A method for regulating Pichia pastoris to reduce acetic acid accumulation during ethanol-induced expression of α-lactalbumin

Abstract

The present invention relates to the field of genetic engineering technology, and in particular to a method for regulating Pichia pastoris to reduce the accumulation of acetic acid during the ethanol-induced expression of α-lactalbumin. The recombinant yeast strain provided by the present invention has a greatly reduced accumulation of acetic acid during the expression of a target protein by ethanol induction, and can achieve essentially no accumulation of acetic acid, thereby achieving a higher biomass and target protein yield, and achieving the highest yield of human α-lactalbumin to date. This provides strain resources and a foundation for the application of the ethanol-induced Pichia pastoris expression system and the development of bioengineered milk proteins. The present invention provides a method for solving the problem of acetic acid accumulation in the process of Pichia pastoris expressing a target protein by ethanol induction. This method provides an effective strategy for the promotion and application of the ethanol-induced expression system and for improving the yield of the target protein expressed by ethanol induction, and is of great significance for the production of proteins such as α-lactalbumin.

Description

Technical Field

[0001] The present invention relates to the technical field of genetic engineering, in particular to a method for regulating Pichia pastoris to reduce acetic acid accumulation during the process of ethanol-induced expression of alpha-lactalbumin. Background Art

[0002] Pichia pastoris ( 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 starting properties, which is suitable for high-level induced expression of foreign genes; (4) Pichia pastoris has a preference for aerobic growth and is suitable for high-density cell culture, which 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] Currently, Pichia pastoris mostly uses a methanol-inducible system to express exogenous proteins. However, expression systems using methanol as an inducer and sole carbon source present significant risks in industrial and food production. Due to its flammability, explosiveness, and toxicity, methanol is considered a potential threat to safe production in industrial production. To mitigate the negative impact of methanol to some extent, the ESAD expression system, which uses ethanol as an inducer and sole carbon source, was invented. However, in actual applications, the ESAD system is prone to the accumulation of large amounts of acetic acid when ethanol is continuously fed into the fermenter, leading to cell death and interruption of fermentation. Therefore, there is an urgent need to address the problem of large amounts of acetic acid accumulation during ethanol induction. Summary of the Invention

[0004] The invention provides a method for regulating Pichia pastoris to reduce acetic acid accumulation during the process of ethanol-induced expression of alpha-lactalbumin.

[0005] To address the problem of significant acetic acid accumulation during ethanol induction in the Pichia pastoris ESAD expression system using ethanol as the inducer and sole carbon source, this invention incorporates an ATP repurposing tool into the Pichia pastoris ESAD system, promoting the conversion of acetic acid into acetyl-CoA. This not only completely resolves the acetic acid accumulation issue but also provides more energy for the fermentation process. In practical applications, this significantly improves protein expression and the feasibility of high-intensity fermentation in the ESAD system.

[0006] Specifically, the present invention provides the following technical solutions.

[0007] In a first aspect, the present invention provides a recombinant yeast strain comprising an exogenous gene encoding polyphosphate kinase and an exogenous target protein expression system capable of being induced to express by ethanol.

[0008] The present invention discovered that introducing exogenous polyphosphate kinase into a yeast strain can promote the conversion of acetic acid produced during ethanol induction into acetyl-CoA, thereby achieving ATP replenishment. This not only solves the problem of acetic acid accumulation, but also provides more energy for the fermentation process, can relieve the inhibition of acetic acid on bacterial growth and protein expression, and significantly increase biomass and the expression of target protein.

[0009] Preferably, the polyphosphate kinase is derived from Staphylococcus epidermidis ( Staphylococcus epidermidis ).

[0010] Polyphosphate kinase is found in a variety of microorganisms. The present invention found that compared with polyphosphate kinases from other sources, polyphosphate kinase derived from Staphylococcus epidermidis is more suitable for functioning in yeast, especially Pichia pastoris, and more effectively promotes the conversion of acetic acid into acetyl-CoA, which is more beneficial for increasing the expression level of the target protein.

[0011] Preferably, the polyphosphate kinase has the amino acid sequence shown in SEQ ID NO.1.

[0012] Based on the amino acid sequence of the polyphosphate kinase described above, those skilled in the art can obtain the sequence of the gene encoding it. Due to codon degeneracy, gene sequences encoding the same amino acid sequence are not unique. All nucleotide sequences encoding the polyphosphate kinase described above are within the scope of protection of the present invention. To further facilitate the expression of the polyphosphate kinase, those skilled in the art can perform codon optimization as needed.

[0013] Preferably, the nucleotide sequence of the exogenous gene encoding polyphosphate kinase is shown as SEQ ID NO.2.

[0014] The above-mentioned foreign gene encoding polyphosphate kinase exists in the form of an expression cassette to utilize the expression of the foreign gene.

[0015] Preferably, the recombinant yeast strain comprises an expression cassette of an exogenous gene encoding polyphosphate kinase, which is capable of expressing the polyphosphate kinase under ethanol induction conditions; the expression cassette comprises a promoter that controls the transcription of the exogenous gene encoding polyphosphate kinase and the exogenous gene encoding polyphosphate kinase.

[0016] The present invention has conducted a large number of screenings of promoters that control the transcription of exogenous genes encoding polyphosphate kinases and found that compared with other promoters (such as AOX1, DAS2, FDH1, GAP, ICL1, etc.), the ADH2 promoter is more conducive to expressing polyphosphate kinase under ethanol-induced conditions, and is thus more conducive to reducing acetic acid accumulation during the fermentation process. It is more effective in increasing the expression level of the target protein.

[0017] Preferably, the promoter for controlling the transcription of the exogenous gene encoding polyphosphate kinase is preferably ADH2 promoter. The sequence of the ADH2 promoter is shown in SEQ ID NO.16.

[0018] The expression cassette of the exogenous gene encoding polyphosphate kinase described above further comprises a terminator. In principle, the present invention has no particular limitation on the choice of the terminator, and any sequence capable of terminating gene transcription in yeast is applicable.

[0019] In some embodiments of the present invention, the terminator is an AOX1 terminator.

[0020] Preferably, the expression cassette of the exogenous gene encoding polyphosphate kinase is integrated into the genome of the recombinant yeast strain.

[0021] In principle, the present invention has no particular limitation on the integration site, and optional integration sites include Int6 (ChrII:17928−17947).

[0022] The above-mentioned exogenous target protein expression system capable of being induced to express by ethanol comprises an ethanol-inducible promoter and at least one copy of an exogenous target protein encoding gene.

[0023] In some embodiments of the present invention, the target protein is α-lactalbumin. The amino acid sequence of α-lactalbumin is shown in SEQ ID NO. 3. The coding gene sequence of α-lactalbumin is preferably shown in SEQ ID NO. 4, which is a codon-optimized sequence.

[0024] Preferably, the copy number of the target protein encoding gene is 1-10; more preferably 2-5.

[0025] In some embodiments of the present invention, the yeast strain has three copies of the α-lactalbumin encoding gene integrated into its genome.

[0026] Preferably, the exogenous target protein expression system capable of being induced to express by ethanol comprises a first expression cassette and a second expression cassette; wherein, the first expression cassette comprises an ethanol-inducible promoter, a LacI encoding gene, and a Mit1AD encoding gene; and the second expression cassette comprises 5 to 9 copies of a LacO encoding gene, a yeast promoter capable of being activated by Mit1AD, a signal peptide, and a target protein encoding gene.

[0027] In a specific embodiment of the present invention, a LacI-Mit1AD fusion activator and multiple copies of the activator are used under the control of an ethanol-inducible promoter. lacO - yeast constitutive promoter constitutes a hybrid promoter as the core element of the expression system, which controls the expression of the LacI-Mit1AD activator by ethanol induction and the expression of the LacI- lacO The system controls the expression of the target protein encoding gene regulated by the hybrid promoter, thereby achieving efficient expression of the target protein encoding gene by inducing ethanol.

[0028] Preferably, the ethanol-inducible promoter is the ICL1 promoter (its sequence is preferably shown as SEQ ID NO. 5). The yeast promoter that can be activated by Mit1AD is the core sequence of the AOX1 promoter, core AOX1 (its sequence is preferably shown as SEQ ID NO. 6).

[0029] Preferably, the signal peptide is a serum albumin hybrid signal peptide (the coding sequence of which is preferably shown as SEQ ID NO.7).

[0030] Preferably, the amino acid sequence of the LacI protein is shown in SEQ ID NO. 8. The gene sequence encoding the LacI protein is shown in SEQ ID NO. 9.

[0031] Preferably, the amino acid sequence of Mit1AD protein is shown as SEQ ID NO. 10. The gene sequence encoding Mit1AD protein is shown as SEQ ID NO. 11.

[0032] Preferably, the sequence of lacO is shown as SEQ ID NO.12.

[0033] In some embodiments of the present invention, the exogenous target protein expression system capable of being induced to express by ethanol comprises a first expression cassette and a second expression cassette; wherein, the first expression cassette comprises the following elements in sequence from the 5'-3' direction: ICL1 promoter, LacI encoding gene and Mit1AD encoding gene; the second expression cassette comprises the following elements in sequence from the 5'-3' direction: 5 copies of the LacO encoding gene 5LacO, core AOX1, serum albumin hybrid signal peptide SA Pro and α-lactalbumin encoding gene LALBA.

[0034] The above expression system can achieve efficient expression of α-lactalbumin, which is beneficial for increasing α-lactalbumin production. The ethanol-induced α-lactalbumin expression system, combined with the introduction of exogenous polyphosphate kinase, significantly reduces acetic acid accumulation in the yeast strain during ethanol-induced α-lactalbumin expression, significantly increasing α-lactalbumin production.

[0035] In the present invention, the yeast is preferably Pichia pastoris; more preferably Pichia pastoris.

[0036] Preferably, the recombinant yeast strain significantly reduces the accumulation of acetic acid during the ethanol-induced expression of the target protein. The recombinant yeast strain can substantially not accumulate acetic acid when ethanol is the sole carbon source.

[0037] In a second aspect, the present invention provides the use of the above-mentioned recombinant yeast strain in expressing a target protein by inducing ethanol or in constructing a production strain for expressing a target protein by inducing ethanol.

[0038] Preferably, the target protein is α-lactalbumin.

[0039] In a third aspect, the present invention provides a method for preparing α-lactalbumin, comprising: culturing the recombinant yeast strain described above to express α-lactalbumin, and recovering the α-lactalbumin in the culture; wherein the target protein in the recombinant yeast strain is α-lactalbumin.

[0040] The above-mentioned culture includes the step of adding ethanol to induce expression and the step of adding sodium hexametaphosphate.

[0041] Preferably, in the ethanol induction expression stage, the method comprises: producing the fermentation mixture at a volume of 6-12 mL·(L·h) relative to the initial fermentation volume. -1 Ethanol was added at a rate of 0.5-1.2 mL / h / L, and 45-55 g / L of sodium hexametaphosphate was added at a rate of 0.8-1.2 mL / h / L.

[0042] Preferably, the initial fermentation volume is 7-8 mL·(L·h) -1 Ethanol was added at a rate of 1.5-2.5 h, and then the flow rate was adjusted to 10-12 mL·(L·h) relative to the initial fermentation volume. -1 Until the end of cultivation.

[0043] In some embodiments of the present invention, a method for preparing α-lactalbumin is provided, which is carried out using a fermenter or a large-scale bioreactor and comprises the following steps:

[0044] (1) activating the recombinant yeast strain on a solid culture medium;

[0045] (2) Transfer the activated bacteria from step (1) to the seed culture medium and culture for 16-24 h until its OD 600 When the value reaches 18-22, seed solution is obtained;

[0046] (3) The seed liquid of step (2) was inoculated at a ratio of 5%-10% of the initial fermentation volume to a volume of 4.2-4.4 mL·L -1 Fermentation was carried out in a fermentation basal salt medium (pH 5.0-5.2) containing trace element solution (PTM1), at a temperature of 29-32°C and with dissolved oxygen maintained above 18-22%.

[0047] (4) After the dissolved oxygen rebounds, start feeding 50-70% w / v glycerol, containing 11-13 mL of trace element solution (PTM1) per liter of glycerol feed; set the feed rate to 17.5-18.5 mL·L -1 Glycerol feeding was performed for 3–6 h with the initial fermentation volume;

[0048] (5) Stop adding glycerol and induce the induction of the fermentation with a pure ethanol solution containing 11-13 mL of trace element solution (PTM1) per liter, setting the feed rate to 3.5-3.7 mL·h per liter of original fermentation volume. -1 During the first 2-3 hours, ethanol will accumulate in the fermenter, and dissolved oxygen values will become unstable as the culture adapts to the ethanol.

[0049] (6) After maintaining a low ethanol feed rate for at least 1 hour, double the feed rate to 7.1-7.5 mL·(L·h) -1 Initial fermentation volume can further increase the rate of product formation; simultaneously add 48-52 g / L of sodium hexametaphosphate at 0.9-1.1 mL / h / L; feed rate in the first 2 hours is 7.1-7.5 mL·(L·h) -1 , and then increase the ethanol feed rate to approximately 10.7-11.1 mL·(L·h) per liter of initial fermentation volume -1 , remained unchanged for the rest of the fermentation; the entire ethanol fed-batch phase lasted about 65-75 h.

[0050] Preferably, the trace element solution (PTM1) comprises the following components: 63.0-67.0 g·L -1 FeSO4·7H2O, 215.0-225.0 g·L -1 ZnCl, 5.0-7.0 g L -1 CuSO4·5H2O, 2.5-3.5 g·L-1 MnSO4·H2O, 20.0-21.0 g·L -1 CoCl, 0.15-0.25 g L -1 MoNa2O4·2H2O, 0.15-0.25 g·L -1 D-biotin, 0.08-0.10 g L -1 KI, 0.01-0.03 g·L -1 H3BO3; 4.8-5.2 mL concentrated H2SO4.

[0051] Preferably, the fermentation basal salts medium (BSM) comprises the following components: 35.0-45.0 g·L -1 Glycerol, 16-20 g·L -1 K2SO4, 14.5-15.5 g·L -1 MgSO4·7H2O, 4.10-4.20 g·L - 1 KOH, 0.9-1.0 g L -1 CaSO4, 26-28 mL phosphoric acid, 4.2-4.4 mL·L -1 PTM1.

[0052] Preferably, the culture medium used for activation and seed culture is YPD medium.

[0053] During the above fermentation process, the cell wet weight can increase to 450-500 g·L -1 ; The final fermentation volume will be approximately twice the initial fermentation volume.

[0054] In some embodiments of the present invention, a method for preparing α-lactalbumin is provided, which is carried out using a shake flask and comprises the following steps:

[0055] (1) Pick a single colony of the recombinant yeast strain and place it in a primary seed liquid medium (preferably YPD) and culture it at 29-32°C and 200-220 rpm for 16-18 hours;

[0056] (2) Transfer the primary seed solution to the secondary seed solution medium (preferably YPD) and ferment at 29-32°C and 200-220 rpm for 22-26 hours;

[0057] (3) Collect all the cells by centrifugation and transfer them to an induction expression medium (preferably BMEY), add 0.05-0.1 g / L sodium hexametaphosphate, and ferment at 26-28°C and 200-220 rpm for 60-80 hours, adding 0.5-1.5% (v / v) ethanol every 20-26 hours. Then collect the fermentation supernatant.

[0058] In a fourth aspect, the present invention provides a method for reducing acetic acid accumulation in Pichia pastoris during ethanol-induced expression of a target protein, the method comprising: introducing an exogenous gene encoding polyphosphate kinase into the Pichia pastoris.

[0059] The present invention proves through experimental verification that the accumulation of acetic acid can be significantly reduced and the yield of the target protein can be increased by introducing an exogenous gene encoding polyphosphate kinase into Pichia pastoris that is induced to express the target protein using ethanol.

[0060] Preferably, the polyphosphate kinase is derived from Staphylococcus epidermidis ( Staphylococcus epidermidis ).

[0061] Preferably, the polyphosphate kinase has the amino acid sequence shown in SEQ ID NO.1.

[0062] In the above method, the exogenous gene encoding polyphosphate kinase is preferably introduced in the form of an expression cassette.

[0063] Preferably, the method comprises: introducing an expression cassette comprising an exogenous gene encoding polyphosphate kinase into the Pichia pastoris, wherein the expression cassette is capable of expressing the polyphosphate kinase under ethanol induction conditions;

[0064] The expression cassette comprises a promoter for controlling the transcription of an exogenous gene encoding polyphosphate kinase and an exogenous gene encoding polyphosphate kinase, and the promoter is preferably an ADH2 promoter.

[0065] Preferably, the expression cassette of the exogenous gene encoding polyphosphate kinase further comprises a terminator; the terminator is preferably an AOX1 terminator.

[0066] Preferably, the expression cassette of the exogenous gene encoding polyphosphate kinase is integrated into the genome of the recombinant yeast strain.

[0067] Preferably, the target protein is α-lactalbumin.

[0068] Preferably, the Pichia pastoris expresses the target protein using an ethanol-inducible expression system. An expression system capable of inducing the expression of the target protein using ethanol is integrated into the Pichia pastoris genome.

[0069] Preferably, the ethanol-inducible expression system comprises a first expression cassette and a second expression cassette; wherein, the first expression cassette comprises an ethanol-inducible promoter, a LacI encoding gene, and a Mit1AD encoding gene; and the second expression cassette comprises 5 to 9 copies of a LacO encoding gene, a yeast constitutive promoter, a signal peptide, and a target protein encoding gene.

[0070] In a specific embodiment of the present invention, an expression system with a LacI-Mit1AD fusion activator controlled by an ethanol-inducible promoter and a hybrid promoter composed of multiple copies of the lacO-yeast constitutive promoter as core elements is used. The expression of the LacI-Mit1AD activator is controlled by ethanol induction, and the expression of the target protein encoding gene regulated by the hybrid promoter is controlled by the LacI-LacO system, thereby achieving efficient expression of the target protein encoding gene by ethanol induction.

[0071] Preferably, the ethanol-inducible promoter is the ICL1 promoter (its sequence is preferably shown as SEQ ID NO. 5). The yeast constitutive promoter is the core sequence of the AOX1 promoter, core AOX1 (its sequence is preferably shown as SEQ ID NO. 6).

[0072] Preferably, the signal peptide is a serum albumin hybrid signal peptide (the sequence of which is preferably shown as SEQ ID NO. 7).

[0073] In some embodiments of the present invention, the ethanol-inducible expression system comprises a first expression cassette and a second expression cassette; wherein, the first expression cassette comprises the following elements in sequence from the 5'-3' direction: ICL1 promoter, LacI encoding gene and Mit1AD encoding gene; the second expression cassette comprises the following elements in sequence from the 5'-3' direction: 5 copies of LacO encoding gene 5LacO, core AOX1, serum albumin hybrid signal peptide SA Pro and α-lactalbumin encoding gene LALBA.

[0074] The beneficial effects of the present invention include at least: the recombinant yeast strain provided by the present invention significantly reduces the accumulation of acetic acid during ethanol-induced expression of a target protein, achieving essentially no acetic acid accumulation, achieving high biomass and target protein yields, and achieving the highest yield of human α-lactalbumin to date. This provides a strain resource and foundation for the application of the ethanol-inducible Pichia pastoris expression system and the development of bioengineered milk proteins. The present invention also provides a method for resolving the problem of acetic acid accumulation during ethanol-induced expression of a target protein in Pichia pastoris. This method provides an effective strategy for promoting the application of the ethanol-inducible expression system and increasing the yield of target proteins expressed using ethanol-inducible expression, and is of great significance for the production of proteins such as α-lactalbumin. BRIEF DESCRIPTION OF THE DRAWINGS

[0075] In order to more clearly illustrate the technical solutions in the present invention or the prior art, a brief introduction is given below to the drawings required for use in the embodiments or the description of the prior art. Obviously, the drawings described below are some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0076] Figure 1 OD is the growth condition of the ESAD-LA3 strain in Example 2 of the present invention using ethanol as an inducer and the sole carbon source using the traditional fermentation method 600 The results of the liquid chromatography determination of the acetic acid accumulation were shown.

[0077] Figure 2 These are the shake flask results demonstrating the promoting effect of co-expressing SePPK and adding sodium hexametaphosphate to the culture medium on acetic acid consumption in Example 3 of the present invention. The horizontal axes correspond to the experimental groups of strains ESAD-LA3 (Control), ESAD-LA3-ADH2-PPK (ADH2-PPK), ESAD-LA3-AOX1-PPK (AOX1-PPK), ESAD-LA3-DAS2-PPK (DAS2-PPK), ESAD-LA3-FDH1-PPK (FDH1-PPK), ESAD-LA3-GAP-PPK (GAP-PPK), and ESAD-LA3-ICL1-PPK (ICL1-PPK), respectively.

[0078] Figure 3 These are the monitoring results and SDS-PAGE gel electrophoresis results of the experimental group of the fermentation scale-up experiment of the strain ESAD-LA3-ADH2-PPK in Example 4 of the present invention, in which 10 g / L sodium hexametaphosphate was added.

[0079] Figure 4 These are the monitoring results and SDS-PAGE gel electrophoresis results of the experimental group of the fermentation scale-up experiment of the strain ESAD-LA3-ADH2-PPK in Example 4 of the present invention, in which 50 g / L sodium hexametaphosphate was added.

[0080] Figure 5 These are the monitoring results and SDS-PAGE gel electrophoresis results of the experimental group of the ESAD-LA3-ADH2-PPK fermentation tank amplification experiment in Example 4 of the present invention, with 100 g / L sodium hexametaphosphate added. DETAILED DESCRIPTION

[0081] In a specific embodiment of the present invention, a recombinant Pichia pastoris strain is provided. The strain uses the recombinant Pichia pastoris ESAD-LA3 (GS115-ICL1-LacI-Mit1AD -pPIC3.5K-5LacO-cAOX1-SA Pro-LALBA3, containing multiple (3) copies of the α-lactalbumin gene) that can secrete and express α-lactalbumin as a starting strain. Polyphosphokinase (SePPK, derived from Staphylococcus epidermidis) is co-expressed in the starting strain to efficiently replenish ATP using ADP and PPi in the cell. This, to a certain extent, solves the problem of the large accumulation of acetic acid rapidly generated by ethanol assimilation during fermentation, which is difficult to convert into acetyl-CoA due to a lack of sufficient ATP supply. Specifically, they co-expressed SePPK with the ADH2 promoter, which can achieve high levels of expression under ethanol as the sole carbon source. Furthermore, by adding 50 g / L of sodium hexametaphosphate (SHMP) at a rate of 0.8-1.2 mL / h / L (which generates ATP under the action of SePPK), they completely eliminated the problem of acetic acid accumulation during fermentation. Finally, the engineered yeast strain ESAD-LA3-ADH2-PPK was scaled up for production in a 3L fermenter, increasing α-lactalbumin production to 1.3 g / L.

[0082] In a specific embodiment of the present invention, a method for constructing the above-mentioned recombinant Pichia pastoris strain is also provided, the method comprising the steps of introducing a polyphosphate kinase expression cassette into the recombinant Pichia pastoris ESAD-LA3 capable of secreting and expressing α-lactalbumin, specifically comprising the following steps:

[0083] First, the ADH2-SePPK-AOX1T expression cassette (from 5' to 3', the ADH2 promoter sequence, the SePPK gene sequence, and the AOX1 terminator sequence) was constructed using fusion PCR. This expression cassette was inserted into the yeast genome at the Int6 site (ChrII:17928−17947). Fusion PCR was then used to add 500-bp homology arms to the front and back of the expression cassette to generate the final donor DNA. Simultaneously, a Crispr plasmid carrying the sgRNA and Cas9 gene corresponding to Int6 was constructed, designated Crispr-Int6. Finally, the donor DNA and Crispr-Int6 plasmid were simultaneously introduced into ESAD-LA3 by electroporation, generating the strain ESAD-LA3-ADH2-PPK.

[0084] A specific embodiment of the present invention provides a method for producing α-lactalbumin using the recombinant Pichia pastoris strain described above. This method can be applied in either shake flask or fermentor fermentation. In shake flask fermentation, ethanol is used for induction, and sodium hexametaphosphate is added at a low concentration (0.05-0.1 g / L). In fermentor fermentation, ethanol is fed, and 50 g / L of sodium hexametaphosphate is fed at a rate of 0.8-1.2 mL / h / L. This method addresses the problem of acetic acid accumulation in ethanol-induced Pichia pastoris expression systems due to the continuous feeding of high amounts of ethanol as the sole carbon source, ensuring good strain growth during fermentation. In fermentor fermentation, the target protein, α-lactalbumin, was expressed at a level of 1.3 g / L, with no acetic acid accumulation.

[0085] To make the objectives, technical solutions, and advantages of the present invention more clear, the technical solutions of the present invention will be clearly and completely described below in conjunction with the accompanying drawings. Obviously, the embodiments described are only some of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative efforts shall fall within the scope of protection of the present invention.

[0086] In the following examples, the medium formula used for seed culture and fermentation of recombinant Pichia pastoris is as follows:

[0087] The seed culture medium is YPD: 1% yeast extract, 2% peptone, and 2% glucose. The ethanol fermentation medium (BMEY) is formulated as follows: 10 g / L yeast extract, 20 g / L peptone, 3 g / L K₂HPO₄·3H₂O, 11.8 g / L KH₂PO₄, and 894 mL ultrapure water. Sterilize at 121°C for 20 min. After the temperature reaches 60°C, add 100 mL of 10× YNB, 1 mL of 500× B (0.02% biotin), and 5 mL of ethanol in a clean hood. PTM1 trace salts solution: 65.0 g·L -1 FeSO4·7H2O, 220.0 g·L -1 ZnCl, 6.0 g·L -1 CuSO4·5H2O, 3.0 g·L -1 MnSO4·H2O, 20.5 g·L -1 CoCl, 0.2 g L -1 MoNa2O4·2H2O, 0.2 g·L -1 D-biotin, 0.09 g·L -1 KI, 0.02 g·L-1 H3BO3; 5.0 mL concentrated H2SO4. BSM (Basal salts medium): 40.0 g·L -1 Glycerol, 18 g·L - 1 K2SO4, 14.9 g·L -1 MgSO4·7H2O, 4.13 g·L -1 KOH, 0.93 g·L -1 CaSO4, 27 mL·L -1 Phosphoric acid, 4.4 mL·L -1 PTM1. Glycerol feed medium: Prepare a 50% (w / v) glycerol solution with deionized water, sterilize it, and add 12 mL·L of filter-sterilized -1 PTM1 solution.

[0088] The shake flask fermentation method used in the following examples comprises the following steps:

[0089] (1) Pick a single colony and place it in 5 mL of primary seed liquid medium (YPD) at 30 o C, culture at 220 rpm for 16-18 hours;

[0090] (2) Take 100 μL of the first seed solution and transfer it to 50 mL of the second seed solution medium (YPD). o C. Fermentation at 220 rpm for 24 hours;

[0091] (3) Collect all the cells by centrifugation, transfer to 50 mL of induction expression medium (BMEY), and add 0.1 g / L or 1 g / L sodium hexametaphosphate. o C. Fermentation was carried out at 220 rpm for 72 h, with 1% (v / v) ethanol (as an inducer and the sole carbon source) added every 24 h. The fermentation supernatant was then collected, purified, and quantified.

[0092] The fermentation tank fermentation method used in the following examples includes the following steps:

[0093] (1) The recombinant Pichia pastoris strain was streaked onto YPD solid medium and incubated in a 30°C constant temperature incubator for about 3 days until a clearly visible single colony was formed;

[0094] (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 hours until its OD 600 The value reaches 20;

[0095] (3) Place the fermentation basal salt medium (BSM medium) without PTM1 in the fermenter, then sterilize the fermenter. After sterilization and cooling, set the temperature to 30°C, stir and aerate to the operating conditions (1200 rpm and 1.0 vvm), and adjust the pH value of the fermentation basal salt medium to 5.0 with ammonia water. Aseptically add 4.4 mL·L -1 The trace element solution (PTM1) is added to the above-mentioned fermentation basal salt medium without PTM1, thereby obtaining the fermentation basal salt medium (BSM medium). The seed solution obtained in step (2) is then inoculated into the fermentation tank at a ratio of 5%-10% of the initial fermentation volume, and oxygen is added as needed to maintain the dissolved oxygen level above 20% (1.5 vvm).

[0096] (4) After the dissolved oxygen rebounded, a 50% w / v glycerol feed was started, containing 12 mL of PTM1 trace elements per liter of glycerol feed. The feed rate was set to 18.15 mL·L -1 The initial fermentation volume was glycerol fed for about 4 hours or more;

[0097] (5) Stop adding glycerol and induce the induction input with a pure ethanol solution containing 12 mL of PTM1 per liter, setting the feed rate to 3.6 mL·h per liter of initial fermentation volume. -1 During the first 2-3 hours, ethanol will accumulate in the fermenter, and dissolved oxygen values will become unstable as the culture adapts to the ethanol.

[0098] (6) After maintaining the low ethanol feed rate for at least 1 hour, increase the feed rate to 7.3 mL·(L·h) -1 The initial fermentation volume can further increase the rate of product formation. At the same time, 50 g / L of sodium hexametaphosphate was added at a rate of 1 ml / h / L. The feed rate in the first 2 hours was 7.3 mL·(L·h) -1 , and then the ethanol feed rate was increased to approximately 10.9 mL·(L·h) per liter of initial fermentation volume. -1 The entire ethanol fed-batch phase lasts about 70 h, and the cell wet weight can increase to 450-500 g·L -1 The final fermentation volume will be approximately double the initial fermentation volume.

[0099] The sample detection method used in the following examples includes the following steps: First, the fermentation broth was centrifuged, the supernatant was collected, and the supernatant was passed through a 0.45 µm water filter. Subsequently, the supernatant was purified using a His-Binding-resin purification column. The purified sample was collected and subjected to SDS-PAGE electrophoresis. Protein quantification was performed using a Bradford kit.

[0100] Example 1 Construction of ethanol-induced α-lactalbumin production strain ESAD-LA3

[0101] First, an endogenous ethanol-inducible promoter was amplified from the Pichia pastoris genome. ICL1 , the plasmid vector PICZ_B (sequence shown in SEQ ID NO.13) AOX1 Promoter replaced with promoter ICL1 Then, the gene sequence of LacI-Mit1AD fusion protein was inserted into the multiple cloning site to obtain PICZ_B- ICL1-LacI-Mit1AD At the same time, a plasmid with plasmid PIC3.5K (sequence shown in SEQ ID NO.14) as the vector backbone was constructed. In this round of construction, AOX1 Promoter core sequence core AOX1 Keep, and replace the rest with 5 LacO Repeat sequence, followed by core AOX1 Insert sequence in sequence SAPro (serum albumin hybrid signal peptide), LALBA (human α-lactalbumin encoding gene) 、 6His , get PIC3.5K- 5LacO-coreAOX1-SA Pro-LALBA-6His After the above plasmid PICZ_B- ICL1-LacI-Mit1AD and PIC3.5K- 5LacO-coreAOX1-SA Pro-LALBA-6His Afterwards, the plasmid was linearized using restriction enzymes EcoRI and SalI, and then the linearized recombinant plasmid was integrated into the histidine-deficient Pichia pastoris strain GS115 using electroporation. 5LacO- coreAOX1-SA Pro-LALBA-6His Transform Pichia pastoris competent cells to obtain a large number of transformants. Use 4 mg / mL high-concentration geneticin plate culture medium to screen high-copy transformants, and perform RT-qPCR verification on transformants that are more likely to have high copies, thereby obtaining 5LacO-coreAOX1-SA Pro-LALBA-6His The transformant with a copy number of 3 was named ESAD-LA3.

[0102] Example 2 Determination of acetic acid accumulation level during scale-up culture of strain ESAD-LA3 using ethanol induction in a fermenter

[0103] The strain ESAD-LA3 constructed in Example 1 was fermented using the aforementioned fermentation method without adding sodium hexametaphosphate. The results showed that ( Figure 1Using traditional fermentation methods, due to the high ethanol feed rate, the bacterial count in the fermenter decreased significantly after 46 hours, and significant acetic acid accumulation was observed in the fermentation broth at 58 hours, disrupting normal fermentation. Ultimately, at the end of the fermentation at 108 hours, the acetic acid content in the fermentation broth reached 134 g / L.

[0104] Example 3 Verification of the Promoting Effect of Co-expression of SePPK and Addition of Sodium Hexametaphosphate to the Culture Medium on Acetate Consumption at the Shake Flask Level

[0105] Based on ESAD-LA3, expression cassettes for expressing the SePPK gene using promoters ADH2 (SEQ ID NO.16), AOX1 (SEQ ID NO.17), DAS2 (SEQ ID NO.18), FDH1 (SEQ ID NO.19), GAP (SEQ ID NO.20), and ICL1 (SEQ ID NO.21) were integrated into the Pichia pastoris genome. First, fusion PCR was used to construct the ADH2-SePPK-AOX1T, AOX1-SePPK-AOX1T, DAS2-SePPK-AOX1T, FDH1-SePPK-AOX1T, GAP-SePPK-AOX1T, and ICL1-SePPK-AOX1T expression cassettes (from 5' to 3', representing the promoter sequence, SePPK gene sequence, and AOX1 terminator sequence, respectively). The genomic insertion site for the expression cassettes was selected as Int6 (ChrII:17928-17947). Fusion PCR was then used to add 500-bp homology arms to the front and back of the fusion expression cassettes to generate the final donor DNA. Simultaneously, a Crispr plasmid carrying the sgRNA and Cas9 gene corresponding to Int6 was constructed, designated Crispr-Int6 (sequence shown in SEQ ID NO. 15). Finally, the donor DNA and Crispr-Int6 plasmid were simultaneously introduced into ESAD-LA3 by electroporation, and the strains ESAD-LA3-ADH2-PPK, ESAD-LA3-AOX1-PPK, ESAD-LA3-DAS2-PPK, ESAD-LA3-FDH1-PPK, ESAD-LA3- GAP -PPK, and ESAD-LA3- ICL1-PPK were obtained respectively. The above six strains were cultured and fermented in shake flasks. In order to make it easier for the fermentation strains to accumulate acetic acid in the shake flasks, the amount of ethanol added in the fermentation was increased from 0.5% (v / v) to 4% (v / v) and 5 g / L of ammonium sulfate was added. In order to explore whether the addition of sodium hexametaphosphate promotes the consumption of acetic acid and to explore the approximate range of the optimal sodium hexametaphosphate concentration, control experimental groups were designed with no sodium hexametaphosphate, 0.1 g / L sodium hexametaphosphate, and 1 g / L sodium hexametaphosphate. After the fermentation, samples were taken for OD 600 The amount of acetic acid accumulated in the fermentation broth was determined and accurately measured using liquid chromatography. Figure 2 As shown, considering the cell growth recovery and acetic acid accumulation, the ADH2 Overexpression of SePPK by the promoter can most efficiently replenish ATP and solve the problems of acetate accumulation and weakened growth to the greatest extent.

[0106] Example 4 Fermentation tank scale-up experiment on strain ESAD-LA3-ADH2-PPK

[0107] By analyzing the experimental results of the shake flask fermentation and comprehensively analyzing the growth status and acetic acid accumulation of the strain, ESAD-LA3-ADH2-PPK was selected as the strain for subsequent experiments. Subsequently, a fermentation scale-up experiment was conducted on ESAD-LA3-ADH2-PPK. Since the shake flask experiment showed that the experimental group with 0.1 g / L sodium hexametaphosphate addition had generally better growth and generally lower acetic acid accumulation, the difference between the biomass in the shake flask and the biomass in the fermenter was calculated, and a fermentation scale-up experiment was designed to add 10 g / L ( Figure 3 )、50 g / L( Figure 4 )、100 g / L( Figure 5 Finally, in the experimental group fed with 50 g / L sodium hexametaphosphate, no acetic acid accumulated during the entire fermentation cycle, and 1.3 g / L of α-lactalbumin was obtained.

[0108] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or make equivalent replacements for some of the technical features therein. However, these modifications or replacements do not deviate the essence of the corresponding technical solutions from the spirit and scope of the technical solutions of the various embodiments of the present invention.

Claims

1. A recombinant yeast strain, characterized in that The recombinant yeast strain comprises an expression cassette of an exogenous gene encoding polyphosphate kinase, which is capable of expressing the polyphosphate kinase under ethanol induction conditions; the recombinant yeast strain further comprises an exogenous target protein expression system capable of being induced to express by ethanol; Wherein, the expression cassette comprises a promoter for controlling the transcription of an exogenous gene encoding polyphosphate kinase and an exogenous gene encoding polyphosphate kinase, and the promoter is an ADH2 promoter; The amino acid sequence of the polyphosphate kinase is shown in SEQ ID NO.1; The yeast is Pichia pastoris ( Pichia pastoris ).

2. The recombinant yeast strain according to claim 1, characterized in that The expression cassette of the exogenous gene encoding polyphosphate kinase further comprises a terminator.

3. The recombinant yeast strain according to claim 2, characterized in that The terminator is the AOX1 terminator.

4. The recombinant yeast strain according to claim 1, characterized in that The expression cassette of the exogenous gene encoding polyphosphate kinase is integrated into the genome of the recombinant yeast strain.

5. The recombinant yeast strain according to any one of claims 1 to 4, characterized in that The exogenous target protein expression system capable of being induced to express by ethanol comprises an ethanol-inducible promoter and at least one copy of an exogenous target protein encoding gene.

6. The recombinant yeast strain according to claim 5, characterized in that The target protein is α-lactalbumin.

7. The recombinant yeast strain according to claim 5, characterized in that The copy number of the target protein encoding gene is 1-10.

8. The recombinant yeast strain according to claim 7, characterized in that The copy number of the target protein encoding gene is 2-5.

9. The recombinant yeast strain according to claim 5, characterized in that The exogenous target protein expression system capable of being induced to express by ethanol comprises a first expression cassette and a second expression cassette; Wherein, the first expression cassette comprises an ethanol-inducible promoter, a LacI encoding gene and a Mit1AD encoding gene; The second expression cassette contains 5 to 9 copies of the LacO encoding gene, a yeast promoter that can be activated by Mit1AD, a signal peptide, and a target protein encoding gene.

10. The recombinant yeast strain according to any one of claims 6 to 8, characterized in that The exogenous target protein expression system capable of being induced to express by ethanol comprises a first expression cassette and a second expression cassette; Wherein, the first expression cassette comprises an ethanol-inducible promoter, a LacI encoding gene and a Mit1AD encoding gene; The second expression cassette contains 5 to 9 copies of the LacO encoding gene, a yeast promoter that can be activated by Mit1AD, a signal peptide, and a target protein encoding gene.

11. The recombinant yeast strain according to claim 9, characterized in that The ethanol-inducible promoter is the ICL1 promoter; and / or the yeast promoter that can be activated by Mit1AD is the core sequence core AOX1 of the AOX1 promoter.

12. The recombinant yeast strain according to claim 10, characterized in that The ethanol-inducible promoter is the ICL1 promoter; and / or the yeast promoter that can be activated by Mit1AD is the core sequence core AOX1 of the AOX1 promoter.

13. The recombinant yeast strain according to claim 9 or 11, characterized in that The signal peptide is a serum albumin hybrid signal peptide.

14. The recombinant yeast strain according to claim 10 or 12, characterized in that The signal peptide is a serum albumin hybrid signal peptide.

15. The recombinant yeast strain according to any one of claims 1 to 4, 6 to 9, and 11 to 12, characterized in that: The acetic acid accumulation of the recombinant yeast strain during the process of ethanol-induced expression of the target protein is significantly reduced.

16. Use of the recombinant yeast strain according to any one of claims 1 to 15 in expressing a target protein by induction with ethanol or in constructing a production strain for expressing a target protein by induction with ethanol.

17. The use according to claim 16, characterized in that The target protein is α-lactalbumin.

18. A method for preparing α-lactalbumin, characterized in that: The method comprises: culturing the recombinant yeast strain according to any one of claims 1 to 15 to make it express α-lactalbumin, and recovering the α-lactalbumin in the culture; Wherein, the target protein in the recombinant yeast strain is α-lactalbumin.

19. The method according to claim 18, characterized in that The culture comprises the step of adding ethanol to induce expression and the step of adding sodium hexametaphosphate.

20. The method according to claim 19, characterized in that During the ethanol induction expression phase, the fermentation volume was 6-12 mL·(L·h) -1 Ethanol was added at a rate of 0.5-1.2 mL / h / L, and 45-55 g / L of sodium hexametaphosphate was added at a rate of 0.8-1.2 mL / h / L.

21. The method according to claim 20, characterized in that First, relative to the initial fermentation volume of 7-8 mL·(L·h) -1 Ethanol was added at a rate of 1.5-2.5 h, and then the flow rate was adjusted to 10-12 mL·(L·h) relative to the initial fermentation volume. -1 Until the end of cultivation.

22. A method for reducing acetic acid accumulation in Pichia pastoris during ethanol-induced expression of a target protein, characterized in that: The method comprises: introducing an exogenous gene encoding polyphosphate kinase into the Pichia pastoris; Wherein, the promoter controlling the transcription of the exogenous gene encoding polyphosphate kinase is the ADH2 promoter; The amino acid sequence of the polyphosphate kinase is shown in SEQ ID NO.1; The Pichia pastoris is Pichia pastoris ( Pichia pastoris ).

23. The method according to claim 22, characterized in that The method comprises: introducing an expression cassette containing an exogenous gene encoding polyphosphate kinase into the Pichia pastoris, wherein the expression cassette is capable of expressing the polyphosphate kinase under ethanol induction conditions; The expression cassette comprises an ADH2 promoter for controlling the transcription of an exogenous gene encoding polyphosphate kinase and an exogenous gene encoding polyphosphate kinase.

24. The method according to claim 23, wherein The expression cassette of the exogenous gene encoding polyphosphate kinase further comprises a terminator.

25. The method according to claim 24, characterized in that The terminator is the AOX1 terminator.

26. The method according to claim 22, characterized in that The expression cassette of the exogenous gene encoding polyphosphate kinase is integrated into the genome of the yeast strain.

27. The method according to any one of claims 22 to 26, characterized in that The target protein is α-lactalbumin.

Images