Method for improving secretory expression efficiency of sweet protein in pichia pastoris

Abstract

The invention relates to the technical field of protein engineering or genetic engineering, and discloses a method for improving the secretory expression efficiency of sweet protein in pichia pastoris. The invention discloses pichia pastoris for secretory expression of sweet protein. The pichia pastoris simultaneously expresses a sweet protein gene, a secretory signal peptide gene and a gene expression regulatory element. The pichia pastoris is subjected to two-stage fermentation culture, the sweet protein can be successfully prepared, and the yield is 4g / L or above. The technical scheme provided by the invention has an extremely high effect of improving the secretory expression efficiency of the sweet protein.

Description

Technical Field

[0001] The invention relates to the technical field of protein engineering or gene engineering, and in particular to a method for improving the secretion expression efficiency of sweet protein in Pichia pastoris. Background Art

[0002] Sweet protein Brazzein is isolated from the fruit of the wild plant Pentadiplandra brazzeana Baillon in West Africa, and its yield accounts for about 0.2% of the total weight of the fruit. The protein is mainly composed of 54 amino acid residues. The relative molecular mass of Brazzein is 6.5kDa, and its sweetness is about 2000 times that of the same mass of sucrose. It also has good water solubility. The molecular structure is one α-helix and three antiparallel β-folds, connected by four pairs of disulfide bonds, which enables it to fold correctly at 85°C and the sweet taste does not disappear. Therefore, it has great potential as a sucrose substitute. However, due to reasons such as the source of raw materials and purification costs, the preparation amount of sweet protein Brazzein cannot meet the needs of various fields. At present, there are many ways to produce sweet protein Brazzein using prokaryotic or eukaryotic hosts, but the yield is unsatisfactory. In the prior art, the highest yield of sweet protein at the fermentation tank level is about 1g / L. Summary of the invention

[0003] Purpose of the invention: The technical problem to be solved by the present invention is to provide a method for improving the secretion expression efficiency of the sweet protein Brazzein in Pichia pastoris in view of the shortcomings of the prior art methods for improving the secretion expression efficiency of the sweet protein Brazzein.

[0004] In order to solve the above technical problems, the present invention discloses a method for improving the secretion expression efficiency of sweet protein in Pichia pastoris. The method includes the following technical solutions:

[0005] The present invention provides a Pichia pastoris that secretes and expresses a sweet protein, which simultaneously expresses a sweet protein gene, a secretion signal peptide gene and a gene expression regulatory element; wherein the nucleotide sequence of the sweet protein gene is shown in SEQ ID No. 2; the nucleotide sequence of the secretion signal peptide gene is selected from any one of SEQ ID No. 3 to 10; the nucleotide sequence of the gene expression regulatory element is selected from any one of SEQ ID No. 11 to 13. The nucleotide sequence of the sweet protein gene is a sequence optimized according to the codon preference of Pichia pastoris.

[0006] Preferably, the nucleotide sequence of the secretory signal peptide gene is selected from any one of SEQ ID No. 3 to 6, and more preferably SEQ ID No. 4. The nucleotide sequence of the gene expression regulatory element is shown in SEQ ID No. 11.

[0007] Wherein, the starting bacteria of Pichia pastoris that secretes and expresses sweet protein is Pichia pastoris GS115.

[0008] Wherein, the sweet protein gene is expressed by an inducible promoter; the inducible promoter includes P AOX1 .

[0009] In a second aspect, the present invention provides a method for constructing the Pichia pastoris that secretes and expresses the sweet protein described in the first aspect, comprising the following steps: amplifying a secretion signal peptide gene and a sweet protein gene, and cloning them into a first starting vector to obtain a first recombinant vector; amplifying a gene expression regulatory element, and cloning them into a second starting vector to obtain a second recombinant vector; and introducing the first recombinant vector and the second recombinant vector into Pichia pastoris to obtain the recombinant Pichia pastoris.

[0010] Wherein, the first starting vector is pPIC9K; the second starting vector is pPICZA.

[0011] In a third aspect, the present invention provides use of the Pichia pastoris that secrete and express sweet protein as described in the first aspect in producing sweet protein.

[0012] In a fourth aspect, the present invention provides a method for improving the secretion and expression efficiency of sweet proteins in Pichia pastoris, using the Pichia pastoris secreting and expressing sweet proteins described in the first aspect to perform two-stage fermentation culture, comprising the following steps:

[0013] (1) Strain growth stage: The Pichia yeast expressing the sweet protein is inoculated into the growth medium and cultured at 28-32°C until the logarithmic growth phase; preferably at 30°C. Further preferably, the OD 600 is 100.

[0014] (2) Methanol induction stage: Methanol is added to the bacterial solution obtained in step (1) for induction, and the culture is continued at 28-32°C for 96-144 hours, preferably at 30°C for 96-120 hours.

[0015] Wherein, in step (1), the growth medium is a buffered complete medium BMGY containing glycerol; the fermentation pH of the strain growth stage is 5-6; preferably, the BMGY formula comprises: 20 g / L peptone, 10 g / L yeast extract powder, 3.4 g / L yeast nitrogen source without amino acids and ammonium sulfate, 10 g / L ammonium sulfate, 20 g / L glycerol, 0.02%-0.12% v / v biotin, KH 2 PO 4 11.8g / L, K 2 HPO 4 2.29 g / L; the fermentation pH during the growth stage of the strain is 5.0. Further preferably, biotin is 0.02% to 0.04% v / v.

[0016] Wherein, in step (2), during the methanol induction stage, the added volume of methanol is 0.5% to 12% of the volume of the fermentation broth; preferably 3% to 4%. Further preferably, the volume of the fermentation broth is the volume of the initial fermentation medium. More preferably, in a 50L fermenter, the initial fermentation medium volume is 25L, and during the methanol induction stage, methanol is continuously added at a rate of 10mL / h until the fermentation is completed. The fermentation time of the methanol induction stage is further preferably 96h.

[0017] In step (2), the fermentation pH in the methanol induction stage is 5 to 6. Preferably, the fermentation pH in the methanol induction stage is 6.0.

[0018] Beneficial effects:

[0019] The present invention provides a Pichia pastoris that secretes and expresses a sweet protein, which simultaneously expresses a sweet protein gene, a secretion signal peptide gene, and a gene expression regulatory element. The Pichia pastoris is cultured by two-stage fermentation to successfully prepare the sweet protein with a yield of more than 4 g / L. The technical solution provided by the present invention has the effect of extremely improving the secretion expression efficiency of the sweet protein. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] The present invention will be further described in detail below in conjunction with the accompanying drawings and specific embodiments, and the above and / or other advantages of the present invention will become more clear.

[0021] Figure 1 is the plasmid map of pPIC9K-Bra constructed in Example 1;

[0022] Figure 2 The sweet protein expression level of the strain in Example 1 at different methanol concentrations, biotin concentrations, pH or temperatures;

[0023] Figure 3 The protein expression levels of the recombinant strains after different signal peptides were inserted into the proteins in Example 2;

[0024] Figure 4 This is the recombinant plasmid map obtained by inserting gene expression regulatory elements into plasmid pPICZA.

[0025] Figure 5 is the protein expression level of the strain after expressing different gene expression regulatory elements in Example 3;

[0026] Figure 6 is the relevant data of the fermentation process in Example 4, wherein Figure 6 a is a schematic diagram of the fermentation process. Figure 6 b is the OD 600 The protein yields of the induced expression were obtained by adding methanol at 100 and 200 values. Figure 6 c is the protein yield when the pH value during the induction period is 4, 5, 6, and 7, respectively. Figure 6 d is OD 600 =100 when it entered the methanol induction period and the fermentation diagram when the pH value of the induction period was 6.0. DETAILED DESCRIPTION

[0027] In order to make the above-mentioned objects, features and advantages of the present invention more obvious and easy to understand, the specific implementation methods of the present invention are described in detail below in conjunction with the embodiments of the specification.

[0028] In the following description, many specific details are set forth to facilitate a full understanding of the present invention, but the present invention may also be implemented in other ways different from those described herein, and those skilled in the art may make similar generalizations without violating the connotation of the present invention. Therefore, the present invention is not limited to the specific embodiments disclosed below.

[0029] Secondly, the term "one embodiment" or "embodiment" as used herein refers to a specific feature, structure, or characteristic that may be included in at least one implementation of the present invention. The term "in one embodiment" that appears in different places in this specification does not necessarily refer to the same embodiment, nor does it refer to a separate or selective embodiment that is mutually exclusive with other embodiments.

[0030] In the following examples, the methanol-inducible secretory plasmid pPIC9K is a commercial vector with a promoter P AOX1 And signal peptide α signal peptide (α-factor).

[0031] Example 1

[0032] This embodiment involves optimizing the base sequence encoding the sweet protein Brazzein (Bra) by codon preference of Pichia pastoris and synthesizing it artificially, inserting it into the methanol-inducible secretory plasmid pPIC9K of Pichia pastoris, and using double restriction enzyme digestion technology to achieve the insertion of Bra into the methanol-inducible secretory plasmid pPIC9K of Pichia pastoris. The endonucleases used for double restriction enzyme digestion are EcoRⅠ and NotⅠ, wherein the promoter P of pPIC9K itself AOX1 The sequence of the sweet protein Brazzein is shown in SEQ ID No. 1, and the sequence of the sweet protein Brazzein is shown in SEQ ID No. 2. After double enzyme digestion, the recombinant plasmid pPIC9K-Bra is obtained. The plasmid map is shown in Figure 1 shown.

[0033] After linearizing the plasmid pPIC9K-Bra using SalⅠ enzyme, the Bra gene was inserted into the genome of Pichia pastoris GS115 by electroporation with the voltage set to 1500V and the resistance set to 200Ω. 1mL of 1mol / L sorbitol was quickly added to the electroporation cup and pre-cultured at 200rpm in a 30℃ shaker for 3h before spreading on an MD plate. A high-copy strain was screened out using a YPD plate containing 4mg / mL G418 as the starting strain GS115-pPIC9K-Bra.

[0034] The products cultured in shake flasks were verified by SDS-PAGE. The specific conditions of shake flask culture were as follows: the prepared gene-transformed strain GS115-pPIC9K-Bra and the original strain GS115 were cultured in YPD medium at 30°C for 12 h, and then inoculated with 5% (V / V) in BMGY medium and cultured for 12 h until the logarithmic growth phase (OD 600 10), then transferred to BMMY medium and continued to culture for 120h at 30℃, methanol was added every 24h, and the volume of methanol added each time was 0.5% of the volume of the fermentation liquid. The bacterial liquid after induced expression was centrifuged and the supernatant was collected to measure the protein secretion expression efficiency. The formulas of BMGY medium and BMMY medium are shown in Table 1 and Table 2.

[0035] Table 1 BMGY medium formula

[0036]

[0037] Table 2 BMMY medium formula

[0038]

[0039] The method for determining the expression level of the sweet protein Brazzein in the present invention can be as follows:

[0040] Accurately weigh 0.05g of crystallized bovine serum albumin on an analytical balance into a beaker, add a small amount of distilled water to dissolve, and transfer to a 50mL volumetric flask. Rinse the residual liquid in the beaker with a small amount of distilled water for several times, pour the rinse liquid into the volumetric flask, and finally dilute to the scale with distilled water to complete the preparation of standard protein, in which the concentration of bovine serum albumin is 1g / L.

[0041] The method for drawing the standard curve is to take 6 test tubes respectively, number them, add reagents according to Table 3 and mix them evenly.

[0042] Table 3 Standard curve concentration drawing method

[0043] Tube number 1 2 3 4 5 6 Sample (mL) 0 0.4 0.8 1.2 1.6 2 Distilled water (mL) 2 1.6 1.2 0.8 0.4 0 Protein concentration (g / L) 0 0.2 0.4 0.6 0.8 1.0

[0044] The samples were subjected to SDS-PAGE protein electrophoresis and imaged using an optical density scanner. The SDS-PAGE images were analyzed using computer software Image J to obtain the absolute gray value of the standard protein to be tested. A standard curve was drawn with protein concentration as the horizontal axis and absolute gray value as the vertical axis.

[0045] The sample detection method is to use SDS-PAGE protein electrophoresis, use optical density scanner to image, analyze the SDS-PAGE image through computer software Image J, obtain the absolute gray value of the protein of the sample to be tested, and obtain the protein concentration based on the standard curve.

[0046] In this example, the methanol concentration (0.5% to 3%) added every 24 hours, the initial biotin concentration in the BMMY culture medium (0.02% to 0.12%), the initial pH of the BMMY culture medium (4 to 7), and the culture temperature (28 to 32° C.) were adjusted to measure the protein expression levels under different methanol concentrations, biotin concentrations, temperatures, and pH conditions. The methanol concentration, biotin concentration, temperature, and pH were adjusted and optimized in sequence to obtain the expression conditions of the protein below the optimal expression level.

[0047] Depend on Figure 2 Available, inducible promoter P AOX1 The optimal conditions for fermentation production of Bra are 1% v / v methanol concentration each time, 0.04% v / v biotin concentration in BMMY medium, 30°C optimal culture temperature, and 5.0 optimal pH of BMMY medium. Under the above optimal conditions, the expression level of sweet protein can reach 122 mg / L.

[0048] Example 2

[0049] The signal peptide of strain GS115-pPIC9K-Bra constructed in Example 1 was optimized, and eight signal peptides were used to replace the α-signal peptide of Saccharomyces cerevisiae (α-factor, such as Figure 1The primers ost1-proα-F / R, eSIG-F / R, Lysozyme-F / R, ost1-F / R, αΔ57-70-F / R, dan4-F / R, dddk-F / R, and msb2-F / R in Table 5 were used to amplify the gene sequences of the signal peptides ost1-proα, eSIG, Lysozyme, ost1, αΔ57-70, dan4, dddk, and msb2 (as shown in Table 4) using the synthesized signal peptide gene sequence or the Pichia pastoris gene sequence as a template. After being ligated to the linearized pPIC9K-Bra vector digested with AflⅡ and EcoRⅠ, the α signal peptide in the plasmid was replaced, and the plasmids pPIC9K-ost1-proα-Bra, pPIC9K-eSIG-Bra, pPIC9K-Lysozyme-Bra, pPIC9K-αΔ57-70-Bra, pPIC9K-ost1-Bra, pPIC9K-dan4-Bra, pPIC9K-dddk-Bra, and pPIC9K-msb2-Bra were constructed.

[0050] The signal peptides used in the above construction process and their corresponding sequences are shown in Table 4.

[0051] Table 4 Signal peptides and their corresponding sequences

[0052]

[0053]

[0054] The optimization of the signal peptide is signal peptide insertion, and the specific operation method is as follows: design primers to insert the signal peptide sequence into the AflⅡ and EcoRⅠ restriction sites of the Pichia pastoris methanol-inducible secretion plasmid pPIC9K-Bra to achieve the insertion of the signal peptide. The signal peptide primers and their corresponding sequences are shown in Table 5.

[0055] Table 5 Primer sequences used for signal peptide insertion

[0056] Primer name Sequence (5’→3’) ost1-proα-F gcttcactcaacaacaaaaCTTAAGaaacgatgaggcaggtttggt ost1-proα-R cacttttttgcatttgtccatGAATTCtacgtaagcttcagcctctct eSIG-F cgcttcactcaacaacaaaaCTTAAGaaacgatgagatccttgttgatt eSIG-R ttttttgcatttgtccatGAATTCtacgtaaactttacccaaagcagc Lysozyme-F cttcactcaacaacaaaaCTTAAGaaacgatgctcgggaagaacg Lysozyme-R cacttttttgcatttgtccatGAATTCtacgtagccctggcagatacc ost1-F cgcttcactcaacaacaaaaCTTAAGaaacgatgaggcaggtttggt ost1-R cacttttttgcatttgtccatGAATTCtacgtaagcagaagacacgtt αΔ57-70-F cgcttcactcaacaacaaaaCTTAAGaaacgatgagatttccttcaatttttactgc αΔ57-70-R ttcttacacttgtccatGAATTCtacgtaagcttcagcctctcttttctc dan4-F cttcactcaacaacaaaaCTTAAGaaacgatgttcctcaaaagtctcctt dan4-R cttcttacacttgtccatGAATTCtacgtactttggtgcatcttgtacat dddk-F cttcactcaacaacaaaaCTTAAGaaacgatgttcaacctgaaaactatt dddk-R cttcttacacttgtccatGAATTCtacgtaggcaacagcgatcgatgcaa msb2-F cttcactcaacaacaaaaCTTAAGaaacgatgattaatttaaactccttt msb2-R cttcttacacttgtccatGAATTCtacgtatcttttagcaaggtcgtctt

[0057] The strains after replacing different signal peptides were fermented in shake flasks according to the fermentation conditions optimized in Example 1. The protein expression amount was determined by referring to Example 1. The protein expression conditions were the conditions optimized in Example 1, i.e., 1% v / v methanol concentration, 0.04% v / v biotin concentration, 30°C, pH 5.0. The Bra expression levels of the strains after replacing different signal peptides are shown in Figure 1. Figure 3 As shown. Figure 3It can be seen that the protein expression level is the highest after the eSIG signal peptide is inserted, and the secretory expression level of Bra can be increased to 246.4 mg / L, which is 2.02 times that of the starting strain GS115-pPIC9K-Bra.

[0058] It can be further concluded that the signal peptide designed in this example can promote the expression level of the protein to a certain extent, that is, it achieves a certain degree of promotion effect on protein expression.

[0059] Example 3

[0060] The strain optimized with the signal peptide in Example 2 was subjected to optimization of gene expression regulatory elements, and genes of three gene expression regulatory elements, PDI, Hac1 and BiP, were added to the genome of the strain respectively to achieve overexpression of gene expression regulatory elements by the strain. The sequences corresponding to the three gene expression regulatory elements, PDI, Hac1 and BiP, are shown in SEQ ID Nos. 11 to 13.

[0061] The three gene expression regulatory elements were amplified by PCR using the primers PDI-F / R, Hac1-F / R, and BiP-F / R described in Table 6 to obtain PDI, Hac1, and BiP gene fragments, respectively. The specific method for inserting the gene expression regulatory elements is as follows: using the Pichia pastoris GS115 genome as a template to amplify the gene expression regulatory element gene fragment, and then ligating the gene expression regulatory element gene fragment to the commercial vector pPICZA after restriction endonucleases EcoRⅠ and NotⅠdigestion to construct the corresponding plasmids pPICZA-PDI, pPICZA-Hac1, and pPICZA-BiP. The plasmid maps are shown in FIG. Figure 4 As shown, the gene expression regulatory element primers and their corresponding sequences are shown in Table 6.

[0062] Table 6 Primer sequences used for protein expression regulatory element insertion

[0063] Primer name Sequence (5’→3’) PDI-F acaactaattattcgaaGAATTCatgcaattcaactggaatatt PDI-R gacgctcacgacgagctttaaCTGCAGcatcatcatcatcatcattg Hac1-F acaactaattattcgaaGAATTCatgcccgtagattcttctcat Hac1-R caatgatgatgatgatgatgCTGCAGtcacctgatcgctatgcatgt BiP-F acaactaattattcgaaGAATTCatgctgtcgttaaaaccatct BiP-R caatgatgatgatgatgatgCTGCAGctacaactcatcatgatcata

[0064] Then, the constructed plasmid was linearized by restriction endonuclease NheⅠ, and the purified linear plasmid was mixed evenly with the competent state of Pichia pastoris GS115-pPIC9K-eSIG-Bra constructed in Example 2 in an electroporation cup, and allowed to stand on ice for 30 min. The linear plasmid was inserted into the Pichia pastoris genome by electroporation (1500 V, 200 Ω), 1 mL of pre-cooled 1 mol / L sorbitol was quickly added, and pre-cultured at 30 ° C for 3 h, and then spread on a YPD plate containing 100 μg / mL bleomycin and grown for 3 days. The obtained strains (GS115-pPIC9K-eSIG-Bra / pPICZA-PDI, GS115-pPIC9K-eSIG-Bra / pPICZA-Hac1, GS115-pPIC9K-eSIG-Bra / pPICZA-BiP) were fermented in shake flasks according to the optimal methanol concentration, biotin concentration, temperature and pH measured in Example 1. The expression level after the signal peptide was inserted was measured. The carbon source was methanol. The protein expression level was measured after 120 hours of culture. The results were as follows: Figure 5 As shown. Figure 5 It can be seen that the strain after signal peptide optimization contains the gene of the inserted gene expression regulatory element. At the same time, different gene expression regulatory elements have different degrees of effect on the production of target proteins. Among them, PDI improves the secretory expression of Bra. Ultimately, the secretory expression level of Bra can be increased to 305 mg / L at the shake flask level, which is 1.24 times that of the starting strain GS115-pPIC9K-eSIG-Bra (CK).

[0065] Example 4

[0066] The strain GS115-pPIC9K-eSIG-Bra / pPICZA-PDI constructed in Example 3 was used to detect the expression of Bra: a single colony with good growth condition was selected and inoculated into 500mL YPD medium and cultured at 30°C for 24h as the primary seed liquid, and the primary seed liquid was transferred to BMGY medium and cultured at 30°C for 24h as the secondary seed liquid. The secondary seed liquid was inoculated into a 50L fermenter at a 10% v / v inoculation amount. The culture medium in the fermenter was BMGY medium with an initial biotin content of 0.04% v / v. The remaining raw materials were the same as those shown in Table 1 of Example 1. The fermentation process was as follows: Figure 6 As shown in a.

[0067] The initial liquid volume of the fermentation process is 25L, the temperature is controlled at 30°C, pH 5.0, and the ventilation ratio is 1.0vvm. 50% ammonia water is used to adjust the pH of the fermentation liquid. During the fermentation process, the stirring is gradually increased from 200rpm to 850rpm to keep the dissolved oxygen (DO) in the fermenter above 20%. If necessary, an appropriate amount of defoaming agent is added to control the fermentation foam. The fermentation process of methanol-type Pichia pastoris includes a batch fermentation stage, a fed-batch fermentation stage, and a methanol induction stage. In the batch fermentation stage, the bacteria consume the carbon source in the initial culture medium. When the DO in the fermenter rises, proving that the carbon source in the initial culture medium is exhausted, the fed-batch fermentation stage is entered, and a 50% v / v glycerol aqueous solution is added at a rate of 150mL / h. When the bacteria concentration in the culture medium reaches the range of the yeast logarithmic growth phase (OD 600 =100 or 200), stop the addition of glycerol, starve the cells for 4 h, at which time the DO in the fermenter is maintained above 80%, and finally enter the methanol induction stage, adjust the biotin concentration, pH and temperature of the fermentation broth to the optimal fermentation conditions determined in Example 1, continue to add methanol to the culture broth at a rate of 10 mL / h, and take samples every 6 h to detect the expression of Bra.

[0068] The biotin concentration, pH and temperature optimized in the shake flask were used to produce Bra in a 50L fermenter. During the growth period of the strain, the OD of the bacteria in the fermenter was adjusted to 600 =100 or 200, methanol induced the secretion expression of Bra. The secretion expression of Bra protein was detected by SDS-PAGE, and its expression level was determined. The results are as follows Figure 6 As shown in the figure, the secretory expression of Bra in the fermenter was different from that in the shake flask, and the protein expression level reached the highest level 96h after methanol induction. 600 =100, 200, methanol was used to induce Bra expression, and the highest protein expression levels were 3.913 g / L and 2.618 g / L, respectively. Figure 6 b. Therefore, when the OD of the bacteria in the fermentation broth is 600 When the concentration reached 100, the Bra expression level induced by methanol addition was the highest, reaching 3.913 g / L.

[0069] In OD 600 On the basis of achieving optimization, the pH of the methanol induction period was optimized. During the methanol induction period, the pH values ​​in the fermentation tank were set to 4, 5, 6, and 7 respectively to induce the secretion expression of Bra. The secretion expression of Bra protein was detected by SDS-PAGE, and its expression level was determined. The results are as follows Figure 6As shown in c, when the pH value in the fermentation tank was 4, 5, 6, and 7, methanol was used to induce the expression of Bra, and the highest protein expression levels were 3.08 g / L, 3.89 g / L, 4.59 g / L, and 2.86 g / L, respectively. Among them, when the pH value in the fermentation broth reached 6.0 during the methanol induction period, the Bra expression level induced by methanol addition was the highest, reaching 4.59 g / L. The specific fermentation process yield is shown in the figure Figure 6 As shown in d.

[0070] Depend on Figure 6 It can be seen that during the fermentation process, the increase in the secretion expression of Bra with time is not a linear relationship. This may be because in the early stage of methanol induction, the DO in the fermentation broth can be controlled above 20%, but in the middle and late stages of induction, methanol accumulates in the fermentation broth and some toxic by-products are produced, which affects the secretion expression of Bra.

[0071] The present invention provides a method and idea for improving the secretion and expression efficiency of sweet proteins in Pichia pastoris. There are many methods and approaches to implement the technical solution. The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the protection scope of the present invention. All components not specified in this embodiment can be implemented by existing technologies.

Claims

1. A Pichia pastoris that secretes and expresses a sweet protein, characterized in that: The Pichia yeast simultaneously expresses a sweet protein gene, a secretion signal peptide gene and a gene expression regulatory element; Wherein, the nucleotide sequence of the sweet protein gene is shown as SEQ ID No.2; The nucleotide sequence of the secretory signal peptide gene is selected from any one of SEQ ID No. 3 to 10; The nucleotide sequence of the gene expression regulatory element is selected from any one of SEQ ID No.11-13.

2. The Pichia pastoris according to claim 1, characterized in that The nucleotide sequence of the secretion signal peptide gene is selected from any one of SEQ ID No.3-6; the nucleotide sequence of the gene expression regulatory element is shown in SEQ ID No.

11.

3. The Pichia pastoris according to claim 1, characterized in that The starting bacteria is Pichia pastoris GS115.

4. The Pichia pastoris according to claim 1, characterized in that The sweet protein gene is expressed by an inducible promoter; the inducible promoter includes P AOX1 .

5. The method for constructing Pichia pastoris that secretes and expresses sweet protein according to any one of claims 1 to 4, characterized in that: The method comprises the following steps: amplifying a sweet protein gene and a secretion signal peptide gene, cloning them into a first starting vector to obtain a first recombinant vector; amplifying a gene expression regulatory element, cloning them into a second starting vector to obtain a second recombinant vector; The first recombinant vector and the second recombinant vector are introduced into a Pichia pastoris starter strain to obtain the Pichia pastoris that secretes and expresses the sweet protein.

6. The construction method according to claim 5, characterized in that: The first starting vector is pPIC9K; the second starting vector is pPICZA.

7. Use of the Pichia pastoris secretory expression of sweet protein according to any one of claims 1 to 4 in the production of sweet protein.

8. A method for improving the secretion expression efficiency of sweet protein in Pichia pastoris, characterized in that: The two-stage fermentation culture using the Pichia yeast expressing the sweet protein secreted by any one of claims 1 to 4 comprises the following steps: (1) Strain growth stage: inoculating the Pichia pastoris secreting and expressing the sweet protein into the growth medium and culturing at 28-32° C. until the logarithmic growth phase; (2) Methanol induction stage: Methanol was added to the bacterial solution obtained in step (1) for induction, and the culture was continued at 28-32°C for 96-144 hours.

9. The method according to claim 8, characterized in that In step (1), the growth medium is a buffered complete medium BMGY containing glycerol; the fermentation pH during the strain growth stage is 5-6.

10. The method according to claim 8, characterized in that In step (2), during the methanol induction stage, the volume of methanol added is 0.5% to 12% of the volume of the fermentation liquid; and the fermentation pH during the methanol induction stage is 5 to 6.

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