A novel vector for expression of foreign proteins in yeast

Abstract

This invention belongs to the field of genetic engineering and microbial expression technology, specifically disclosing a novel vector for expressing exogenous proteins in yeast. The vector is named pPHZAa, and its nucleotide sequence is shown in SEQ ID NO.2. The pPHZAa vector comprises: a methanol-inducible expression cassette, a histidine deficiency compensation gene PpHIS4, a bleomycin resistance expression cassette BleoR, and a pBR322 / pUC replicon. The methanol-inducible expression cassette includes an AOX1 promoter, an AOX1 terminator, and a secretion guide sequence. The optimized elements of the vector provided by this invention work together to produce a synergistic effect, significantly improving the overall expression level and secretion efficiency of exogenous proteins in yeast.

Description

Technical Field

[0001] This invention belongs to the field of genetic engineering and microbial expression technology, specifically relating to a novel vector for expressing exogenous proteins in yeast. Background Technology

[0002] Pichia pastoris ( Pichia pastoris Due to its powerful methanol-inducible alcohol oxidase (AOX1) promoter, clear genetic background, and mature fermentation process, Pichia pastoris has become one of the mainstream eukaryotic systems for expressing exogenous proteins. Currently, commercially available Pichia pastoris expression vectors (such as pPIC9K and pPICZaA) are widely used in scientific research and industrial production. However, existing commercial vectors still have significant limitations. For example, although the pPIC9K vector contains the histidine auxotroph selection marker (HIS4), its plasmid is large (approximately 9.3 kb), resulting in low efficiency of electroporation transformation of host cells when carrying large exogenous gene fragments. Furthermore, the antibiotic concentration required for multi-copy selection using G418 is as high as 3 mg / ml, leading to high costs and significant inhibition of host growth. On the other hand, while the pPICZaA vector contains a bleomycin resistance marker at a low selection concentration, it lacks a histidine compensation gene. The transformed histidine-deficient host bacteria (such as GS115) cannot grow normally in basal inorganic salt medium, making high-density fermentation difficult and limiting its industrial application. In addition, the transcription initiation strength of the natural AOX1 promoter is limited, and the natural terminator and secretory signal peptide need to be improved in terms of mRNA stability and protein secretion efficiency. These factors together restrict the further increase of exogenous protein production.

[0003] Therefore, there is an urgent need to develop a novel Pichia pastoris expression vector that can combine small plasmid size, high efficiency in transformation, low-cost screening markers, and the ability to support high-density fermentation of host bacteria, and achieve efficient expression and secretion of exogenous proteins through optimization of key expression elements. Summary of the Invention

[0004] This invention aims to provide a novel vector pPHZAa for the expression of exogenous proteins in yeast. Through multi-element synergistic optimization, this vector system can significantly improve the expression level and secretion efficiency of exogenous proteins (especially secretory proteins), and the host bacteria can achieve high-density fermentation in inorganic salt culture medium. It has the advantages of high conversion efficiency, low screening cost, and wide applicability.

[0005] Specifically, the present invention provides the following technical solutions: This invention provides a vector for expressing exogenous proteins in yeast, the vector being named pPHZAa, with a nucleotide sequence as shown in SEQ ID NO.2; the pPHZAa vector comprises: a methanol-inducible expression cassette, a histidine deficiency compensation gene PpHIS4, a bleomycin resistance expression cassette BleoR, and a pBR322 / pUC replicon; the methanol-inducible expression cassette comprises an AOX1 promoter, an AOX1 terminator, and a secretion guide sequence.

[0006] Furthermore, the AOX1 promoter can be replaced with the PA3 promoter, the PA3 nucleic acid sequence of which is shown in SEQ ID NO.5.

[0007] Furthermore, the AOX1 terminator can be replaced by TA1, TA2 or TA4 terminators, the TA1 nucleic acid sequence is shown in SEQ ID NO.7, the TA2 nucleic acid sequence is shown in SEQ ID NO.8, and the TA4 nucleic acid sequence is shown in SEQ ID NO.10.

[0008] Furthermore, the secretion guide sequence can be replaced with α-factor mut1, α-factor mut2 or OST1mut guide sequences, where the α-factor mut1 nucleic acid sequence is shown in SEQ ID NO.11, the α-factor mut2 nucleic acid sequence is shown in SEQ ID NO.12, and the OST1 mut nucleic acid sequence is shown in SEQ ID NO.14.

[0009] Furthermore, the vector contains a PA3 promoter, a TA1 terminator, and an α-factor mut1 guide sequence, and the nucleotide sequence of the vector is shown in SEQ ID NO.16.

[0010] Furthermore, the present invention provides the application of any of the vectors described herein in expressing exogenous proteins after conversion into yeast, wherein the yeast is Pichia pastoris or Saccharomyces cerevisiae.

[0011] Preferably, the Pichia pastoris includes, but is not limited to, Pichia pastoris GS115.

[0012] The novel carrier provided by this invention has the following significant effects: 1. The plasmid vector backbone consists of: a methanol-induced expression cassette, a histidine deficiency compensation gene, a resistance gene expression cassette, and a replicon. The replicon uses the pBR322 / pUC high-copy replicon to ensure high-efficiency plasmid amplification in *E. coli*. The histidine deficiency compensation gene uses the PpHIS4 gene from pPIC9K, which can compensate for the normal growth of histidine-deficient yeast in inorganic salt media. The resistance gene expression cassette uses the BleoR expression cassette from pPICZaA. Both plasmid amplification in *E. coli* and gene expression in yeast can be screened using bleomycin.

[0013] 2. The difference between the pPIC9K vector and the pPIC9K vector is that the pPIC9K vector has two resistance regions and the plasmid is larger, at 9276 bp. When carrying a large exogenous gene, the electroporation efficiency of the linearized plasmid is low. In addition, the G418 high copy screening requires a high antibiotic concentration, usually reaching 3 mg / ml. The plasmid pPHZAa of this patent has only one resistance region, at 6547 bp, which is relatively small. The linearized plasmid has high electroporation efficiency, and the bleomycin concentration for high copy screening only needs to reach 0.3 mg / ml.

[0014] 3. Unlike the pPICZaA vector, the pPICZaA vector does not contain the PpHIS4 gene, which cannot compensate for the host bacterial histidine deficiency, resulting in the inability of the selected high-expression strains to ferment at high density in inorganic salt media. The pPHZAa vector combines the advantages of both pPIC9K and pPICZaA vectors while avoiding their disadvantages.

[0015] 4. The methanol-induced expression cassette includes: an optimized methanol oxidase promoter (AOX1mut promoter), an optimized methanol oxidase terminator (AOX1mut terminator), and an optimized secretion guidance sequence. The optimized methanol oxidase promoter (AOX1mut promoter) has stronger initiation strength than the conventional AOX1 promoter, effectively increasing the expression level of the target protein. The optimized methanol oxidase terminator (AOX1mut terminator) effectively improves post-transcriptional mRNA stability and increases intracellular mRNA concentration.

[0016] 5. The optimized secretion guide sequence can effectively improve the secretion expression rate of the target protein. Attached Figure Description

[0017] Figure 1 This is a schematic diagram of the pPICZaA carrier structure.

[0018] Figure 2 This is a schematic diagram of the pPIC9K carrier structure.

[0019] Figure 3 A schematic diagram of the pPHZAa carrier structure of this invention.

[0020] Figure 4 This is a schematic diagram of the carrier's functional areas, where A is the pPICZaA carrier and B is the pPIC9K carrier.

[0021] Figure 5 Structure diagram of the vector PHZAa-PA3-mut1-TA1.

[0022] Figure 6 Map showing the expression region of exogenous protein in the vector PHZAa-PA3-mut1-TA1. Detailed Implementation

[0023] The following will clearly and completely describe the concept and technical effects of this application in conjunction with embodiments, so as to fully understand the purpose, features and effects of this application. For the experimental methods, purchased goods, unless specific conditions are specified, shall be carried out under conventional conditions or conditions recommended by the manufacturer. Reagents or instruments used, unless the manufacturer is specified, can be commercially available conventional products. Unless otherwise defined herein, the scientific and technical terms used in connection with this invention should have the meaning commonly understood by one of ordinary skill in the art. Exemplary methods and materials are described below; however, similar or equivalent methods and materials described herein can also be used in the practice and testing of this disclosure.

[0024] In this invention, BMMY medium is a standard culture medium for Pichia pastoris, and "biotin" in the medium refers to "vitamin B7". Unless otherwise specified, all "%" values ​​used in this invention are mass percentages.

[0025] Example 1 1. Synthesis of the starting vector See Figure 1-4 The pPICZaA vector was synthesized by Suzhou Genewise Biotechnology Co., Ltd., and double-digested with BbvCI / AleI. The plasmid pPIC9K was also double-digested with BbvCI / AleI, and a 4577bp fragment was recovered. The synthesized sequence and the recovered fragment were ligated to form the first-generation pPHZAa vector, which is 6559bp in length and has the full sequence SEQ ID NO.2.

[0026] The plasmid vector backbone consists of a methanol-induced expression cassette, a histidine deficiency compensation gene, a resistance gene expression cassette, and a replicon. The replicon uses the pBR322 / pUC high-copy replicon (GenBank: J01749.1 2534..3122) to ensure high-efficiency plasmid amplification in *E. coli*. The histidine deficiency compensation gene uses the PpHIS4 gene from pPIC9K (GenBank: X56180.1) to compensate for the normal growth of histidine-deficient yeast in inorganic salt media. The resistance gene expression cassette uses the BleoR (GenBank: LT726836.1 420..1592) expression cassette from pPICZaA. Plasmid amplification in *E. coli* and gene expression in yeast were both screened using bleomycin. Full-sequence sequencing by Beijing Qingke Biotechnology Co., Ltd. confirmed that the constructed vector was completely consistent with the design.

[0027] The recombinant vector pPHZAa of this invention is a small plasmid with a single antibiotic region, with a full length of 6559 bp (SEQ ID NO.2). Compared to the commercial pPIC9K vector (9276 bp), its size is significantly reduced. The linearized plasmid significantly improves the transformation efficiency of yeast host cells via electroporation, which is beneficial for efficiently obtaining high-copy-count integrated recombinant strains. The vector carries the bleomycin resistance expression cassette BleoR, requiring only a bleomycin concentration of 0.3 mg / ml for high-copy screening; while the existing pPIC9K vector uses G418 screening, requiring an antibiotic concentration of 3 mg / ml. This invention significantly reduces the amount of screening antibiotic used, saving fermentation production costs, and simultaneously mitigating the inhibitory effect of high-concentration antibiotics on yeast cell growth.

[0028] This invention integrates the PpHIS4 histidine deficiency compensation gene from the pPIC9K vector with the bleomycin resistance system of the pPICZaA vector into the pPHZAa vector. This overcomes the shortcomings of pPICZaA, such as the lack of a histidine compensation gene and the inability of transformed strains to ferment at high density in inorganic salt media. Simultaneously, it avoids the drawbacks of pPIC9K, such as excessively large plasmid size, low transformation efficiency, and excessively high G418 selection concentration, combining the advantages of both commercially available vectors. The PpHIS4 gene compensates for the growth defects of histidine-deficient yeasts, allowing the recombinant strains to grow normally in inorganic salt media, meeting the requirements of industrial high-density fermentation. Furthermore, the vector employs the pBR322 / pUC high-copy replicon, ensuring efficient amplification of the recombinant plasmid in *E. coli*, facilitating large-scale plasmid preparation and molecular modification.

[0029] 2. Synthesis of reporter gene vectors A lipase gene from Candida antarctica was screened from the Uniprot database, with the gene number P41365. After removing the signal peptide, the gene was synthesized by Beijing Qingke Biotechnology Co., Ltd. and constructed into the SnaBI / AvrII space of the pPHZAa vector. The resulting plasmid was named pPHZAa-CALB.

[0030] 3. Methods for yeast strain culture, transformation, and enzyme activity testing (1) Commonly used culture media YPD medium: 2.0% peptone, 1.0% yeast extract, 2.0% glucose.

[0031] YPD-G418 solid medium: 2.0% peptone, 1.0% yeast extract, 2.0% glucose, 2.0% agar, sterilized at 115℃ for 20 min, then add G-418 to a final concentration of 3 mg / mL.

[0032] YPD-Zeocin solid medium: 2.0% peptone, 1.0% yeast extract, 2.0% glucose, 2.0% agar, sterilized at 115℃ for 20 min, then bleomycin was added to a final concentration of 0.3 mg / mL.

[0033] MD medium: 1.34% YNB (containing ammonium sulfate), 2% glucose, 4×10⁻⁶ -7 mg / mL biotin.

[0034] BMGY medium: 2.0% peptone, 1.0% yeast extract, 1.34% YNB (containing ammonium sulfate), 1.0% glycerol, 100 mM potassium phosphate buffer (pH 6.0), 4 × 10⁻⁶ ppm -7 mg / mL biotin.

[0035] BMMY medium: 2.0% peptone, 1.0% yeast extract, 1.34% YNB (containing ammonium sulfate), 0.5% methanol, 100 mM potassium phosphate buffer (pH 6.0), 4 × 10⁻⁶ ppm -7 mg / mL biotin.

[0036] If the above culture medium is a solid culture medium, then add 2% agar to the original culture medium.

[0037] (2) Preparation and electroconversion of Pichia pastoris competent cells A. Preparation of competent cells a. Pick a single colony of Pichia pastoris from a fresh plate and inoculate it into a 250 mL Erlenmeyer flask containing 25 mL YPD. Incubate at 30 °C and 220 rpm for 24 h with shaking. b. Take 0.3 mL of culture and inoculate it into a 250 mL Erlenmeyer flask containing 30 mL of fresh YPD medium. Incubate at 30℃ and 220 rpm for 7-8 h with shaking until the OD600 reaches 1.3-1.5. c. Transfer 1.0 mL of culture to a 1.5 mL EP tube and centrifuge at 2,000 rpm for 5 min to collect cells; d. Resuspend the cells in 1.5 mL of pre-cooled sterile water, centrifuge at 2,000 rpm for 5 min to collect the cells; e. Resuspend the cells in 1.5 mL of pre-cooled sterile water, centrifuge at 2,000 rpm for 5 min to collect the cells; f. Resuspend the cells in 1.5 mL of pre-cooled 1 M sorbitol and collect the cells by centrifugation at 2,000 rpm for 5 min; g. Resuspend cells in 0.1 mL of pre-cooled 1 M sorbitol; h. The prepared competent cells can be stored at 4°C for one week.

[0038] B. Plasmid linearization a. Sal I linearization of pPHZAa plasmid: Take 20 μL of pPHZAa plasmid, 3 μL of Sac I enzyme, 3 μL of 10× buffer, and 4 μL of sterile water, mix well, and digest at 37℃ for 4 hours. After digestion, perform nucleic acid electrophoresis and recover the linearized fragment using a gel recovery kit to determine the concentration of the linearized plasmid.

[0039] To improve the transformation efficiency of Pichia pastoris, the amount of linearized plasmid needs to reach 5–20 μg.

[0040] C. Electrocution a. Take 80 μL of competent Pichia pastoris cells and mix with 5–20 mg of linearized DNA (dissolved in 5–10 μL of TE solution), transfer to a pre-cooled 0.2 cm electroporation vessel, mix well and incubate on ice for 5 min; b. Electric shock using an electroconversion device (1800 V); c. Immediately add 1 mL of pre-cooled 1 M sorbitol to the cup, and transfer the contents to a sterile 1.5 mL centrifuge tube; d. Divide into 2-3 equal parts and apply to MD / YPD-G418 / YPD-Zeocin plates; e. Incubate at 30°C until recombinant yeast grows (approximately 2-3 days).

[0041] (3) Shaking flask screening Single colonies were picked and inoculated into 250 mL Erlenmeyer flasks containing 50 mL of BMGY medium. The flasks were incubated at 30 °C and 220 rpm for 24 h on a shaker. After standing for 1 h, the supernatant was discarded, and the supernatant was transferred to 50 mL of BMMY medium. The culture was continued, with methanol added every 24 h to a final concentration of 1.0%. Expression was induced for 48–96 h. Samples were centrifuged at 10,000 rpm for 5 min, and the supernatant was used to characterize the expression level of the target protein using an activity assay.

[0042] (4) CALB lipase activity assay method Enzyme activity is defined as the amount of enzyme required to generate 1 μmol of p-nitrophenol per minute in 100mM pH 7.0 potassium phosphate buffer at 30℃, using 4-nitrophenylacetate as a substrate.

[0043] Preparation of substrate solution: Accurately weigh 7.28 mg of 4-nitrophenylacetic acid ester, add 4 ml of DMSO reagent, dissolve thoroughly, and set aside for use on the same day. If the initial OD... 410 If the concentration is greater than 0.5, the DMSO reagent needs to be replaced and the solution prepared again.

[0044] The amount of enzyme used can be adjusted according to the actual reaction conditions to ensure OD... 410 The change value per minute is between 0.05 and 0.20. If the change is too rapid, the enzyme solution needs to be diluted before measurement.

[0045] The activity assay system (4 ml cuvette) consists of: 2650 μl of 100 mM pH 7.0 potassium phosphate buffer; 300 μl of substrate solution; and 50 μl of enzyme solution. Add the potassium phosphate buffer and substrate solution to the cuvette, preheat to 30°C, add the enzyme solution, mix immediately, place in a spectrophotometer, and react for 5 minutes. Record the OD per minute. 410 The value.

[0046] Enzyme activity calculation: Discard the data from the first minute and take the OD data from the last 4 minutes. 410 Change in ΔOD 410 The enzyme activity is calculated using the following formula.

[0047] Enzyme activity (U / ml) = (3000 ΔOD 410 (dilution factor) / (50) 4 12.36) 4. AOX1 promoter optimization Four variants of the AOX1 promoter were designed using promoter analysis software and named PA1, PA2, PA3, and PA4, with sequences shown in Table 1. These variants were synthesized by Beijing Qingke Biotechnology Co., Ltd., and the AOX1 promoter in the vector pPHZAa-CALB was replaced by double digestion with BglII / BstBI. The resulting vectors were named pPHZAa-PA1-CALB, pPHZAa-PA2-CALB, pPHZAa-PA3-CALB, and pPHZAa-PA4-CALB, respectively. The five vectors were transformed into GS115 competent cells. The strains with the highest enzyme activity were selected from each strain by shaking flask. The five strains were then simultaneously subjected to shake-flask fermentation for comparison. Enzyme activity data after 96 hours of induction are shown in Table 2.

[0048] Table 1. Nucleic acid sequences of AOX1 promoter variants PA1 SEQ ID NO.3 PA2 SEQ ID NO.4 PA3 SEQ ID NO.5 PA4 SEQ ID NO.6 Table 2 Comparison of enzyme activities of AOX1 promoter variants in shake flasks

[0049] Referring to Table 2, shake-flask experiments confirmed that optimizing promoters PA3 and PA4 significantly increased the expression level of the target protein, with PA3 showing the best effect. Using CALB lipase as a reporter gene, the PA3 promoter induced enzyme activity to reach 4.62±0.19 U / ml after 96 h, an increase of approximately 62% compared to the natural AOX1 promoter (2.85±0.21 U / ml). This demonstrates that the optimized promoter possesses stronger transcriptional activation capabilities, providing a powerful driver for the efficient expression of exogenous genes.

[0050] 5. Optimization of AOX1 Terminator Based on bioinformatics data, a large number of AOX1 terminators that can improve mRNA stability were generated using a model. The four with the highest scores were selected, as shown in Table 3. These terminators were synthesized by Beijing Qingke Biotechnology Co., Ltd., and the AOX1 terminators in the vector pPHZAa-PA3-CALB were replaced by NotI / ClaI double digestion. The synthesized vectors were named pPHZAa-PA3-CALB-TA1, pPHZAa-PA3-CALB-TA2, pPHZAa-PA3-CALB-TA3, and pPHZAa-PA3-CALB-TA4. After SalI linearization, the four vectors were transformed into GS115 competent cells. The strains with the highest enzyme activity were selected from the shake-flask samples. The four strains were then compared with the control strain pPHZAa-PA1-CALB in shake-flask fermentation. The enzyme activity data after 96 hours of induction are shown in Table 4.

[0051] Table 3 AOX1 Terminator Variant Sequences TA1 SEQ ID NO.7 TA2 SEQ ID NO.8 TA3 SEQ ID NO.9 TA4 SEQ ID NO.10 Table 4 Comparison of enzyme activities in shake flasks of AOX1 terminator variants

[0052] Referring to Table 4, the results show that the TA1 and TA2 terminators performed best. The enzyme activity of the TA1 combination strain reached 6.77±0.27 U / ml, which is about 60% higher than the natural AOX1 terminator control (4.21±0.17 U / ml). This effect is due to the fact that the optimized terminator can enhance the stability of transcript mRNA, reduce intracellular mRNA degradation, maintain a higher intracellular mRNA concentration, thereby prolonging the translation window and continuously increasing the total amount of protein synthesis.

[0053] 6. Optimization of secretion signal-guided sequences Based on bioinformatics data, a large number of secretion signal guide sequences were generated using a model. Five of these sequences were selected for testing. The amino acid and nucleic acid sequences are shown in Table 5. These sequences were synthesized by Beijing Qingke Biotechnology Co., Ltd., and the secretion signal guide sequences in the vector pPHZAa-PA3-CALB-TA1 were replaced by double digestion with BstBI / PsiI. Vectors were then synthesized separately. After linearization with SalI, the vectors were transformed into GS115 competent cells. The strains with the highest enzyme activity were selected from the shake flasks. The resulting strains were then compared with the control strain pPHZAa-PA3-CALB-TA1 in shake flask fermentation. The enzyme activity data after 72 hours of induction are shown in Table 6.

[0054] Table 5 Optimization of Secretion Signal-Guided Sequences

[0055] Table 6. Comparison of shake-flask enzyme activities after optimization of secretion signal-guided sequences.

[0056] As shown in Table 6, the data indicate that α-factor mut1, α-factor mut2, and OST1 mut all significantly increased the expression level of the reporter gene. Among them, the α-factor mut1 signal peptide showed the best effect, with an enzyme activity of 8.32±0.28 U / ml after 72 h of induction, which is more than 62% higher than that of wild-type α-factor (5.13±0.34 U / ml). The optimized secretion signal peptide can more efficiently guide the target protein to be secreted extracellularly, shorten the protein's residence time in the cell, reduce the endoplasmic reticulum load, reduce intracellular protease degradation, and ultimately achieve a simultaneous increase in the secretion rate and total expression level of exogenous protein.

[0057] 7. Comparison of different carriers Synthesized by Beijing Qingke Biotechnology Co., Ltd., the initial pPHZAa vector was replaced with one containing a PA3 promoter, a TA1 terminator, and an α-factor mut1 guide sequence to construct an enhanced vector pPHZAa-PA3-mut1-TA1, the nucleotide sequence of which is shown in SEQ ID NO.16. Vectors pPHZAa-CALB and pPHZAa-PA3-mut1-TA1-CALB were then constructed. After SalI linearization, the vectors were transformed into GS115 competent cells, and enzyme activity data after 72 hours of shaking flask fermentation were compared. The results are shown in Table 7.

[0058] Table 7 Comparison of enzyme activity in shake flasks after optimization

[0059] In summary, this invention systematically optimizes the core regulatory elements of the methanol-induced expression cassette: a series of AOX1 promoter variants were designed and obtained, among which the PA3 promoter has the strongest transcriptional activation ability. Using CALB lipase as a reporter gene, the enzyme activity reached 4.62±0.19 U / ml after 96 hours of induction, which is significantly improved compared with the natural AOX1 promoter (2.85±0.21 U / ml); multiple optimized AOX1 terminator variants were screened and obtained, among which the TA1 terminator can effectively improve post-transcriptional mRNA stability and increase intracellular mRNA abundance. When combined with the PA3 promoter, the enzyme activity increased to 6.77±0.27 U / ml; the secretion guide sequence was modified, among which the α-factor mut1 signal peptide can accelerate the secretion rate of the target protein and reduce intracellular protein accumulation, and the enzyme activity reached 8.32±0.28 U / ml after 72 hours of induction. The various optimized elements work together to produce a synergistic gain effect. The optimized pPHZAa-PA3-mut1-TA1 significantly improves the overall expression level and secretion efficiency of exogenous proteins compared with the original vector pPHZAa.

[0060] The recombinant vector provided by this invention has a standardized multiple cloning site, exhibiting good versatility and adaptability to various exogenous target gene clones. It is not only suitable for the Pichia pastoris GS115 host strain but also compatible with other histidine auxotrophic Pichia pastoris strains, enabling its widespread application in the eukaryotic expression of various exogenous proteins, such as industrial enzymes and pharmaceutical proteins. The entire expression system simultaneously offers multiple advantages, including high transformation efficiency, low-cost resistance screening, support for high-density fermentation, strong transcriptional drive, high mRNA stability, and efficient protein secretion, demonstrating promising prospects for industrial application.

[0061] The above description is merely a preferred embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any equivalent substitutions or modifications made by those skilled in the art within the scope of the technology disclosed in the present invention, based on the technical solution and inventive concept of the present invention, should be covered within the scope of protection of the present invention.

Claims

1. A vector for expressing exogenous proteins in yeast, characterized in that, The vector is named pPHZAa, and its nucleotide sequence is shown in SEQ ID NO.

2. The pPHZAa vector contains: a methanol-inducible expression cassette, a histidine deficiency compensation gene PpHIS4, a bleomycin resistance expression cassette BleoR, and a pBR322 / pUC replicon. The methanol-inducible expression cassette contains an AOX1 promoter, an AOX1 terminator, and a secretion guide sequence.

2. The carrier according to claim 1, characterized in that, The AOX1 promoter can be replaced with the PA3 promoter, the PA3 nucleic acid sequence of which is shown in SEQ ID NO.

5.

3. The carrier according to claim 2, characterized in that, The AOX1 terminator can be replaced by TA1, TA2 or TA4 terminators. The TA1 nucleic acid sequence is shown in SEQ ID NO.7, the TA2 nucleic acid sequence is shown in SEQ ID NO.8, and the TA4 nucleic acid sequence is shown in SEQ ID NO.

10.

4. The carrier according to claim 3, characterized in that, The secretion guide sequence can be replaced by α-factor mut1, α-factor mut2 or OST1 mut guide sequence, where the α-factor mut1 nucleic acid sequence is shown in SEQ ID NO.11, the α-factor mut2 nucleic acid sequence is shown in SEQ ID NO.12, and the OST1 mut nucleic acid sequence is shown in SEQ ID NO.

14.

5. The carrier according to claim 4, characterized in that, The vector contains a PA3 promoter, a TA1 terminator, and an α-factor mut1 guide sequence, and the nucleotide sequence of the vector is shown in SEQ ID NO.

16.

6. The use of the vector according to any one of claims 1-5 in expressing exogenous proteins after transformation into yeast, characterized in that, The yeast mentioned is Pichia pastoris or Saccharomyces cerevisiae.

7. The application according to claim 6, wherein the Pichia pastoris includes, but is not limited to, Pichia pastoris GS115.

Images