Method for inducing pichia pastoris to express human alpha-lactalbumin by using ethanol

Abstract

The invention relates to a method for inducing pichia pastoris to express human alpha-lactalbumin by using ethanol, and belongs to the technical field of genetic engineering. Recombinant pichia pastoris using ethanol as an inducer for heterologous expression of human alpha-lactalbumin is constructed, three different expression vectors are used in the construction process, the first expression vector uses an ethanol inducible promoter to express fusion polypeptide of DNA binding protein and a transcription activator, and the second expression vector uses an ethanol inducible promoter to express fusion polypeptide of human alpha-lactalbumin. The second expression vector sequentially expresses a protein binding sequence, a methanol inducible promoter, an alpha signal peptide and human alpha-lactalbumin, the third expression vector overexpresses a signal recognition particle subunit participating in a protein targeting endoplasmic reticulum, and the yield of the constructed recombinant pichia pastoris shake flask fermentation human alpha-lactalbumin is 23.3 mg / L; the yield of the human alpha-lactalbumin reaches 1.1 g / L in a 3L fermentation tank, and methanol is not required to be added as an inducer in the fermentation process, so that the problems of toxicity and safety possibly caused by methanol are avoided, and efficient production of the human alpha-lactalbumin is realized.

Description

Technical Field

[0001] The invention relates to the technical field of genetic engineering, in particular to a method for inducing Pichia pastoris to express human alpha-lactalbumin by using ethanol. Background Art

[0002] The Pichia pastoris (also known as Komagataella phaffii) expression system is one of the most commonly used expression systems in the industrial and pharmaceutical fields and is widely used for the expression of foreign proteins. As a unicellular eukaryotic organism, Pichia pastoris has many advantages: (1) Compared with prokaryotic expression systems, Pichia pastoris is more conducive to protein folding, processing and post-translational modification; (2) Compared with eukaryotic expression systems such as baculovirus and mammalian tissue culture, Pichia pastoris is faster, simpler and cheaper, and has a higher level of foreign protein expression; (3) The promoter of the alcohol oxidase gene of Pichia pastoris has strong inducibility and strong activation, which is suitable for high-level induced expression of foreign genes; (4) Pichia pastoris has a preference for aerobic growth, which is suitable for high-density cell culture and is conducive to large-scale industrial production; (5) The amount of endogenous protein secreted by Pichia pastoris is extremely small, and the secreted foreign protein can account for up to 90% of the total secreted protein, which is conducive to subsequent purification.

[0003] Methanol is the most commonly used inducer in the Pichia pastoris expression system. The methanol-induced alcohol oxidase promoter (AOX1) can express alcohol oxidase in large quantities, thereby driving the expression of exogenous proteins. However, the use of methanol also has some disadvantages. When methanol is used as the sole carbon source for Pichia pastoris, although it can induce the expression of exogenous proteins, it may also produce certain pressure on yeast cells, thereby affecting cell growth and protein expression efficiency. The concentration, timing and method of addition of methanol will affect the expression level of exogenous proteins, so these parameters need to be carefully controlled, which increases the complexity and cost of the operation. In addition, methanol is a toxic substance, and excessive intake may lead to serious consequences such as blindness. Therefore, when using the Pichia pastoris expression system to produce oral drugs or food proteins, the use of methanol as an inducer is often not accepted by people.

[0004] α-lactalbumin (α-La) accounts for about 22% of the protein in human milk and 3.5% of the protein in bovine milk. It has multiple functions such as regulating milk production, cytolytic activity, inducing cell growth inhibition and cell apoptosis. α-La helps regulate cytolytic activity, induce cell growth and inhibit cell apoptosis, as well as relieve stress, improve sleep and improve memory. All these functions of improving mood, sleep and memory depend on the higher tryptophan content in α-La. Since human α-La is one of the main protein components in human milk, and its amino acid similarity with bovine α-La is only 74%, using human α-La in infant formula and increasing the dosage can make infant formula on the market closer to human milk, thus narrowing the gap between formula feeding and breastfeeding.

[0005] Although the expression of human α-La in Pichia pastoris has been achieved, it still faces the problems of low secretion yield and the toxicity of methanol as an inducer. Summary of the invention

[0006] In order to solve the above technical problems, the present invention provides a method for inducing Pichia pastoris to express human α-lactalbumin using ethanol. The present invention constructs a recombinant Pichia pastoris that uses ethanol as an inducer to heterologously express human α-lactalbumin. Three different expression vectors are used in the construction process, wherein the first expression vector uses an ethanol-inducible promoter to express a fusion polypeptide of a DNA binding protein and a transcription activator, the second expression vector sequentially expresses a protein binding sequence, a methanol-inducible promoter, an α signal peptide and human α-lactalbumin, and the third expression vector overexpresses a signal recognition particle subunit involved in protein targeting to the endoplasmic reticulum. The constructed recombinant Pichia pastoris can be used to produce human α-lactalbumin by methanol-free fermentation.

[0007] The first object of the present invention is to provide a recombinant Pichia pastoris expressing human α-lactalbumin, wherein the recombinant Pichia pastoris contains a first expression cassette, a second expression cassette and a third expression cassette, wherein:

[0008] The first expression cassette uses an ethanol-inducible promoter to express a fusion polypeptide of a DNA binding protein and a transcription activator;

[0009] The second expression cassette sequentially expresses a protein binding sequence, a methanol-inducible promoter, an α signal peptide, and human α-lactalbumin, wherein the protein binding sequence is LacO;

[0010] The third expression cassette overexpresses a signal recognition particle subunit, which is involved in targeting proteins to the endoplasmic reticulum.

[0011] Furthermore, the recombinant Pichia pastoris uses Pichia pastoris GS115 as a chassis strain.

[0012] Furthermore, in the first expression cassette, the ethanol-inducible promoter is ICL1, the DNA binding protein is LacI, and the transcription activator is Mit1AD.

[0013] Furthermore, in the second expression cassette, the copy number of the protein binding sequence is 5.

[0014] Furthermore, in the second expression cassette, the pre region of the α signal peptide is replaced with a serum albumin signal peptide, a killer protein signal peptide, an Ost1 signal peptide or an Epx1 signal peptide.

[0015] Preferably, in the second expression cassette, the pre region of the α signal peptide is replaced with a serum albumin signal peptide

[0016] Furthermore, the copy number of the second expression cassette in the recombinant Pichia pastoris is 1-5.

[0017] Preferably, the copy number of the second expression cassette in the recombinant Pichia pastoris is 3.

[0018] The second object of the present invention is to provide an expression vector combination for constructing the above-mentioned recombinant Pichia pastoris, wherein the expression vector combination comprises a first expression vector, a second expression vector and a third expression vector, wherein:

[0019] The first expression vector uses an ethanol-inducible promoter to express a fusion polypeptide of a DNA binding protein and a transcription activator;

[0020] The second expression vector sequentially expresses a protein binding sequence, a methanol-inducible promoter, an α signal peptide, and human α-lactalbumin, wherein the protein binding sequence is LacO;

[0021] The third expression vector overexpresses a signal recognition particle subunit, and the signal recognition particle subunit is involved in protein targeting to the endoplasmic reticulum.

[0022] The third object of the present invention is to provide a method for inducing Pichia pastoris to express human α-lactalbumin using ethanol, comprising the following steps:

[0023] Step S1, constructing the above-mentioned recombinant Pichia pastoris to obtain a seed solution;

[0024] Step S2, adding fermentation basal salt culture medium and PTM1 trace element solution into the fermentation tank, inoculating the seed solution into the fermentation tank, and keeping the dissolved oxygen above 20%;

[0025] Step S3, feeding with glycerol after dissolved oxygen rebounds, and then inducing with ethanol solution;

[0026] Step S4, alternately performing ethanol feeding and glycerol feeding until the fermentation is completed.

[0027] Furthermore, PTM1 trace element solution is added to both the glycerol and the ethanol.

[0028] Preferably, the amount of PTM1 trace element solution added to glycerol and ethanol is 12 mL / L.

[0029] Furthermore, during step S4, the concentration of acetic acid in the fermentation broth is monitored, and when acetic acid accumulates, ethanol feeding is stopped and glycerol feeding is performed.

[0030] Beneficial effects of the present invention:

[0031] The Pichia yeast strain constructed by the present invention uses ethanol as an inducer to secrete and express secretory human α-La, and the yield in a shake flask fermentation is 23.3 mg / L, and the yield in a 3L fermenter reaches 1.1 g / L, and the fermentation process does not need to add methanol as an inducer, avoiding the toxicity and safety problems that may be caused by methanol, and realizing the efficient production of human α-lactalbumin. It lays a theoretical foundation for the large-scale methanol-free production of human α-La, promotes the development of methanol-free production technology of human α-lactalbumin in Pichia yeast, and provides new progress for infant formula milk powder simulating breast milk and the development of bioengineered milk. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] In order to make the content of the present invention more clearly understood, the present invention is further described in detail below according to specific embodiments of the present invention in conjunction with the accompanying drawings, wherein

[0033] Figure 1 This is a graph of the SDS-PAGE verification results in Example 1 of the present invention, wherein lane 1 is a marker, lane 2 is a shake flask fermentation product of the wild-type strain X33, lane 3 is a shake flask fermentation product of the recombinant strain X33-LA, and lane 4 is a shake flask fermentation product of the recombinant strain GS115-ESAD-LA;

[0034] Figure 2 This is a diagram showing the quantitative expression results of α-LA in the strain in Example 1 of the present invention;

[0035] Figure 3 is a diagram showing the growth status of the strain expressing α-LA in Example 1 of the present invention;

[0036] Figure 4: This is a SDS-PAGE verification result diagram of the hybrid signal peptide screening experiment in Example 2 of the present invention, wherein lane 1 in A is a marker, lane 2 is a shake flask fermentation product of the X33 strain, lane 3 is a shake flask fermentation product of the ESAD-LA strain, and lanes 3-10 are shake flask fermentation products of ESAD-2Pro-LA to ESAD-8Pro-LA strains, respectively; lane 1 in B is a marker, lane 2 is a shake flask fermentation product of the X33 strain, lane 3 is a fermentation product of the ESAD-LA strain, and lanes 3-10 are shake flask fermentation products of ESAD-9Pro-LA to ESAD-15Pro-LA strains, respectively;

[0037] Figure 5 are the quantitative comparison results of the hybrid signal peptide screening experiment in Example 2 of the present invention, which are the changes in expression levels of the recombinant strains ESAD-2Pro-LA to ESAD-15Pro-LA compared with ESAD-LA;

[0038] Figure 6 This is the SDS-PAGE result of the purified products of the SRP subunit and receptor function verification experiment in Example 3 of the present invention, wherein lane 1 is the marker, lane 2 is the shake flask fermentation product of the X33 strain, lane 3 is the shake flask fermentation product of the ESAD-LA strain, and lanes 4-9 are shake flask fermentation products of the ESAD-4Pro-LA-SRP1 to ESAD-4Pro-LA-SRP6 strains, respectively;

[0039] Figure 7 is a quantitative result diagram of the SRP subunit and receptor function verification experiment in Example 3 of the present invention;

[0040] Figure 8 This is a result diagram showing the effects of SRP subunits and receptors on the growth of recombinant strains in Example 3 of the present invention;

[0041] Fig. 9 This is a quantitative result diagram of the multi-copy recombinant strain function verification experiment in Example 4 of the present invention;

[0042] Fig.10 This is a graph showing the growth status of the multi-copy recombinant strain in Example 4 of the present invention. DETAILED DESCRIPTION

[0043] The present invention is further described below in conjunction with the accompanying drawings and specific embodiments so that those skilled in the art can better understand the present invention and implement it, but the embodiments are not intended to limit the present invention.

[0044] The cultivation involved in the embodiment is as follows:

[0045] YPD medium: 1% yeast extract, 2% peptone, 2% glucose;

[0046] Methanol fermentation medium (BMMY): yeast extract 10g / L, peptone 20g / L, K2HPO4·3H2O 3g / L, KH2PO4 11.8g / L, ultrapure water 895mL, sterilize at 121℃ for 20min, after the temperature drops to 60℃, add 100mL 10*YNB, 1mL 500*B (0.02% biotin), and 5mL methanol in the clean bench;

[0047] Ethanol fermentation medium (BMEY): yeast extract 10g / L, peptone 20g / L, K2HPO4·3H2O 3g / L, KH2PO4 11.8g / L, ultrapure water 895mL, sterilize at 121℃ for 20min, after the temperature drops to 60℃, add 100mL 10*YNB, 1mL 500*B (0.02% biotin), 5mL ethanol in the clean bench

[0048] PTM1 trace elements (PTM1 Trace Salts) solution: 65.0g / L FeSO4·7H2O, 220.0g / L ZnCl, 6.0g / L CuSO4·5H2O, 3.0g / L MnSO4·H2O, 20.5g / L CoCl, 0.2g / L MoNa2O4·2H2O, 0.2g / L D-biotin, 0.09g / L KI, 0.02g / L H3BO3, 5.0mL concentrated H2SO4.

[0049] Basal salts medium (BSM): 40.0 g / L glycerol, 18 g / L K2SO4, 14.9 g / LMgSO4·7H2O, 4.13 g / L KOH, 0.93 g / L CaSO4, 27 mL phosphoric acid, 4.4 mL PTM1.

[0050] Glycerol feed medium: Prepare 70% (w / v) glycerol solution with deionized water, sterilize it and add 12 mL / L of filter-sterilized PTM1 solution.

[0051] The shake flask fermentation process is:

[0052] (1) Pick a single colony and add it to 5 mL of primary seed liquid medium (YPD medium) and culture it at 30°C and 220 rpm for 16-18 hours;

[0053] (2) 100 μL of the first-level seed solution was transferred to 50 mL of the second-level seed solution medium (YPD) and fermented at 30°C and 220 rpm for 24 h;

[0054] (3) All the cells were collected by centrifugation, transferred to 50 mL of induction expression medium (BMMY / BMEY), and fermented at 28°C and 220 rpm for 72 h.

[0055] Sample detection method: After the fermentation liquid is centrifuged, the supernatant is taken and filtered through a 0.45 μm water filter membrane, purified using a His-Binding-resin purification column, and the purified sample is collected for SDS-PAGE electrophoresis. Protein quantification uses a BradFord kit.

[0056] Example 1: Comparison of α-La expression levels of Pichia pastoris recombinant strains induced by methanol and ethanol

[0057] Construction of a recombinant Pichia pastoris strain that produces α-La induced by methanol: The codon-modified α-La gene LALBA (nucleotide sequence as shown in SEQ ID NO.1) was synthesized after the α-factor secretion signal peptide (nucleotide sequence as shown in SEQ ID NO.2) of the plasmid pPICZαA, the correctly sequenced recombinant plasmid was extracted, and then 5-10 μg of the plasmid was linearized using the restriction endonuclease Sec I, and the linearized recombinant plasmid was integrated into the genome of the Pichia pastoris wild-type strain X33 by electroporation, and the constructed recombinant strain was named X33-LA.

[0058] Construction of a recombinant strain of Pichia pastoris that produces α-La under ethanol induction:

[0059] (1) The endogenous ethanol-inducible promoter ICL1 was amplified from the Pichia pastoris genome, the AOX1 promoter in the plasmid vector pPICZB was replaced with the promoter ICL1 (the nucleotide sequence is shown in SEQ ID NO.3), and the gene sequence of the LacI-Mit1AD fusion protein was inserted into the multiple cloning site, wherein the gene encoding the LacI protein was codon-optimized, and the nucleotide sequence was shown in SEQ ID NO.4, and the nucleotide sequence of the gene encoding the Mit1AD protein was shown in SEQ ID NO.5, to obtain the recombinant plasmid pPICZB-ICL1-LacI-Mit1AD;

[0060] (2) Plasmid pPIC3.5K was used as the vector backbone, the core sequence core AOX1 (nucleotide sequence as shown in SEQ ID NO.6) of the AOX1 promoter in the vector backbone was retained, and the rest was replaced with 5 LacO repeat sequences (nucleotide sequence as shown in SEQ ID NO.7), and the α-factor secretion signal peptide α-factor secretion signal peptide (nucleotide sequence as shown in SEQ ID NO.2), the codon-modified α-La gene LALBA (nucleotide sequence as shown in SEQ ID NO.1) and the histidine tag 6-His were inserted after core AOX1 in sequence to obtain the recombinant plasmid pPIC3.5K-5LacO-coreAOX1-LALBA-6His;

[0061] (3) The recombinant plasmids pPICZB-ICL1-LacI-MitAD and pPIC3.5K-5LacO-coreAOX1-LALBA-6His were linearized using restriction endonucleases EcoR I and Sal I, respectively. The linearized recombinant plasmids were integrated into the histidine-deficient Pichia pastoris strain GS115 using electroporation, and the constructed recombinant strain was named GS115-ESAD-LA.

[0062] The single-copy recombinant strains X33-LA and GS115-ESAD-LA were fermented and verified. The unmodified wild-type X33 strain was used as a control. The corresponding α-La samples were obtained using the above-mentioned shake flask fermentation method and verified using SDS-PAGE, and the corresponding bands of α-La were detected.

[0063] SDS-PAGE verification results are as follows Figure 1 As shown in Figure 2, α-lactalbumin was successfully expressed in the recombinant strains X33-LA and GS115-ESAD-LA. The expression levels of α-lactalbumin in different strains are shown in Figure 2. Figure 2 As shown in Figure 2, compared with X33-LA, GS115-ESAD-LA secreted more than twice the amount of α-lactalbumin. Figure 3 As shown in the figure, after 72 hours of fermentation induction, the growth state of GS115-ESAD-LA was better than that of X33 and X33-LA, and the OD 600 Reach 60 or above.

[0064] Example 2: Comparison of α-La expression levels in ethanol-induced Pichia pastoris recombinant strains using 14 different hybrid signal peptides

[0065] The pre region of the α factor secretion signal peptide in the above-constructed plasmid pPIC3.5K-5LacO-coreAOX1-LALBA-6His was replaced with 14 different signal peptides to obtain 14 hybrid signal peptides, and then Escherichia coli was transformed to extract the correctly sequenced recombinant plasmid, and 5-10 μg of the plasmid was linearized using restriction endonuclease Sal I. The linearized recombinant plasmid was integrated into the Pichia pastoris strain GS115 into which the plasmid PICZ_B-ICL1-LacI-MitAD had been integrated by electroporation to obtain 14 recombinant strains with different hybrid signal peptides. The 14 hybrid signal peptides and the corresponding recombinant strains are shown in the following table.

[0066] Table 1 Hybrid signal peptides and corresponding recombinant strains

[0067]

[0068]

[0069] The recombinant strain constructed above was verified by shake flask fermentation and SDS-PAGE, and the corresponding band of α-La was detected. The corresponding α-La samples were quantified, and the quantitative results were compared with the secretion amount of ESAD-LA to explore the changes in expression levels.

[0070] SDS-PAGE verification results are as follows Figure 4 As shown in the figure, α-lactalbumin was almost not expressed in the recombinant strains ESAD-3Pro-LA, ESAD-5Pro-LA and ESAD-15Pro-LA. Compared with ESAD-LA, the secretion expression of α-lactalbumin in the recombinant strain ESAD-4Pro-LA was significantly increased. The quantitative comparison results are shown in Figure 5 As shown, it can also be seen that the expression levels of the recombinant strains ESAD-3Pro-LA, ESAD-5Pro-LA and ESAD-15Pro-LA decreased, while the expression level of the recombinant strain ESAD-4Pro-LA increased.

[0071] Example 3: Comparison of α-La expression levels in ethanol-induced Pichia pastoris recombinant strains overexpressing six different SRP subunits and receptors

[0072] Construction of a recombinant Pichia pastoris strain for ethanol-induced α-La production corresponding to 6 different SRP subunits and receptors: The resistance gene Zeocin in the pGAPZα plasmid was replaced with the hygromycin resistance gene Hyg to obtain the pGAPH plasmid (sequence as shown in SEQ ID NO.22), and 6 different SRP subunits and receptors were respectively inserted behind the promoter of the plasmid backbone pGAPH, followed by transformation of Escherichia coli, from which the correctly sequenced recombinant plasmids were extracted, 5-10 μg of the plasmid was linearized using restriction endonuclease Avr II, and the linearized recombinant plasmid was integrated into the Pichia pastoris strain GS115-ESAD-4Pro-LA by electroporation to obtain a recombinant strain with 6 different SRP subunits and receptors, as shown in the table below.

[0073] Table 2 SRP subunits and receptors and corresponding recombinant strains

[0074]

[0075]

[0076] The obtained single copy strain was verified by shake flask fermentation and SDS-PAGE, the corresponding band of α-La was detected, the obtained α-La sample was quantified, and the growth status of the recombinant strain was detected.

[0077] SDS-PAGE verification results are as follows Figure 6 As shown in Figure 2, the addition of SRP subunits and receptors did not affect the production of α-lactalbumin in the recombinant bacteria. Figure 7 As shown in Figure 2, after the introduction of SRP2 (SRP subunit involved in protein targeting to the endoplasmic reticulum), the α-LA production of the recombinant strain ESAD-4Pro-LA-SPR2 was improved. Figure 8 As shown, the OD of the recombinant strain ESAD-4Pro-LA-SPR2 600 It was slightly lower than that of the unmodified wild strain X33 and the recombinant strain ESAD-LA without the introduction of SRP subunits and receptors, but the impact was not significant.

[0078] Example 4: Comparison of α-La expression levels in recombinant Pichia pastoris strains with different copy numbers

[0079] Construction of recombinant strains of Pichia pastoris with different copy numbers: The plasmid pPICZB-ICL1-LacI-Mit1AD and the plasmid pGAPH-SRP2 were linearized by restriction enzymes and then integrated into the starting strain GS115. Subsequently, a high concentration of linearized plasmid (concentration not less than 500ng / μL) pPIC3.5K-5LacO-coreAOX1-4Pro-LALBA-6His was used to transform Pichia pastoris competent cells to obtain a large number of transformants. High-copy transformants were screened using a 4mg / mL high-concentration geneticin plate medium, and RT-qPCR was performed to verify the transformants that were more likely to be high-copy, thereby determining the transformants with different copy numbers.

[0080] The obtained multi-copy strain was verified by shake flask fermentation and SDS-PAGE, and the corresponding band of α-La was detected. The obtained α-La sample was quantified, and the growth status of the recombinant strain was detected.

[0081] Quantitative results such as Fig. 9 As shown in Figure 2, when the copy number was 3.2, the α-LA production level of the recombinant strain ESAD-4Pro-LA-SPR2 was the highest, reaching 23.3 mg / L. Fig.10 As shown, the different copy numbers of the pPIC3.5K-5LacO-coreAOX1-4Pro-LALBA-6His plasmid did not have a significant effect on the growth of the recombinant strain.

[0082] Example 5: Fermentation tank scale-up fermentation process

[0083] (1) The recombinant ESAD-4Pro-LA-SRP2 strain with a plasmid copy number of 3.2 obtained in Example 4 was streaked on a YPD solid medium and incubated in a 30° C. constant temperature incubator for about 3 days until a clearly visible single colony was formed;

[0084] (2) Select a single colony and transfer it to a 250 mL shake flask containing 30 mL YPD liquid medium. Then, culture it in a constant temperature shaking incubator at 30°C and 220 rpm / min for 16-24 h until its OD 600 The value reaches 20;

[0085] (3) Place the fermentation basal salt medium containing 4% glycerol into a 3L fermentor, then disinfect the fermentor, sterilize and cool, set the temperature to 30°C, stir and aerate to operating conditions (1200rpm and 1.0vvm), and adjust the pH of the fermentation basal salt medium to 5.0 with ammonia water. Aseptically add 4.4mL / L PTM1 trace salt to the fermentation basal salt medium. Then inoculate the seed liquid in the aforementioned shake flask into the fermentor at a ratio of 5-10% of the initial fermentation volume, ensuring that the dissolved oxygen is maintained above 20% (1.5vvm) by adding oxygen as needed;

[0086] (4) After the dissolved oxygen rebounds, start a 50% w / v glycerol feed containing 12 mL of PTM1 trace elements per liter of glycerol feed. Set the feed rate to 18.15 mL / L initial fermentation volume for about four hours or more of glycerol feeding;

[0087] (5) Stop adding glycerol and induce with 12 mL of pure ethanol solution containing PTM1 per liter, setting the feed rate to 3.6 mL / h per liter of original fermentation volume. During the first 2-3 h, ethanol will accumulate in the fermenter, and the dissolved oxygen value will be unstable as the culture adapts to ethanol;

[0088] (6) After maintaining a low ethanol feed rate for at least 1 hour, the feed rate was increased and doubled to 7.3 mL / (L·h) of initial fermentation volume to further increase the rate of product formation. The feed rate was 7.3 mL / (L·h) for the first 2 hours, then the ethanol feed rate was increased to approximately 10.9 mL / (L·h) per liter of initial fermentation volume and maintained constant for the remainder of the fermentation.

[0089] (7) About 20-30 hours after induction, acetic acid begins to accumulate. In order to consume the large amount of acetic acid generated, the ethanol feed is suspended and glycerol is fed at a volume of 18.15 mL / L of fermentation broth. After 4-8 hours, the glycerol feed is stopped and the ethanol feed is restarted;

[0090] (8) During the fermentation process, samples are taken every 4-6 hours to monitor the acetic acid in the fermentation broth. Once a large amount of acetic acid is found to have accumulated, the operation of step 7 is repeated;

[0091] (9) The entire ethanol fed-batch phase lasts about 70 h, and the cell wet weight can increase to 450-500 g / L. The final fermentation volume will be approximately twice the initial fermentation volume. The final yield of α-lactalbumin is 1.1 g / L.

[0092] Obviously, the above embodiments are merely examples for clear explanation and are not intended to limit the implementation methods. For those skilled in the art, other different forms of changes or modifications can be made based on the above description. It is not necessary and impossible to list all the implementation methods here. The obvious changes or modifications derived from these are still within the protection scope of the invention.

Claims

1. A recombinant Pichia pastoris expressing human α-lactalbumin, characterized in that: The recombinant Pichia pastoris contains a first expression cassette, a second expression cassette and a third expression cassette, wherein: The first expression cassette uses an ethanol-inducible promoter to express a fusion polypeptide of a DNA binding protein and a transcription activator; The second expression cassette sequentially expresses a protein binding sequence, a methanol-inducible promoter, an α signal peptide, and human α-lactalbumin, wherein the protein binding sequence is LacO; The third expression cassette overexpresses a signal recognition particle subunit, which is involved in targeting proteins to the endoplasmic reticulum.

2. The recombinant Pichia pastoris according to claim 1, characterized in that: The recombinant Pichia pastoris uses Pichia pastoris GS115 as a chassis strain.

3. The recombinant Pichia pastoris according to claim 1, characterized in that: In the first expression cassette, the ethanol-inducible promoter is ICL1, the DNA-binding protein is LacI, and the transcription activator is Mit1AD.

4. The recombinant Pichia pastoris according to claim 1, characterized in that: In the second expression cassette, the copy number of the protein binding sequence is 5.

5. The recombinant Pichia pastoris according to claim 1, characterized in that: In the second expression cassette, the pre region of the α signal peptide is replaced with a serum albumin signal peptide, a killer protein signal peptide, an Ost1 signal peptide or an Epx1 signal peptide.

6. The recombinant Pichia pastoris according to claim 1, characterized in that: The copy number of the second expression cassette in the recombinant Pichia pastoris is 1-5.

7. An expression vector combination for constructing the recombinant Pichia pastoris according to any one of claims 1 to 6, characterized in that: The expression vector combination comprises a first expression vector, a second expression vector and a third expression vector, wherein: The first expression vector uses an ethanol-inducible promoter to express a fusion polypeptide of a DNA binding protein and a transcription activator; The second expression vector sequentially expresses a protein binding sequence, a methanol-inducible promoter, an α signal peptide, and human α-lactalbumin, wherein the protein binding sequence is LacO; The third expression vector overexpresses a signal recognition particle subunit, and the signal recognition particle subunit is involved in protein targeting to the endoplasmic reticulum.

8. A method for inducing Pichia pastoris to express human α-lactalbumin using ethanol, characterized in that: The following steps are involved: Step S1, constructing the recombinant Pichia pastoris according to any one of claims 1 to 6 to obtain a seed solution; Step S2, adding fermentation basal salt culture medium and PTM1 trace element solution into the fermentation tank, inoculating the seed solution into the fermentation tank, and keeping the dissolved oxygen above 20%; Step S3, after the dissolved oxygen rebounds, glycerol feeding is performed, followed by induction with an ethanol solution; Step S4, alternately performing ethanol feeding and glycerol feeding until the fermentation is completed.

9. The method according to claim 8, characterized in that: PTM1 trace element liquid is added to both the glycerol and the ethanol.

10. The method according to claim 8, characterized in that: During step S4, the concentration of acetate in the fermentation broth is monitored, and when acetate accumulates, ethanol feeding is stopped and glycerol feeding is performed.

Images