Method for reducing acetic acid accumulation in process of ethanol-induced expression of alpha-lactalbumin by regulating pichia pastoris

Abstract

The invention relates to the technical field of gene engineering, in particular to a method for reducing acetic acid accumulation in the process of ethanol-induced expression of alpha-lactalbumin by regulating pichia pastoris. According to the recombinant yeast strain provided by the invention, the accumulation amount of acetic acid is greatly reduced in the process of inducing expression of target protein by adopting ethanol, acetic acid can be basically not accumulated, relatively high biomass and target protein yield can be achieved, and the highest yield of human alpha-lactalbumin up to now is achieved; and a strain resource and a foundation are provided for application of an ethanol-induced pichia pastoris expression system and development of bioengineering milk protein. The invention provides a method for solving the problem of acetic acid accumulation of pichia pastoris in the process of inducing expression of the target protein by using ethanol, and the method provides an effective strategy for popularization and application of an ethanol-induced expression system and improvement of the yield of the target protein induced and expressed by using the ethanol. The method has important significance on production of proteins such as alpha-lactalbumin and the like.

Description

Technical Field

[0001] The invention relates to the technical field of genetic engineering, and in particular to a method for regulating Pichia pastoris to reduce acetic acid accumulation during 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] At present, Pichia pastoris mostly uses a methanol induction system to express exogenous proteins. However, the expression system using methanol as an inducer and as the sole carbon source has huge hidden dangers in industrial production and food production. Since methanol is flammable, explosive and toxic, it is regarded as a potential factor threatening safe production in industrial production. In order to reduce the negative impact of methanol to a certain extent, the expression system ESAD with ethanol as an inducer and the sole carbon source was invented. However, in actual applications, the ESAD system is very likely to produce a large amount of acetic acid accumulation under the condition of continuous feeding of ethanol in the fermenter, which leads to cell death and interruption of fermentation. Therefore, it is urgent to solve the problem of large accumulation of acetic acid during ethanol induction. Summary of the invention

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

[0005] In order to solve the problem of large-scale accumulation of acetic acid in the expression system (ESAD) of Pichia pastoris using ethanol as an inducer and the only carbon source during ethanol induction, the present invention constructs an ATP replenishment tool in the ESAD system of Pichia pastoris to promote the conversion of acetic acid into acetyl-CoA, which not only completely solves the problem of acetic acid accumulation, but also provides more energy for the fermentation process. In practical applications, the protein expression amount and high-intensity fermentation feasibility in the ESAD system are greatly improved.

[0006] Specifically, the present invention provides the technical solution described below.

[0007] In a first aspect, the present invention provides a recombinant yeast strain, wherein the recombinant yeast strain comprises 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 finds that the introduction of exogenous polyphosphate kinase into a yeast strain can promote the conversion of acetic acid produced during ethanol induction into acetyl-CoA, thereby realizing ATP replenishment. This not only solves the problem of acetic acid accumulation, but also provides more energy for the fermentation process, and can relieve the inhibition of acetic acid on bacterial growth and protein expression, thereby significantly increasing biomass and the expression amount of target protein.

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

[0010] Polyphosphate kinase is contained in a variety of microorganisms. The present invention finds that, compared with polyphosphate kinases from other sources, polyphosphate kinase derived from Staphylococcus epidermidis is more suitable for playing a role 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 amount of the target protein.

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

[0012] Based on the amino acid sequence of the polyphosphate kinase, those skilled in the art can obtain the sequence of its coding gene. Based on the degeneracy of codons, the gene sequence encoding the same amino acid sequence is not unique, and all nucleotide sequences capable of encoding the polyphosphate kinase are within the protection scope of the present invention. To facilitate the expression of the polyphosphate kinase, those skilled in the art can optimize the codons thereof as needed.

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

[0014] The above-mentioned foreign gene encoding polyphosphate kinase is present 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 controlling the transcription of the exogenous gene encoding polyphosphate kinase and the exogenous gene encoding polyphosphate kinase.

[0016] The present invention has carried out a large number of screenings on promoters that control the transcription of exogenous genes encoding polyphosphate kinase, 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 induction conditions, and thus is more conducive to reducing acetic acid accumulation during the fermentation process, and its effect of increasing the expression amount of the target protein is better.

[0017] Preferably, the promoter 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; the present invention has no particular limitation on the selection of the terminator in principle, and any sequence that can terminate 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. Optional integration sites include Int6 (ChrII: 17928-17947) and the like.

[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; 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 the 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 as the core element of the hybrid promoter expression system, through ethanol induction to control the expression of LacI-Mit1AD activator, through 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 (its coding sequence 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 coding gene sequence of 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 coding gene sequence of 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 that can be 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 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 to increasing the yield of α-lactalbumin. The above ethanol-induced α-lactalbumin expression system cooperates with the introduction of exogenous polyphosphate kinase, so that the accumulation of acetic acid in the yeast strain during the ethanol-induced α-lactalbumin expression process is greatly reduced, and the yield of α-lactalbumin is greatly increased.

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

[0036] Preferably, the acetic acid accumulation of the recombinant yeast strain during the ethanol-induced expression of the target protein is significantly reduced. The recombinant yeast strain can substantially not accumulate acetic acid when ethanol is the only 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 also includes the step of adding sodium hexametaphosphate.

[0041] Preferably, the ethanol induction expression stage comprises: 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 fermentation volume is 7-8 mL·(L·h) relative to the initial fermentation volume. -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, comprising the following steps: (1) activating the recombinant yeast strain on a solid culture medium; (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; (3) The seed solution from step (2) was inoculated at a ratio of 5%-10% of the initial fermentation volume until 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), the fermentation temperature was 29-32°C, and the dissolved oxygen was maintained above 18-22%; (4) After the dissolved oxygen rebounds, start the 50-70% w / v glycerol feed, 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 The initial fermentation volume was fed with glycerol for 3-6 h; (5) Stop adding glycerol and induce the fermentation with 11-13 mL of trace element solution (PTM1) per liter of pure ethanol solution, setting the feed rate to 3.5-3.7 mL h per liter of original fermentation volume. -1 During the first 2-3 h, ethanol will accumulate in the fermenter and dissolved oxygen values ​​will be unstable as the culture adapts to the ethanol; (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; 48-52 g / L sodium hexametaphosphate is added at 0.9-1.1 mL / h / L; the feed rate is 7.1-7.5 mL·(L·h) in the first 2 hours -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.

[0044] Preferably, the trace element solution (PTM1) comprises the following components: 63.0-67.0 g·L -1 FeSO 4 7H 2 O, 215.0-225.0 g·L -1 ZnCl, 5.0-7.0 g L -1 CuSO4·5H 2 O, 2.5-3.5 g L -1 MnSO 4 ·H 2 O, 20.0-21.0 g·L -1CoCl, 0.15-0.25 g L -1 MoNa 2 O 4 ·2H 2 O, 0.15-0.25 g·L -1 D-biotin, 0.08-0.10 g·L -1 KI, 0.01-0.03 g L -1 H 3 BO 3 ; 4.8-5.2 mL concentrated H 2 SO 4 .

[0045] Preferably, the fermentation basal salts medium (BSM) comprises the following components: 35.0-45.0 g·L -1 Glycerol, 16-20 g·L -1 K 2 SO 4 , 14.5-15.5 g·L -1 MgSO 4 7H 2 O, 4.10-4.20 g·L - 1 KOH, 0.9-1.0 g L -1 CaSO 4 , 26-28 mL phosphoric acid, 4.2-4.4 mL·L -1 PTM1.

[0046] Preferably, the culture medium used for the activation and seed culture is YPD medium.

[0047] During the above fermentation process, the wet weight of cells can increase to 450-500 g·L -1 ; The final fermentation volume will be approximately twice the initial fermentation volume.

[0048] 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: (1) picking a single colony of the recombinant yeast strain into a primary seed liquid culture medium (preferably YPD), and culturing at 29-32° C. and 200-220 rpm for 16-18 hours; (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; (3) Collect all the cells by centrifugation, transfer to an induction expression medium (preferably BMEY), and add 0.05-0.1 g / L sodium hexametaphosphate. 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, and then collect the fermentation supernatant.

[0049] 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.

[0050] The present invention verifies through experiments that the accumulation of acetic acid can be significantly reduced and the yield of the target protein can be increased by introducing a foreign gene encoding polyphosphate kinase into Pichia pastoris that is induced to express the target protein by ethanol.

[0051] Preferably, the polyphosphate kinase is derived from Staphylococcus epidermidis ( Staphylococcus epidermidis ).

[0052] Preferably, the polyphosphate kinase has an amino acid sequence shown in SEQ ID NO.1.

[0053] In the above method, the exogenous gene encoding polyphosphate kinase is preferably introduced in the form of an expression cassette.

[0054] 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; 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.

[0055] Preferably, the expression cassette of the exogenous gene encoding polyphosphate kinase further comprises a terminator; the terminator is preferably an AOX1 terminator.

[0056] Preferably, the expression cassette of the exogenous gene encoding polyphosphate kinase is integrated into the genome of the recombinant yeast strain.

[0057] Preferably, the target protein is α-lactalbumin.

[0058] 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.

[0059] 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.

[0060] 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 multi-copy 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.

[0061] 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).

[0062] Preferably, the signal peptide is a serum albumin hybrid signal peptide (its sequence is preferably as shown in SEQ ID NO.7).

[0063] 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.

[0064] The beneficial effects of the present invention include at least: the accumulation of acetic acid in the recombinant yeast strain provided by the present invention during the process of expressing the target protein by ethanol induction is greatly reduced, and basically no accumulation of acetic acid can be achieved, and a high biomass and target protein yield can be achieved, achieving the highest yield of human α-lactalbumin to date, and providing strain resources and a basis for the application of the ethanol-induced Pichia pastoris expression system and the development of bioengineering milk protein. The present invention provides a method for solving the problem of acetic acid accumulation in the process of expressing the target protein by ethanol induction in Pichia pastoris, which provides an effective strategy for the promotion and application of the ethanol-induced expression system and the improvement of the yield of the target protein expressed by ethanol induction, and is of great significance for the production of proteins such as α-lactalbumin. BRIEF DESCRIPTION OF THE DRAWINGS

[0065] In order to more clearly illustrate the technical solutions in the present invention or the prior art, the following briefly introduces 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 creative work.

[0066] Figure 1 is the OD of the growth of the ESAD-LA3 strain in Example 2 of the present invention using ethanol as an inducer and the sole carbon source in a traditional fermentation method. 600 The results of the liquid chromatography analysis of the acetic acid accumulation.

[0067] Figure 2 The results of verifying the promoting effect of co-expression of SePPK and addition of sodium hexametaphosphate in the culture medium on acetic acid consumption at the shake flask level 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).

[0068] Figure 3 The results are the monitoring results and SDS-PAGE gel electrophoresis results of the experimental group of the fermentation tank amplification experiment of the strain ESAD-LA3-ADH2-PPK in Example 4 of the present invention, in which 10 g / L sodium hexametaphosphate was added.

[0069] Figure 4 These are the monitoring results and SDS-PAGE gel electrophoresis results of the experimental group of the fermentation tank amplification experiment of the strain ESAD-LA3-ADH2-PPK in Example 4 of the present invention, in which 50 g / L sodium hexametaphosphate was added.

[0070] Figure 5 These are the monitoring results and SDS-PAGE gel electrophoresis results of the fermentation tank amplification experiment of the strain ESAD-LA3-ADH2-PPK in Example 4 of the present invention, in which 100 g / L sodium hexametaphosphate was added. DETAILED DESCRIPTION

[0071] 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 copies (3 copies) of α-lactalbumin gene) that can secrete and express α-lactalbumin as a starting strain, and co-expresses polyphosphokinase (SePPK, derived from Staphylococcus epidermidis) in the starting strain to utilize ADP and PPi in the cell to efficiently replenish ATP, thereby solving to a certain extent the problem that acetic acid rapidly generated by ethanol assimilation during fermentation is difficult to be converted into acetyl-CoA due to lack of sufficient ATP supply and accumulates in large quantities. Specifically, the ADH2 promoter, which can be expressed at a high level under the induction condition of ethanol as the only carbon source, was selected to co-express SePPK, and 50 g / L of sodium hexametaphosphate (SHMP, which can generate ATP under the action of SePPK) was added at a rate of 0.8-1.2 mL / h / L to completely solve the problem of acetic acid accumulation during the fermentation process. Finally, the modified yeast strain ESAD-LA3-ADH2-PPK was scaled up in a 3L fermenter to increase the yield of α-lactalbumin to 1.3 g / L.

[0072] 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: introducing a polyphosphate kinase expression frame into the recombinant Pichia pastoris ESAD-LA3 capable of secreting and expressing α-lactalbumin, specifically comprising the following steps: First, the ADH2-SePPK-AOX1T (ADH2 promoter sequence, SePPK gene sequence, AOX1 terminator sequence from 5'-3' direction) expression cassette was constructed using fusion PCR technology. The insertion site of the expression cassette in the yeast strain genome was Int6 (ChrII:17928-17947); then, 500bp of homology arms were added to the front and back ends of the expression cassette by fusion PCR technology to obtain the final donor DNA. At the same time, a Crispr plasmid with sgRNA and Cas9 gene corresponding to Int6 was constructed and named Crispr-Int6. Finally, the donor DNA and Crispr-Int6 plasmid were simultaneously introduced into ESAD-LA3 by electroporation to obtain the strain ESAD-LA3-ADH2-PPK.

[0073] In a specific embodiment of the present invention, a method for preparing α-lactalbumin is provided, which uses the above-mentioned recombinant Pichia yeast strain to ferment and produce α-lactalbumin. The method can be applied in shake flask fermentation or fermentation in a fermenter; wherein, in shake flask fermentation, ethanol induction is used, and sodium hexametaphosphate at a lower concentration (0.05-0.1 g / L) is added; in fermentation in a fermenter, ethanol is fed, and 50 g / L of sodium hexametaphosphate is fed at a rate of 0.8-1.2 mL / h / L. The method solves the problem of acetic acid accumulation in the fermentation of the ethanol-induced Pichia yeast expression system due to continuous feeding of more ethanol as the sole carbon source, and the strain grows well during the fermentation process. In fermentation in a fermenter, the expression level of the final target protein α-lactalbumin reached 1.3 g / L, and acetic acid was not accumulated throughout the process.

[0074] In order to make the purpose, technical solution and advantages of the present invention clearer, the technical solution of the present invention will be clearly and completely described below in conjunction with the drawings of the present invention. Obviously, the described embodiments are part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0075] In the following examples, the medium formula used for seed culture and fermentation of recombinant Pichia pastoris is as follows: The seed liquid culture medium formula is YPD medium: 1% yeast extract, 2% peptone, 2% glucose. The ethanol fermentation medium (BMEY) formula is: yeast extract 10 g / L, peptone 20 g / L, K 2 HPO 4 ·3H 2 O 3 g / L, KH 2 PO 4 11.8 g / L, ultrapure water 894 mL, sterilize at 121℃ for 20 min, and when the temperature reaches 60℃, add 100 mL of 10×YNB, 1 mL of 500×B (0.02% biotin), and 5 mL of ethanol in the clean bench. PTM1 trace salts solution: 65.0 g·L -1 FeSO 4 7H 2 O, 220.0 g·L -1 ZnCl, 6.0 g L -1 CuSO4·5H 2 O, 3.0 g L -1 MnSO 4 ·H 2 O, 20.5 g·L-1 CoCl, 0.2 g L -1 MoNa 2 O 4 ·2H 2 O, 0.2 g L -1 D-biotin, 0.09 g·L -1 KI, 0.02 g·L -1 H 3 BO 3 ; 5.0 mL concentrated H 2 SO 4 BSM (Basal salts medium): 40.0 g·L -1 Glycerol, 18 g·L - 1 K 2 SO 4 、14.9 g·L -1 MgSO4·7H 2 O, 4.13 g·L -1 KOH, 0.93 g·L -1 CaSO 4 , 27 mL·L -1 Phosphoric acid, 4.4 mL·L -1 PTM1. Glycerol feed medium: Prepare 50% (w / v) glycerol solution with deionized water, sterilize it, and add 12 mL·L of filtered sterilized -1 PTM1 solution.

[0076] The shake flask fermentation method used in the following examples comprises the following steps: (1) Pick a single colony and place it in 5 mL of primary seed medium (YPD) at 30 o C, 220 rpm for 16-18 hours; (2) Take 100 µL of the first seed solution and transfer it to 50 mL of the second seed solution medium (YPD) and incubate at 30 o C, fermentation at 220 rpm for 24 hours; (3) Collect all the cells by centrifugation and transfer them 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 only carbon source) added every 24 h. The fermentation supernatant was then collected, purified and quantified.

[0077] The fermentation tank fermentation method used in the following examples comprises the following steps: (1) The recombinant Pichia pastoris strain was streaked on YPD solid medium and incubated in a 30°C constant temperature incubator for about 3 days until a clearly visible single colony was formed; (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; (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, thus obtaining the fermentation basal salt medium (BSM medium). Subsequently, the seed solution obtained in step (2) is 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 above 20% (1.5 vvm); (4) After the dissolved oxygen rebounded, start the 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 -1 The initial fermentation volume was glycerol fed for about 4 hours or more; (5) Stop adding glycerol and induce the feed with 12 mL of pure ethanol solution containing PTM1 per liter, setting the feed rate to 3.6 mL h per liter of initial fermentation volume. -1 During the first 2-3 h, ethanol will accumulate in the fermenter and dissolved oxygen values ​​will be unstable as the culture adapts to the ethanol; (6) After maintaining the low ethanol feed rate for at least 1 hour, double the feed rate to 7.3 mL·(L·h) -1 Initial fermentation volume can further increase the rate of product formation. At the same time, 50 g / L sodium hexametaphosphate was added at 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 , which remains unchanged for the rest of the fermentation. 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 twice the initial fermentation volume.

[0078] The sample detection method used in the following examples includes the following steps: First, the fermentation broth is centrifuged and the supernatant is taken, filtered through a 0.45 µm water filter, and then purified using a His-Binding-resin purification column, and the purified sample is collected for SDS-PAGE electrophoresis. Protein quantification uses a BradFord kit.

[0079] Example 1 Construction of ethanol-induced α-lactalbumin producing strain ESAD-LA3 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 by 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 Core sequence of promoter core AOX1 Keep, and replace the rest with 5 LacO Repeat sequence, followed by core AOX1 Insert the sequence later 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 After that, the plasmid was linearized using restriction enzymes EcoRI and SalI, and then the linearized recombinant plasmid was integrated into the histidine-deficient strain GS115 of Pichia pastoris 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 be high-copy, thereby obtaining 5LacO-coreAOX1-SA Pro-LALBA-6His The transformant with a copy number of 3 was named ESAD-LA3.

[0080] Example 2 Determination of acetic acid accumulation level during scale-up culture of ESAD-LA3 strain induced by ethanol in a fermenter The strain ESAD-LA3 constructed in Example 1 was fermented using the aforementioned fermentation method without adding sodium hexametaphosphate. The results showed that ( Figure 1 ), when using the traditional fermentation method, due to the large amount of ethanol added, the amount of bacteria in the fermenter decreased significantly after 46 hours, and a large amount of acetic acid could be clearly detected in the fermentation liquid at 58 hours, which resulted in the inability to proceed normally. Finally, at the end of the fermentation at 108 hours, the acetic acid content in the fermentation liquid reached 134 g / L.

[0081] Example 3 Verification of the promoting effect of co-expression of SePPK and addition of sodium hexametaphosphate to the culture medium on acetic acid consumption at the shake flask level Based on ESAD-LA3, the expression frames 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, the fusion PCR technology was used to construct the ADH2-SePPK-AOX1T, AOX1-SePPK-AOX1T, DAS2-SePPK-AOX1T, FDH1-SePPK-AOX1T, GAP-SePPK-AOX1T, ICL1-SePPK-AOX1T (promoter sequence, SePPK gene sequence, AOX1 terminator sequence from 5'-3' direction), and the insertion site of the expression cassette in the genome was selected as Int6 (ChrII:17928-17947), and then the fusion PCR technology was used again to add 500bp homology arms at the front and back ends of the fusion expression cassette to obtain the final donor DNA. At the same time, a Crispr plasmid with sgRNA and Cas9 gene corresponding to Int6 was constructed, named 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 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 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 without adding sodium hexametaphosphate, adding 0.1 g / L sodium hexametaphosphate, and adding 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 Promoter overexpression of SePPK can most efficiently replenish ATP and solve the problems of acetate accumulation and weakened growth to the greatest extent.

[0082] Example 4 Fermentation tank scale-up experiment on strain ESAD-LA3-ADH2-PPK By analyzing the experimental results of the above shake flask fermentation, the growth status and acetic acid accumulation of the strain were comprehensively analyzed, and ESAD-LA3-ADH2-PPK was selected as the strain for subsequent experiments. Subsequently, the fermentation scale-up experiment of ESAD-LA3-ADH2-PPK was carried out. Since the conclusion that the experimental group with 0.1 g / L sodium hexametaphosphate addition had generally good growth and generally low acetic acid accumulation was obtained in the shake flask experiment, 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 with 10 g / L (1 mL / h / L) of ... Figure 3 )、50 g / L( Figure 4 )、100 g / L( Figure 5 Finally, in the experimental group with 50 g / L sodium hexametaphosphate added, no acetic acid accumulated during the entire fermentation cycle, and 1.3 g / L α-lactalbumin was obtained.

[0083] 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 embodiments of the present invention.

Claims

1. A recombinant yeast strain, characterized in that: The recombinant yeast strain comprises an exogenous gene encoding polyphosphate kinase and an exogenous target protein expression system capable of being induced to express by ethanol.

2. The recombinant yeast strain according to claim 1, characterized in that The polyphosphate kinase is derived from Staphylococcus epidermidis ( Staphylococcus epidermidis ); Preferably, the polyphosphate kinase has an amino acid sequence shown in SEQ ID NO.

1.

3. The recombinant yeast strain according to claim 1 or 2, 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 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.

4. The recombinant yeast strain according to claim 3, characterized in that The expression cassette of the exogenous gene encoding polyphosphate kinase further comprises a terminator; the terminator is preferably an AOX1 terminator; Preferably, 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; The target protein is preferably α-lactalbumin; The copy number of the target protein encoding gene is 1-10, preferably 2-5.

6. 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 comprises 5 to 9 copies of a LacO encoding gene, a yeast promoter that can be activated by Mit1AD, a signal peptide, and a target protein encoding gene; Preferably, 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; Preferably, the signal peptide is a serum albumin hybrid signal peptide.

7. The recombinant yeast strain according to any one of claims 1 to 6, characterized in that The yeast is Pichia pastoris; preferably Pichia pastoris ( Pichia pastoris ) Preferably, the accumulation of acetic acid in the recombinant yeast strain during ethanol-induced expression of the target protein is significantly reduced.

8. Use of the recombinant yeast strain according to any one of claims 1 to 7 in expressing a target protein by inducing ethanol or in constructing a production strain for expressing a target protein by inducing ethanol; Preferably, the target protein is α-lactalbumin.

9. 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 7 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.

10. The method according to claim 9, characterized in that The culturing includes the step of adding ethanol to induce expression, and also includes the step of adding sodium hexametaphosphate; Preferably, the ethanol induction expression stage comprises: 6-12 mL·(L·h) relative to the initial fermentation volume -1 Ethanol was added at a rate of 0.8-1.2 mL / h / L, and 45-55 g / L of sodium hexametaphosphate was added at a rate of 0.8-1.2 mL / h / L; Preferably, the fermentation volume is 7-8 mL·(L·h) relative to the initial fermentation volume. -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.

11. 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 a foreign gene encoding polyphosphate kinase into the Pichia pastoris.

12. The method according to claim 11, characterized in that The polyphosphate kinase is derived from Staphylococcus epidermidis ( Staphylococcus epidermidis ); Preferably, the polyphosphate kinase has an amino acid sequence shown in SEQ ID NO.

1.

13. The method according to claim 11 or 12, 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 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.

14. The method according to claim 13, characterized in that The expression cassette of the exogenous gene encoding polyphosphate kinase further comprises a terminator; the terminator is preferably an AOX1 terminator; Preferably, the expression cassette of the exogenous gene encoding polyphosphate kinase is integrated into the genome of the recombinant yeast strain.

15. The method according to any one of claims 11 to 14, characterized in that: The target protein is α-lactalbumin.

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