extension PCR technology to link the SP-Mfp-Chaperone-aaRS gene fragment with the endoplasmic reticulum expansion-related factor encoding gene INO4 fragment to obtain the multi-gene fragment SP-Mfp-Chaperone-aaRS-INO4;

[0011] (2) connecting the multi-gene fragment obtained in step (1) to a plasmid to obtain a recombinant expression vector;

[0012] (3) Introducing the recombinant expression vector obtained in step (2) into yeast to obtain the recombinant yeast.

[0013] In step (1), the recombinant mussel fusion protein encoding gene fragment contains at least one copy of the recombinant mussel fusion protein encoding gene, preferably, the recombinant mussel fusion protein encoding gene fragment contains 3 copies of the recombinant mussel fusion protein encoding gene; the aminoacyl tRNA synthetase encoding gene fragment contains any one or more of lysyl tRNA synthetase encoding gene, tyrosyl tRNA synthetase encoding gene and prolyl tRNA synthetase encoding gene, preferably, the aminoacyl tRNA synthetase encoding gene fragment contains lysyl tRNA synthetase encoding gene, tyrosyl tRNA synthetase encoding gene and prolyl tRNA synthetase encoding gene.

[0014] In step (2), the plasmid is preferably any one of pPIC9K, pPIC3.5K, pPICZαA and pHIL-S1, and most preferably pPIC9K. The above plasmids can be purchased from the market.

[0015] In step (3), the yeast is any one of Pichia pastoris, Saccharomyces cerevisiae and baker's yeast, preferably Pichia pastoris, more preferably Pichia pastoris GS115.

[0016] The invention also discloses the application of the recombinant yeast in fermenting and producing recombinant mussel fusion protein.

[0017] The recombinant yeast is inoculated into a fermentation medium for fermentation; the fermentation medium contains any one or more of lysine, tyrosine and proline, preferably, the fermentation medium contains lysine, tyrosine and proline.

[0018] Wherein, the fermentation medium contains an inductive carbon source and a non-inhibitory carbon source; the inductive carbon source is methanol, and the non-inhibitory carbon source is sorbitol.

[0019] The fermentation conditions are as follows: temperature of 25-30°C, rotation speed of 180-220 rpm, initial OD of the recombinant yeast in the fermentation medium 600 The value is 3.5 to 5.5, and the fermentation time is 144 to 168 hours; preferably, the fermentation conditions are: temperature 28 ° C, rotation speed 200 rpm, the initial OD of the recombinant yeast in the fermentation medium is 600 The value is 5.0 and the fermentation time is 168h.

[0020] The present invention strengthens yeast expression of recombinant mussel fusion protein through multiple genetic engineering methods and fermentation process regulation methods. By selecting an appropriate signal peptide, its encoding gene is overlapped and fused with the recombinant mussel fusion protein encoding gene to construct an efficient recombinant expression plasmid to achieve efficient secretion of the recombinant mussel fusion protein; a molecular chaperone co-expression strategy is used to ensure the correct folding of the recombinant mussel fusion protein; in addition, the copy number of the recombinant mussel fusion protein gene is increased (1 to 6 copies), and the expression level of the recombinant mussel fusion protein is improved by optimizing the gene copy number; according to the codon preference of the chassis yeast, the gene sequence of the aminoacyl tRNA synthetase is optimized and transferred into the yeast genome to improve the binding efficiency of the corresponding amino acid and tRNA, improve the efficiency of tRNA transport of amino acids, and thus improve the expression efficiency of the recombinant protein; at the same time, a gene expression vector for increasing the surface area of ​​the endoplasmic reticulum is constructed to optimize the expression of endoplasmic reticulum membrane-related genes to expand the endoplasmic reticulum membrane and improve the secretion efficiency of the recombinant mussel fusion protein.

[0021] Fermentation process regulation: This method uses a non-inhibitory carbon source (sorbitol) and an inducible carbon source (methanol) to co-feed during the fermentation of recombinant yeast to increase the activity of the methanol-inducible promoter. Sorbitol, as a non-inhibitory carbon source, increases the bacterial concentration without affecting methanol-induced expression and can effectively reduce the damage of reactive oxygen species to yeast (Chao L, Jin-Song G, Chang S, et al. Increasing gene dosage and chaperones co-expression facilitate the efficient dextranase expression in Pichia pastoris[J]. LWT, 2023, 181), making the expression of recombinant mussel fusion protein more efficient. At the same time, amino acids such as lysine (Lys), tyrosine (Tyr), and proline (Pro) are added to the fermentation medium to increase the yield of recombinant protein synthesized by yeast.

[0022] In summary, the method for enhancing yeast expression of recombinant mussel fusion protein based on a multi-engineering strategy proposed in the present invention will significantly promote the production efficiency of recombinant mussel fusion protein and provide new technical support and solutions for applications in related fields.

[0023] Beneficial effects:

[0024] 1. The present invention significantly improves the expression efficiency of the recombinant mussel fusion protein and the yield of the recombinant mussel fusion protein by optimizing the recombinant expression plasmid, increasing the copy number of the recombinant mussel fusion protein encoding gene and regulating the fermentation process.

[0025] 2. The present invention adopts multiple genetic engineering strategies (such as molecular chaperone co-expression, signal peptide co-expression and endoplasmic reticulum expansion) to ensure the correct folding and post-translational modification of the recombinant mussel fusion protein, thereby improving its biological activity and function, improving protein quality, and ensuring the stability and effectiveness of the recombinant mussel fusion protein product.

[0026] 3. The present invention improves the secretion efficiency of the recombinant mussel fusion protein in yeast by co-expressing signal peptides and optimizing the protein expression system, making the recombinant mussel fusion protein easy to extract and subsequently process, thereby reducing production costs.

[0027] 4. The recombinant yeast of the present invention has broad application potential and can be used to produce a variety of recombinant proteins. It is suitable for multiple fields such as biomedicine, daily chemical cosmetics, and materials science, and promotes the development of new biomaterials, primer carriers, and high-end consumer products. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] The present invention will be further described below in conjunction with the accompanying drawings and specific embodiments, and the above and / or other advantages of the present invention will become more apparent.

[0029] The present invention will be described in further detail below with reference to the accompanying drawings and specific embodiments:

[0030] Figure 1 This is a graph showing the yield data of recombinant mussel fusion protein produced by fermentation of recombinant Pichia pastoris overexpressing different molecular chaperones in Example 1; wherein, CRP5D represents GS115-mfp1561-CPR5, HACE represents GS115-mfp1561-HAC1, PDI1 represents GS115-mfp1561-PDI1, PDI represents GS115-mfp1561-PDI, ERO1 represents GS115-mfp1561-ERO1, BiP represents GS115-mfp1561-BiP, and mfp-1516 represents GS115-mfp1561.

[0031] Figure 2 This is a graph showing the yield data of the recombinant mussel fusion protein produced by fermenting recombinant Pichia pastoris containing different copy numbers of the recombinant mussel fusion protein encoding gene in Example 2.

[0032] Figure 3 This is a graph showing the yield data of recombinant mussel fusion protein produced by fermentation of recombinant Pichia pastoris overexpressing different aminoacyl-tRNA synthetases in Example 3; wherein, mfp-1516 represents GS115-mfp1561, Tyr-RS represents GS115-mfp1561-Tyr-RS, Lys-RS represents GS115-mfp1561-Lys-RS, Pro-RS represents GS115-mfp1561-Pro-RS, and TLP-RS represents GS115-mfp1561-TLP-RS.

[0033] Figure 4 This is a graph showing the yield data of recombinant mussel fusion protein produced by fermentation of recombinant Pichia pastoris overexpressing different signal peptides in Example 4; wherein, mfp-1516 represents GS115-mfp1561, α-MF represents GS115-α-MF-mfp1561, αOPT represents GS115-αopt-mfp1561, αCC represents GS115-αCC-mfp1561, NCW2 represents GS115-NCW2-mfp1561, and Ost1 represents GS115-Ost1-mfp1561.

[0034] Figure 5This is a graph showing the yield data of the recombinant mussel fusion protein produced by fermentation of recombinant Pichia pastoris overexpressing endoplasmic reticulum expansion-related factors in Example 5; wherein, mfp-1516 represents GS115-mfp1561, and endoplasmic reticulum modification represents GS115-mfp1561-INO4.

[0035] Figure 6 This is a graph showing the yield data of the recombinant mussel fusion protein produced by the recombinant Pichia pastoris GS115-mfp1561 in fermentation medium supplemented with different exogenous amino acids in Example 6; wherein, mfp-1516 represents the fermentation medium without the addition of exogenous amino acids, Tyr represents the fermentation medium supplemented with tyrosine, Lys represents the fermentation medium supplemented with lysine, Pro represents the fermentation medium supplemented with proline, and TLP represents the fermentation medium supplemented with tyrosine, lysine, and proline.

[0036] Figure 7 This is a graph showing the yield data of the recombinant mussel fusion protein produced by simultaneously overexpressing a molecular chaperone, a signal peptide, an endoplasmic reticulum expansion-related factor, and an aminoacyl-tRNA synthetase in Example 7 and containing three copies of the recombinant mussel fusion protein encoding gene on the genome in a fermentation medium supplemented with exogenous amino acids; in the figure, mfp-1516 represents the fermentation of GS115-mfp1561 in a fermentation medium without the addition of exogenous amino acids, PDI1 represents the fermentation of GS115-mfp1561-PDI1 in a fermentation medium without the addition of exogenous amino acids, OST1 represents the fermentation of GS115-Ost1-mfp1561 in a fermentation medium without the addition of exogenous amino acids, and Pro represents the fermentation of GS115-mfp1561 in a fermentation medium supplemented with proline. The results show that the GS115-mfp1561 was fermented in a fermentation medium with tyrosine, lysine and proline, TLP represents the fermentation of GS115-mfp1561 in a fermentation medium with the simultaneous addition of tyrosine, lysine and proline, TLP-RS represents the fermentation of GS115-mfp1561-TLP-RS in a fermentation medium without the addition of exogenous amino acids, INO4 represents the fermentation of GS115-mfp1561-INO4 in a fermentation medium without the addition of exogenous amino acids, copy number 3 represents the fermentation of GS115-(3x)mfp1561 in a fermentation medium without the addition of exogenous amino acids, and the combination strategy represents the fermentation of GS115-Ost1-(3x)mfp1561-PDI1-TLR-RS-INO4 in a fermentation medium with the simultaneous addition of tyrosine, lysine and proline. DETAILED DESCRIPTION

[0037] The present invention is further described below with reference to the following examples. It should be understood that the following embodiments are only used to illustrate the present invention, rather than to limit the present invention.

[0038] Specific techniques or conditions not specified in the examples were carried out according to those described in the literature in the field or according to the product instructions. Reagents or instruments used without manufacturer specified were all conventional products available through regular channels.

[0039] The construction methods of the recombinant plasmid pPIC9K-mfp1561 and the recombinant Pichia pastoris GS115-mfp1561 used in the following examples are as follows:

[0040] The plasmid pPIC9K (purchased from Takara, catalog number: 1040A) was digested with restriction endonuclease EcoRI and recovered from the gel. The linearized plasmid pPIC9K was ligated with the Mfp-1561 encoding gene (synthesized by General Biotechnology (Anhui) Co., Ltd., whose nucleotide sequence is shown in SEQ ID No. 22) using a one-step cloning kit. The ligation product was transformed into Escherichia coli DH5α for amplification. After screening and sequencing, the recombinant plasmid pPIC9K-mfp1561 was successfully obtained.

[0041] Plasmid pPIC9K-mfp1561 was digested with restriction endonuclease SacI (purchased from Takara, Cat. No. 1078A) and gel-recovered. 500 ng of linearized plasmid (no more than 10 μL, otherwise the plasmid must be concentrated) was added to 90 μL of thawed Pichia pastoris GS115 competent cells. Mix thoroughly, incubate on ice for 5 minutes, and transfer to a pre-chilled 2 mm electroporation cuvette. Electroporation parameters were set to 1.5 kV and 4 ms. After electroporation, the competent cells were quickly added with pre-chilled 1 M sorbitol solution (for auxotrophic selection) and incubated at 30°C for 2–4 hours. An appropriate amount of the incubated culture was spread onto MD selection plates (histidine-deficient plates) and cultured in a 30°C incubator for 2–3 days until white colonies appeared. This resulted in recombinant Pichia pastoris GS115-mfp1561. The preparation method of Pichia pastoris GS115 competent host bacteria is as follows: inoculate a single colony of Pichia pastoris GS115 into YPD liquid medium and culture it overnight at 30℃. Transfer the overnight culture liquid to 50mL YPD liquid medium until the initial OD value of the bacteria in the medium is 600 Between 0.2 and 0.5, culture at 30℃ for 4 to 6 hours. 600The pH value is approximately 1.2 to 1.8. Transfer the bacterial solution to a 50 mL sterile centrifuge tube, centrifuge at 4000 rpm for 10 minutes, and discard the supernatant. Add 30 mL of pre-cooled sterile water to resuspend the bacteria, centrifuge at 4000 rpm for 5 minutes, discard the supernatant, and repeat the resuspending and centrifugation of the bacteria. Wash the bacteria with 30 mL, 15 mL, 7.5 mL, and 3.75 mL of pre-cooled sterile 1M sorbitol aqueous solution in sequence, and finally resuspend the bacteria with 1 mL of pre-cooled 1M sorbitol solution and distribute them into sterile 1.5 mL EP tubes, 90 μL per tube, and freeze in a -80°C ultra-low temperature freezer.

[0042] The formula of the YPD liquid culture medium used in the following examples is: 10 g / L yeast powder, 20 g / L tryptone, and 20 g / L glucose.

[0043] The formula of BMGY medium used in the following examples is: yeast powder 10 g / L, bacteriological peptone 20 g / L, KH2PO4 11.8 g / L, K2HPO4 2.29 g / L, yeast nitrogen base (YNB) 13.4 g / L, biotin 0.4 mg / L, and glycerol 10 g / L.

[0044] The formula of BMMY medium (fermentation medium) used in the following examples is: yeast powder 10 g / L, bacteriological peptone 20 g / L, KH2PO4 11.8 g / L, K2HPO4 2.29 g / L, YNB 13.4 g / L, biotin 0.4 mg / L, methanol 10 mL / L.

[0045] The formula of the MD screening plate (histidine-deficient plate) used in the following examples is: glucose 20 g / L, agar powder 20 g / L, YNB 13.4 g / L, and biotin 0.4 mg / L.

[0046] Example 1 Optimization of molecular chaperones

[0047] The genes encoding the molecular chaperones CPR5, HAC1, PDI1, PDI, ERO1, and BiP were amplified by PCR using the Pichia pastoris GS115 genome as a template using primers 1-F / 1-R, 2-F / 2-R, 3-F / 3-R, 4-F / 4-R, 5-F / 5-R, and 6-F / 6-R, respectively. The methanol promoter gene AOX1′ was amplified by PCR using primers AOX1′-F / AOX1′-R from the plasmid pPIC9K-mfp1561. After recovering the PCR products of the above gene fragments, the AOX1 gene fragment was linked to the CPR5, HAC1, PDI1, PDI, ERO1 and BiP encoding genes respectively by overlapping PCR technology using primers AOX1′-F / 1-R, AOX1′-F / 2-R, AOX1′-F / 3-R, AOX1′-F / 4-R, AOX1′-F / 5-R and AOX1′-F / 6-R to obtain linearized gene fragments AOX1′-CPR5, AOX1′-HAC1, AOX1′-PDI1, AOX1′-PDI, AOX1′-ERO1 and AOX1′-BiP. The plasmid pPIC9K-mfp1561 was then digested with NotⅠ (purchased from Takara, product number: 1166A) restriction enzyme and recovered from the gel. The linearized plasmid pPIC9K-mfp1561 was ligated with AOX1′-CPR5, AOX1′-HAC1, AOX1′-PDI1, AOX1′-PDI, AOX1′-ERO1, and AOX1′-BiP using a one-step cloning kit. Then, the ligation products were transformed into Escherichia coli DH5α for amplification. After screening and identification, the recombinant plasmids pPIC9K-mfp1561-CPR5, pPIC9K-mfp1561-HAC1, pPIC9K-mfp1561-PDI1, pPIC9K-mfp1561-PDI, pPIC9K-mfp1561-ERO1 and pPIC9K-mfp1561-BiP were successfully obtained.

[0048] The above recombinant plasmids were extracted from Escherichia coli DH5α and linearized with restriction endonuclease SacⅠ. The linearized recombinant plasmids pPIC9K-mfp1561-CPR5, pPIC9K-mfp1561-HAC1, pPIC9K-mfp1561-PDI1, pPIC9K-mfp1561-PDI, pPIC9K-mfp1561-ERO1 and pPIC9K-mfp1561-BiP were respectively transformed into the competent host strain Pichia pastoris GS115.

[0049] The preparation and transformation of Pichia pastoris GS115 competent host bacteria were as follows: a single colony of Pichia pastoris GS115 was inoculated into YPD liquid medium and cultured overnight at 30°C. The overnight culture solution was transferred to 50 mL YPD liquid medium until the initial OD value of the bacteria in the medium was 600 Between 0.2 and 0.5, culture at 30℃ for 4 to 6 hours. 600 The pH value is approximately 1.2 to 1.8. Transfer the bacterial suspension to a 50 mL sterile centrifuge tube and centrifuge at 4000 rpm for 10 minutes. Discard the supernatant. Resuspend the cells in 30 mL of pre-chilled sterile water and centrifuge at 4000 rpm for 5 minutes. Discard the supernatant and repeat the resuspension and centrifugation. Wash the cells with 30 mL, 15 mL, 7.5 mL, and 3.75 mL of pre-chilled sterile 1M sorbitol solution, respectively. Finally, resuspend the cells in 1 mL of pre-chilled 1M sorbitol solution and aliquot into sterile 1.5 mL EP tubes, 90 μL per tube, and store in a -80°C freezer. Add 500 ng of the linearized plasmid (no more than 10 μL, otherwise the plasmid needs to be concentrated) to 90 μL of thawed Pichia pastoris GS115 competent cells, mix well, and place on ice for 5 minutes. Transfer the cells to a pre-chilled 2 mm electroporation cuvette. Set the electroporation parameters to 1.5 kV and 4 ms. After electroporation, the competent cells were quickly added with pre-chilled 1 M sorbitol aqueous solution (for nutrient deficiency screening) and incubated at 30°C for 2-4 hours. An appropriate amount of the incubated bacterial solution was spread onto MD screening plates (histidine-deficient plates) and cultured in a 30°C incubator for 2-3 days until white colonies appeared. This yielded recombinant Pichia pastoris GS115-mfp1561-CPR5, GS115-mfp1561-HAC1, GS115-mfp1561-PDI1, GS115-mfp1561-PDI, GS115-mfp1561-ERO1, and GS115-mfp1561-BiP, respectively.

[0050] The above-mentioned recombinant Pichia pastoris was used for shake flask fermentation. The specific fermentation method was as follows: GS115-mfp1561-CPR5, GS115-mfp1561-HAC1, GS115-mfp1561-PDI1, GS115-mfp1561-PDI, GS115-mfp1561-ERO1 and GS115-mfp1561-BiP were inoculated into YPD liquid medium containing geneticin (0.25 mg / mL) and cultured overnight in the dark. Appropriate amounts of bacterial liquid were added to BMGY medium so that the initial OD value of the bacterial cells in the BMGY medium was 0. 600 Cultivate at 0.5-0.8, 30°C, 200 rpm until the bacterial OD 600At 4.0-6.0, the mixture was centrifuged at 8000 rpm for 5 min at 4°C, and the cells were taken out and washed twice with physiological saline to obtain the recombinant Pichia pastoris seed solution. The recombinant Pichia pastoris seed solution was added to BMMY medium (fermentation medium) so that the initial OD value of the cells in the fermentation medium was 600 The fermentation temperature was about 3.5-5.5, 28°C, 200 rpm, and methanol was added every 12 hours (the addition amount was 1% of the fermentation medium volume) to induce fermentation. The total fermentation time was 168 hours. After the fermentation was completed, the yield of recombinant mussel fusion protein in the fermentation broth obtained from each recombinant Pichia pastoris fermentation was measured. The results are as follows: Figure 1 As shown in the results, the co-expression of molecular chaperones significantly promoted the production of recombinant mussel fusion protein in the fermentation system. Among them, the molecular chaperone PDI1 had the best promoting effect. Therefore, the molecular chaperone PDI1 encoding gene was co-expressed with the recombinant mussel fusion protein encoding gene to improve the expression effect of the recombinant mussel fusion protein.

[0051] Table 1 Primer sequences used in this example

[0052]

[0053] Example 2 Optimization of the copy number of recombinant mussel fusion protein

[0054] Take 500 ng of the linearized single-copy recombinant mussel fusion protein expression plasmid pPIC9K-mfp1561 (the volume should not exceed 10 μL; otherwise, the plasmid needs to be concentrated) and add it to 90 μL of thawed Pichia pastoris GS115 competent cells prepared in Example 1. Mix thoroughly, place on ice for 5 minutes, and transfer to a pre-chilled electroporation cuvette (2 mm). Set the electroporation parameters to 1.5 kV and 4 ms. After electroporation, quickly add pre-chilled 1 M sorbitol aqueous solution (for auxotrophic screening) to the competent cells and incubate at 30°C for 2-4 hours. An appropriate amount of the incubated bacterial liquid was spread on an MD screening plate (histidine-deficient plate) and cultured in a 30°C incubator for 2 to 3 days until white colonies appeared. The white colonies were picked and inoculated onto geneticin-resistant YPD plates containing 0.25 mg / mL, 0.5 mg / mL, 0.75 mg / mL, 1.0 mg / mL, 1.25 mg / mL, 1.5 mg / mL, 1.75 mg / mL and 2.0 mg / mL for further screening. The plates were cultured at 30°C and observed. The colonies that grew faster on the plates were selected for qPCR verification of the copy number of the recombinant mussel fusion protein Mfp-1561 encoding gene in their genomes. The primers for the internal reference gene GAPDH (NCBI accession number XM_002491300.1) and the primers for the target protein (Mfp-1561) encoding gene used in the qPCR verification are shown in Table 2.

[0055] Finally, the recombinant mussel fusion protein Mfp-1561 encoding gene 1 copy, 2 copies, 3 copies, 4 copies, 5 copies, 6 copies of the recombinant Pichia pastoris GS115 strain GS115-(1x)mfp1561, GS115-(2x)mfp1561, GS115-(3x)mfp1561, GS115-(4x)mfp1561, GS115-(5x)mfp1561 and GS115-(6x)mfp1561 were obtained. Subsequently, the recombinant Pichia pastoris with different copy numbers were fermented to produce recombinant mussel fusion protein. The specific fermentation method was the same as in Example 1. After the fermentation was completed, the yield of recombinant mussel fusion protein in the fermentation broth obtained from the fermentation of each recombinant Pichia pastoris was measured. The results are as follows. Figure 2 As shown, when the recombinant mussel fusion protein mfp1561 encoding gene is 3 copies, the production of the recombinant mussel fusion protein is the highest.

[0056] Table 2 Primer sequences used in this example

[0057]

[0058] Example 3 Optimization of aminoacyl-tRNA synthetase

[0059] The lysyl-tRNA synthetase gene Lys-RS, tyrosyl-tRNA synthetase gene Tyr-RS, and prolyl-tRNA synthetase gene Pro-RS were amplified by PCR using the Pichia pastoris GS115 genome as a template using primers Lys-RS-F / Lys-RS-R, Tyr-RS-F / Tyr-RS-R, and Pro-RS-F / Pro-RS-R, respectively. The methanol promoter gene AOX1′ was amplified by PCR using primers AOX1′-F / AOX1′-R from plasmid pPIC9K-mfp1561. After recovering the PCR products of the above gene fragments, AOX1 was linked to Lys-RS, Tyr-RS, and Pro-RS, respectively, through overlapping PCR using primers AOX1′-F / Lys-RS-R, AOX1′-F / Tyr-RS-R, and AOX1′-F / Pro-RS-R, to obtain linearized gene fragments AOX1′-Lys-RS, AOX1′-Tyr-RS, and AOX1′-Pro-RS. Overlapping PCR was performed using the linearized gene fragments AOX1′-Tyr-RS and Lys-RS as templates with primers AOX1′-F / Lys-RS-R to obtain the linearized gene fragment AOX1′-TL-RS. Furthermore, overlapping PCR was performed using AOX1′-TL-RS and Pro-RS as templates with primers AOX1′-F / Pro-RS-R to obtain the linearized gene fragment AOX1′-TLR-RS. The plasmid pPIC9K-mfp1561 was then digested with NotⅠ restriction endonuclease and recovered from the gel. The linearized plasmid pPIC9K-mfp1561 was ligated with AOX1′-Lys-RS, AOX1′-Tyr-RS, AOX1′-Pro-RS and AOX1′-TLR-RS, respectively, using a one-step cloning kit. The ligation products were transformed into Escherichia coli DH5α for amplification. After screening and identification, the recombinant plasmids pPIC9K-mfp1561-Lys-RS, pPIC9K-mfp1561-Tyr-RS, pPIC9K-mfp1561-Pro-RS and pPIC9K-AOX1′-TLR-RS were successfully obtained.

[0060] The primer sequences used in this example are shown in Table 3.

[0061] The above-mentioned recombinant plasmids were extracted from Escherichia coli DH5α and linearized with the restriction endonuclease Sac I. The linearized recombinant plasmids pPIC9K-mfp1561-Lys-RS, pPIC9K-mfp1561-Tyr-RS, pPIC9K-mfp1561-Pro-RS, and pPIC9K-mfp1561-TLR-RS were respectively transformed into the competent host bacteria Pichia pastoris GS115. The preparation and transformation of the competent host bacteria Pichia pastoris GS115 and the screening and verification of Pichia pastoris GS115 transformants were the same as in Example 1. If the verification was successful, the recombinant Pichia pastoris GS115-mfp1561-Lys-RS, GS115-mfp1561-Tyr-RS, GS115-mfp1561-Pro-RS, and GS115-mfp1561-TLR-RS were obtained, respectively.

[0062] The above recombinant Pichia pastoris was used for shake flask fermentation. The specific fermentation method was the same as that in Example 1. After the fermentation was completed, the yield of recombinant mussel fusion protein in the fermentation broth obtained by fermentation of each recombinant Pichia pastoris was measured. The results are as follows: Figure 3 As shown, the yield of the recombinant mytilus fusion protein was highest when recombinant Pichia pastoris simultaneously overexpressed lysyl tRNA synthetase, tyrosyl tRNA synthetase, and prolyl tRNA synthetase.

[0063] Table 3 Sequences of primers used in this example

[0064]

[0065] Example 4 Optimization of signal peptide

[0066] The genes encoding the signal peptides α-MF, Ost1, αCC, αopt, and NCW2 were amplified by PCR using the genome of Pichia pastoris GS115 as a template using primers α-MF-F / α-MF-R, Ost1-F / Ost1-R, αCC-F / αCC-R, αopt-F / αopt-R, and NCW2-F / NCW2-R, respectively. The gene encoding the mussel fusion protein Mfp-1561 was amplified by PCR using primers mfp-1561-F / mfp-1561-R, using the plasmid pPIC9K-mfp1561 as a template. After the above PCR products were recovered, the Mfp-1561 encoding gene was linked to the signal peptide α-MF, Ost1, αCC, αopt and NCW2 encoding genes using primers α-MF-F / mfp1561-R, Ost1-F / mfp1561-R, αCC-F / mfp1561-R, αopt-F / mfp1561-R and NCW2-F / mfp1561-R, respectively, to obtain linearized gene fragments α-MF-mfp1561, Ost1-mfp1561, αCC-mfp1561, αopt-mfp1561 and NCW2-mfp1561. The plasmid pPIC9K was then double-digested with BamHI and EcoRI and gel-recovered. The linearized plasmid pPIC9K was ligated with α-MF-mfp1561, Ost1-mfp1561, αCC-mfp1561, αopt-mfp1561, and NCW2-mfp1561 using a one-step cloning kit. The ligation products were transformed into Escherichia coli DH5α for amplification. After screening and identification, the recombinant plasmids pPIC9K-α-MF-mfp1561, pPIC9K-Ost1-mfp1561, pPIC9K-αCC-mfp1561, pPIC9K-αopt-mfp1561, and pPIC9K-NCW2-mfp1561 were obtained. The primer sequences used in this example are shown in Table 4.

[0067] The recombinant plasmids were extracted from Escherichia coli DH5α and linearized with the restriction endonuclease Sac I. The linearized recombinant plasmids pPIC9K-α-MF-mfp1561, pPIC9K-Ost1-mfp1561, pPIC9K-αCC-mfp1561, pPIC9K-αopt-mfp1561, and pPIC9K-NCW2-mfp1561 were respectively transformed into the competent host strain Pichia pastoris GS115. The preparation and transformation of the competent host strain Pichia pastoris GS115, and the screening and verification of Pichia pastoris GS115 transformants were the same as in Example 1. Upon successful verification, recombinant Pichia pastoris GS115-α-MF-mfp1561, GS115-Ost1-mfp1561, GS115-αCC-mfp1561, GS115-αopt-mfp1561 and GS115-NCW2-mfp1561 were obtained respectively.

[0068] The above recombinant Pichia pastoris was used for shake flask fermentation. The specific fermentation method was the same as that in Example 1. After the fermentation was completed, the yield of recombinant mussel fusion protein in the fermentation broth obtained by fermentation of each recombinant Pichia pastoris was measured. The results are as follows: Figure 4 As shown in the figure, the results showed that different signal peptides significantly improved the secretion production effect of Mfp-1561, among which the signal peptide Ost1 had the best promoting effect. Therefore, the signal peptide Ost1 encoding gene was selected to co-express with the recombinant mussel fusion protein encoding gene to improve the expression effect of the recombinant mussel fusion protein.

[0069] Table 4 Primer sequences used in this example

[0070]

[0071]

[0072] Example 5: Verification of the effect of endoplasmic reticulum expansion-related factors on promoting the recombinant mussel fusion protein

[0073] The genome of Pichia pastoris GS115 was used as a template and primers INO4-F / INO4-R were used to amplify the endoplasmic reticulum expansion-related transcription factor INO4 gene. The methanol promoter gene AOX1′ was amplified using plasmid pPIC9K-mfp1561 as a template and primers AOX1′-F / AOX1′-R. After the PCR products of the above gene fragments were recovered, AOX1′ was cleaved with INO4 by overlapping PCR using primers AOX1′-F / INO4-R. The O4 encoding gene was connected to obtain a linearized gene fragment AOX1′-INO4; the plasmid pPIC9K-mfp1561 was then digested with Not I restriction endonuclease and gel-recovered. The linearized plasmid pPIC9K-mfp1561 and AOX1′-INO4 were ligated using a one-step cloning kit. The ligation product was transformed into Escherichia coli DH5α for amplification. After screening and identification, the recombinant plasmid pPIC9K-mfp1561-INO4 was successfully obtained. The primer sequences used in this example are shown in Table 5.

[0074] The recombinant plasmid was extracted from Escherichia coli DH5α and linearized with the restriction endonuclease Sac I. The linearized recombinant plasmid pPIC9K-mfp1561-INO4 was then transformed into the competent host strain Pichia pastoris GS115. The preparation and transformation of the competent host strain Pichia pastoris GS115, as well as the screening and verification of Pichia pastoris GS115 transformants, were the same as in Example 1. Successful verification yielded recombinant Pichia pastoris GS115-mfp1561-INO4.

[0075] The above recombinant Pichia pastoris GS115-mfp1561-INO4 was used for shake flask fermentation. The specific fermentation method was the same as that in Example 1. After the fermentation was completed, the yield of the recombinant mussel fusion protein in the fermentation broth obtained by the recombinant Pichia pastoris fermentation was measured. The results are as follows: Figure 5 As shown, the production of Mfp-1561 in the strain after endoplasmic reticulum modification was significantly increased.

[0076] Table 5 Primer sequences used in this example

[0077]

[0078] Example 6 Verification of the effect of fermentation process regulation

[0079] Recombinant Pichia pastoris GS115-mfp1561 was used for shake flask fermentation. The recombinant Pichia pastoris GS115-mfp1561 seed solution was obtained according to the method of Example 1. The GS115-mfp1561 seed solution was inoculated into a fermentation medium containing 10 g / L lysine, a fermentation medium containing 10 g / L tyrosine, a fermentation medium containing 10 g / L proline, and a fermentation medium containing 10 g / L lysine, 10 g / L tyrosine, and 10 g / L proline. The initial OD value of the bacteria in the fermentation medium was controlled to be around 3.5-5.5. The fermentation was carried out at 28°C and 200 rpm. Methanol was added every 12 hours (the addition amount was 1% of the volume of the fermentation medium) and the fermentation was carried out for 168 hours. After the fermentation was completed, the yield of the recombinant mussel fusion protein in the fermentation broth obtained by each recombinant Pichia pastoris fermentation was measured. The results are as follows: Figure 6 As shown in the results, the addition of single amino acids and multiple amino acids to the fermentation medium had a good effect on improving the expression of Mfp-1561. Among them, the simultaneous addition of lysine, tyrosine and proline to the fermentation medium had a more significant effect on improving the production of recombinant mussel fusion protein.

[0080] Example 7: Enhanced yeast expression of recombinant mussel fusion protein based on a multi-engineering strategy

[0081] Based on Examples 1 to 6, the gene encoding the molecular chaperone PDI1, the gene encoding the signal peptide Ost1, the three aminoacyl-tRNA synthetase genes Lys-RS, Tyr-RS, and Pro-RS, and the endoplasmic reticulum expansion-related factor encoding gene INO4 were selected and co-expressed with the expression plasmid pPIC9K-(3x)mfp1561 containing three copies of the recombinant mussel fusion protein gene. The recombinant yeast obtained by the construction was fermented. During the fermentation, lysine, tyrosine, and proline were mixed and added to the fermentation medium. At the same time, 50 g / L sorbitol was added as a non-inhibitory carbon source to increase the bacterial concentration without affecting methanol-induced expression. It can also effectively reduce the damage of reactive oxygen species to the yeast, thereby achieving enhanced yeast expression of the recombinant mussel fusion protein based on a multi-engineering strategy. The construction of the recombinant yeast and the fermentation method thereof are as follows:

[0082] The recombinant plasmid pPIC9K-mfp1561-PDI1 constructed in Example 1 was used as a template and primers AOX1″-F / 3-R were used to amplify the linearized AOX1″-PDI1 gene fragment by PCR;

[0083] The recombinant plasmid pPIC9K-mfp1561-TLR-RS constructed in Example 3 was used as a template and primers AOX1″-F / Pro-RS-R were used to amplify the linearized AOX1″-TLR-RS gene fragment by PCR;

[0084] The recombinant plasmid pPIC9K-mfp1561-INO4 constructed in Example 5 was used as a template and primers AOX1″-F / INO4-R were used to amplify the linearized AOX1″-INO4 gene fragment by PCR;

[0085] Using the genome of the recombinant Pichia pastoris GS115-(3x)mfp1561 containing three copies of the recombinant mussel protein Mfp-1561 encoding gene constructed in Example 2 as a template, primers (3x)1561-F / (3x)1561-R were used to amplify three copies of the Mfp-1561 encoding gene fragment (3x)mfp1561.

[0086] The above gene fragments were then connected by overlapping PCR, and the specific method is as follows:

[0087] The Ost1 coding gene amplified from the Pichia pastoris genome in Example 4 and the (3x)mfp1561 described above were used as templates, and the two were connected by overlapping PCR amplification using primers Ost1-F / mfp1561-R. The largest band was recovered to obtain Ost1-(3x)mfp1561.

[0088] Using Ost1-(3x)mfp1561 and AOX1″-PDI1 genes as templates, the two were connected by overlapping PCR amplification using primers Ost1-F / PDI1-R, and the largest band was recovered to obtain Ost1-(3x)mfp1561-PDI1;

[0089] Using Ost1-(3x)mfp1561-PDI1 and AOX1″-TLR-RS genes as templates, the two were connected by overlapping PCR amplification using primers Ost1-F / Pro-RS-R, and the largest band of the fragment was recovered to obtain Ost1-(3x)mfp1561-PDI1-TLR-RS;

[0090] Using Ost1-(3x)mfp1561-PDI1-TLR-RS and AOX1″-INO4 genes as templates, the two were connected by overlapping PCR amplification using primers Ost1-F / INO4-R, and the largest band was recovered to obtain Ost1-(3x)mfp1561-PDI1-TLR-RS-INO4;

[0091] The plasmid pPIC9K was then double-digested with BamHⅠ and EcoRⅠ and recovered on gel. The linearized plasmid pPIC9K was ligated with Ost1-(3x)mfp1561-PDI1-TLR-RS-INO4 using a one-step cloning kit. The ligation product was transformed into Escherichia coli DH5α for amplification. After screening and identification, the recombinant plasmid pPIC9K-Ost1-(3x)mfp1561-PDI1-TLR-RS-INO4 was successfully obtained.

[0092] The recombinant plasmid pPIC9K-Ost1-(3x)mfp1561-PDI1-TLR-RS-INO4 was extracted from Escherichia coli DH5α and linearized with the restriction endonuclease Sac I. The plasmid was then transformed into the competent host strain Pichia pastoris GS115. The preparation and transformation of the competent host strain Pichia pastoris GS115 and the screening and verification of Pichia pastoris GS115 transformants were the same as in Example 1. If the verification was successful, the recombinant Pichia pastoris GS115-Ost1-(3x)mfp1561-PDI1-TLR-RS-INO4 was obtained.

[0093] The primers used in this example are shown in Table 6.

[0094] The above-mentioned recombinant Pichia pastoris GS115-Ost1-(3x)mfp1561-PDI1-TLR-RS-INO4 was used for shake flask fermentation. The recombinant Pichia pastoris seed solution was obtained according to the method of Example 1, and the recombinant Pichia pastoris seed solution was added to a BMMY medium (fermentation medium) mixed with 10 g / L Tyr, 10 g / L Lys, 10 g / L Pro and 50 g / L sorbitol. The culture was carried out at 28°C and 200 rpm, and methanol was added every 12 hours (the addition amount was 1% of the volume of the fermentation medium). The fermentation was continued for 168 hours. After the fermentation was completed, the yield of the recombinant mussel fusion protein in the fermentation broth obtained by the recombinant Pichia pastoris fermentation was measured. The results are as follows: Figure 7 As shown in the figure, the production of Mfp-1561 in the recombinant Pichia pastoris transformed by multiple engineering strategies was significantly improved, reaching 698 mg / L.

[0095] Table 6 Primer sequences used in this example

[0096]

[0097] The present invention provides a concept and method for enhancing yeast expression of recombinant mussel fusion proteins based on a multi-engineering strategy. Numerous methods and approaches exist for implementing this technical solution. The foregoing description is merely a preferred embodiment of the present invention. It should be noted that those skilled in the art may make various improvements and modifications without departing from the principles of the present invention, and such improvements and modifications are also within the scope of protection of the present invention. Any components not specified in this example may be implemented using existing technologies.

Claims

1. A recombinant yeast for enhanced expression of recombinant mussel fusion protein based on a multi-engineering strategy, characterized in that: The recombinant yeast overexpresses molecular chaperones, signal peptides, aminoacyl tRNA synthetases, endoplasmic reticulum expansion-related factors and recombinant mussel fusion proteins.

2. The recombinant yeast according to claim 1, characterized in that The genome of the recombinant yeast contains at least one copy of the gene encoding the recombinant mussel fusion protein.

3. The recombinant yeast according to claim 1, characterized in that The molecular chaperone is any one of CPR5, HAC1, PDI1, PDI, ERO1 and BiP. The NCBI accession numbers of the genes encoding the molecular chaperones CPR5, HAC1, PDI, ERO1 and BiP are XM_002489874.1, XM_002489994.1, AJ302014.1, XM_002489600.1 and XM_002490982.1, respectively. The nucleotide sequence of the gene encoding the molecular chaperone PDI1 is shown in SEQ ID No. 1; the signal peptide is any one of α-MF, αCC, Ost1, αopt and NCW2, and the nucleotide sequences of the genes encoding the same are shown in SEQ ID Nos. 2 to 6, respectively; the aminoacyl tRNA synthetase is any one or more of lysyl tRNA synthetase, tyrosyl tRNA synthetase and prolyl tRNA synthetase, and the nucleotide sequences of the genes encoding the same are shown in SEQ ID Nos. No.7 to 9; the coding gene of the endoplasmic reticulum expansion-related factor is INO4, and its nucleotide sequence is shown in SEQ ID No.10; the recombinant mussel fusion protein is any one of Mfp-1, Mfp-3, Mfp-5, Mfp-6, Mfp-151 and Mfp-1561, and its amino acid sequences are shown in SEQ ID No.11 to 16, and its nucleotide sequences are shown in SEQ ID No.17 to 22, respectively.

4. The method for constructing a recombinant yeast according to any one of claims 1 to 3, characterized in that: The steps include: (1) connecting a signal peptide encoding gene, a recombinant mussel fusion protein encoding gene fragment, a molecular chaperone encoding gene, an aminoacyl-tRNA synthetase encoding gene fragment, and an endoplasmic reticulum expansion-related factor encoding gene to obtain a multi-gene fragment; (2) connecting the multi-gene fragment obtained in step (1) to a plasmid to obtain a recombinant expression vector; (3) Introducing the recombinant expression vector obtained in step (2) into yeast to obtain the recombinant yeast.

5. The method for constructing a recombinant yeast according to claim 4, characterized in that: In step (1), the recombinant mussel fusion protein encoding gene fragment contains at least one copy of the recombinant mussel fusion protein encoding gene; the aminoacyl tRNA synthetase encoding gene fragment contains any one or more of a lysyl tRNA synthetase encoding gene, a tyrosyl tRNA synthetase encoding gene and a prolyl tRNA synthetase encoding gene.

6. The construction method according to claim 4, characterized in that: In step (3), the yeast is any one of Pichia pastoris, Saccharomyces cerevisiae and baker's yeast.

7. Use of the recombinant yeast according to any one of claims 1 to 3 in fermentation to produce recombinant mussel fusion protein.

8. The use according to claim 7, characterized in that: The recombinant yeast is inoculated into a fermentation medium for fermentation; the fermentation medium contains any one or more of lysine, tyrosine and proline.

9. The use according to claim 8, characterized in that: The fermentation medium contains an inductive carbon source and a non-inhibitory carbon source; the inductive carbon source is methanol, and the non-inhibitory carbon source is sorbitol.

10. The use according to claim 8, characterized in that: The fermentation conditions are: temperature of 25-30°C, rotation speed of 180-220 rpm, initial OD of the recombinant yeast in the fermentation medium 600 The value is 3.5~5.5, and the fermentation time is 144~168h.

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