Method for improving salt, alkali and permeation tolerance of saccharomyces cerevisiae

Abstract

The invention discloses a method for improving tolerance of saccharomyces cerevisiae to salt, alkali and osmotic stress, which comprises the following steps: simultaneously transferring a transcription factor gene LbWIN1 and an enzyme gene LbCER1 gene of lycium ruthenicum into a uracil auxotroph saccharomyces cerevisiae INVSc1 strain by using pESC-URA-GAL1-GAL10 plasmid to obtain a recombinant yeast; according to the invention, a reasonable pESC-URA-GAL1: LbWIN1-GAL10: LbCER1 plasmid construction method is adopted, so that it is ensured that LbWIN1 and LbCER1 genes are inserted behind a specified promoter in a correct direction. A stress tolerance evaluation experiment shows that the tolerance of the recombinant yeast to salt, alkali and osmotic stress is greatly enhanced, the stress tolerance of a fermentation strain taking saccharomyces cerevisiae as chassis cells is promoted, and the fermentation production efficiency is improved. The invention provides a solution for the problem of low tolerance to salt, alkali and osmotic stress of genetic engineering strains commonly encountered in the fermentation industry.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology and relates to a method for improving the salt, alkali and osmotic tolerance of brewer's yeast, and particularly relates to a method for improving the salt, alkali and osmotic stress tolerance of brewer's yeast by introducing LbWIN1 and LbCER1 genes of Lycium barbarum. Background Art

[0002] Saccharomyces cerevisiae is an important chassis cell in the fermentation industry. However, osmotic stress often occurs during the fermentation process as high-concentration products accumulate. In addition, as fermentation proceeds, the composition of the culture medium changes, and salt and alkali stress may occur at any time, inhibiting strain growth or product accumulation and affecting the fermentation effect. Therefore, improving product tolerance as well as salt, alkali and osmotic tolerance is one of the important directions in the development of Saccharomyces cerevisiae breeding.

[0003] Wax inducer 1 / SHN1 (WIN1 / SHN1) is a member of the AP2 / ethylene response factor (AP2 / ERF) transcription factor family and is closely related to plant growth and development, epidermal wax synthesis, drought and cold resistance and other stress responses. Aldehyde decarbonylase (ECERIFERUM1, CER1) is a key enzyme in the decarbonylation pathway, the main pathway for epidermal wax synthesis in plants. It can catalyze the conversion of long-chain even-carbon aldehydes into long-chain odd-carbon alkanes, participate in the synthesis of odd-numbered alkanes and their derivatives, and is closely related to the plant's response to salt and drought stress. A large number of studies have shown that WIN1 can improve plant stress resistance by regulating the expression of the CER1 gene. The LbWIN1 and LbCER1 genes of Lycium barbarum have been cloned and proved to be induced by NaCl and osmotic stress, indicating that LbWIN1 and LbCER1 are involved in plant responses to abiotic stresses such as salt and drought. There have been reports on the use of plant-derived genes to improve yeast tolerance to abiotic stresses such as salt, alkali, and osmosis. For example, Yang Na et al. used the pYES2 plasmid to transfer the thioredoxin (TRX) gene HbTRXo2 from the rubber tree (Hevea brasiliensis) into the uracil auxotrophic yeast INVSc1 strain to improve the resistance of the recombinant yeast to drought and oxidative stress, but reduced its resistance to low temperature stress. Deng Chengju et al. used the pYES2 plasmid to transfer the banana glyoxalase gene MaGLO14 into the uracil auxotrophic yeast INVSc1 strain to improve resistance to NaCl, high temperature, low temperature, drought, and UV stress. However, there have been no reports on the simultaneous transfer of two genes, transcription factor and enzyme genes, to improve the abiotic stress tolerance of Saccharomyces cerevisiae. In addition, there are no reports on whether the LbWIN1 and LbCER1 genes from Lycium barbarum, a plant source, can be simultaneously expressed in yeast, a fungus, and what effects they have on the tolerance of the recombinant yeast to abiotic stresses such as salt, alkali, and osmosis. Summary of the invention

[0004] The purpose of the present invention is to provide a method for improving the tolerance of saccharomyces cerevisiae to salt, alkali and osmotic stress by transferring LbWIN1 and LbCER1 genes of the xerophyte Lycium barbarum to improve the stress resistance of yeast.

[0005] To achieve the above object, the present invention adopts the following technical solution:

[0006] The invention provides a method for improving the salt, alkali and osmotic tolerance of saccharomyces cerevisiae, which comprises co-expressing LbWIN1 and LbCER1 genes of Lycium barbarum in the saccharomyces cerevisiae INVSc1 strain, wherein the transgenic yeast expression plasmid is pESC-URA-GAL1-GAL10.

[0007] As a further preferred embodiment of the technical solution of the present invention, the method comprises the following steps:

[0008] pESC-URA-GAL1:LbWIN1-GAL10:LbCER1 plasmid construction;

[0009] Preparation of competent yeast cells;

[0010] Lithium acetate method was used to transform competent yeast cells;

[0011] Recombinant yeast screening;

[0012] Testing of the tolerance of recombinant yeast to salt, alkali and osmotic stress;

[0013] Evaluation of the tolerance of recombinant yeast to salt, alkali and osmotic stress.

[0014] Furthermore, the pESC-URA-GAL1:LbWIN1-GAL10:LbCER1 plasmid construction method comprises the following steps:

[0015] (1) Extraction of pESC-URA-GAL1-GAL10 plasmid: The LbWIN1 open reading frame with BamH I and Kpn I restriction sites at both ends and the LbCER1 open reading frame with BamH I and Kpn I restriction sites at both ends were amplified by PCR, and the target bands were recovered; the pESC-URA-GAL1-GAL10 plasmid was extracted from the Escherichia coli culture medium;

[0016] (2) Construction of pESC-URA-GAL1:LbWIN1 plasmid: The pESC-URA-GAL1-GAL10 plasmid in step (1) was double-digested with BamH I and Kpn I, and a large fragment of 6544 bp was recovered after complete digestion to obtain a linearized plasmid 1; the LbWIN1 open reading frame amplified by PCR in step (1) was ligated with the linearized plasmid 1 to obtain a recombinant pESC-URA-GAL1:LbWIN1 plasmid, and the plasmid was transformed into competent Escherichia coli DH5α cells;

[0017] (3) Construction of pESC-URA-GAL10:LbCER1 plasmid: The pESC-URA-GAL1-GAL10 plasmid in step (1) was double-digested with NotI and SpeI, and a large fragment of 6544 bp was recovered after complete digestion to obtain a linearized plasmid 2; the LbCER1 open reading frame amplified by PCR in step (1) was connected with the linearized plasmid 2, so that LbCER1 was inserted behind the GAL10 promoter of the linearized plasmid 2 to obtain the pESC-URA-GAL10:LbCER1 plasmid, and the plasmid was transformed into competent Escherichia coli DH5α cells;

[0018] (4) Construction of pESC-URA-GAL1:LbWIN1-GAL10:LbCER1 plasmid: The pESC-URA-GAL10:LbCER1 plasmid was double-digested with BamH I and Kpn I, and the 8425 bp large fragment was recovered to obtain a linearized plasmid 3; the LbWIN1 open reading frame obtained by PCR amplification in step (1) was connected with the linearized plasmid 3, so that LbWIN1 was inserted behind the GAL1 promoter of the linearized plasmid 3, and the pESC-URA-GAL1:LbWIN1-GAL10:LbCER1 plasmid was successfully constructed, and transformed into competent Escherichia coli DH5α cells.

[0019] Furthermore, the method for preparing the competent yeast cells is:

[0020] A single colony of Saccharomyces cerevisiae was picked from the YPD solid medium, inoculated into the YPD liquid medium, and cultured overnight to activate the Saccharomyces cerevisiae; the activated bacterial solution was inoculated into the YPD liquid medium, and cultured with shaking until the OD600 value of the bacterial solution was 0.4-0.6; the yeast cells were washed by centrifugation, and then the yeast cells were slowly resuspended with TE / LiAc solution to obtain competent yeast cells.

[0021] Furthermore, the culture condition of the single colony of Saccharomyces cerevisiae in YPD liquid culture medium is inverted culture at 30° C. for 2 to 4 days.

[0022] Furthermore, the method of transforming competent yeast cells by the lithium acetate method is:

[0023] 0.2 μg of plasmid DNA was taken into 100 μL competent yeast cells, and then 15 μL salmon sperm DNA and 400 μL PEG / LiAc solution were added. After mixing, 70 μL DMSO was added. After heat shock in a water bath, 100 μL 1×TE buffer was added and evenly spread on the yeast culture medium. Inverted culture was carried out at 30°C for 3 to 4 days until a single colony was grown. Saccharomyces cerevisiae transformed with the empty plasmid pESC-URA-GAL1-GAL10 was used as a control.

[0024] Furthermore, the water bath heat shock temperature is 40-45°C, and the heat shock time is 10-20 minutes.

[0025] Furthermore, since the cerevisiae yeast is a uracil auxotrophic cerevisiae INVSc1 strain, the strain cannot grow on the SC-Ura auxotrophic solid culture medium, and only the strain into which the pESC-URA-GAL1-GAL10 plasmid is transferred can grow and form colonies. Therefore, the recombinant yeast screening process of the present invention is: culturing the cerevisiae INVSc1 strain on the SC-Ura auxotrophic solid culture medium, and the recombinant yeast that forms colonies is the successfully transformed recombinant yeast.

[0026] Furthermore, the detection of the tolerance of the recombinant yeast to salt, alkali and osmotic stress specifically includes the following steps:

[0027] (1) The successfully transformed recombinant yeasts were inoculated on yeast culture medium with a bacterial loop and cultured at 30°C for 48 h; a single colony was inoculated on yeast culture medium at 30°C and 200 r / min for 24 h; the yeast cell OD was adjusted with yeast liquid culture medium 600 The value was adjusted to 0.4, and then inoculated into yeast culture medium at a ratio of 1:100, cultured at 30°C and 200 r / min for 24 h, and the recombinant yeast cells were collected by centrifugation; the OD of the Saccharomyces cerevisiae transformed with the empty plasmid pESC-URA-GAL1-GAL10 and the recombinant yeast were adjusted with yeast liquid culture medium 600 Value to 2;

[0028] (2) Salt, alkali and osmotic stress:

[0029] Salt stress: resuspend the recombinant yeast cells in 2-8 M NaCl solution and treat for 1 h;

[0030] Alkaline stress: resuspend the recombinant yeast cells in 4-10% NaHCO3 solution and treat for 1 hour;

[0031] Osmotic stress included PEG6000 stress and sorbitol stress;

[0032] PEG6000 stress: resuspend the recombinant yeast cells in 20-60% PEG6000 solution and treat for 1 h;

[0033] Sorbitol stress: resuspend the recombinant yeast cells in 2-8 M sorbitol solution and treat for 1 h;

[0034] (3) The bacterial solution treated with stress in step (2) is gradiently diluted and inoculated on a yeast culture medium for cultivation, and the colony growth conditions of the recombinant yeast strain and the wild yeast strain are compared.

[0035] Furthermore, the evaluation method of the tolerance of recombinant yeast to salt, alkali and osmotic stress is as follows:

[0036] When Saccharomyces cerevisiae transformed with the empty plasmid pESC-URA-GAL1-GAL10 did not grow on yeast culture medium at a certain dilution concentration after stress treatment, the recombinant Saccharomyces cerevisiae after stress treatment grew colonies, even at high dilutions, indicating that the recombinant Saccharomyces cerevisiae had enhanced tolerance to stress.

[0037] By adopting the above technical solution, the beneficial effects achieved by the present invention are:

[0038] The present invention uses pESC-URA-GAL1-GAL10 plasmid to simultaneously transfer the transcription factor gene LbWIN1 and the enzyme gene LbCER1 into the uracil auxotrophic yeast INVSc1 strain to obtain recombinant yeast; through a reasonable pESC-URA-GAL1:LbWIN1-GAL10:LbCER1 plasmid construction method, it ensures that the LbWIN1 and LbCER1 genes are correctly inserted behind the specified promoter. The stress tolerance evaluation experiment shows that the tolerance of the recombinant yeast to salt, alkali and osmotic stress is greatly enhanced, which promotes the adversity tolerance of the fermentation strain with Saccharomyces cerevisiae as the chassis cell and improves the fermentation production efficiency. BRIEF DESCRIPTION OF THE DRAWINGS

[0039] Figure 1 These are the results of the salt, alkali and osmotic stress tolerance tests of the recombinant yeast. DETAILED DESCRIPTION

[0040] The implementation process of the method for improving the salt, alkali and osmotic tolerance of brewer's yeast of the present invention is described in detail below.

[0041] In the following implementation process of the present invention, the flat wolfberry seeds were collected from Yujie wolfberry introduction base in Jingtai County, Baiyin City, Gansu Province in August 2014; 1 / 2 Hoagland nutrient solution includes 1mmol / LKNO3, 0.25mmol / L NH4H2PO4, 0.125mmol / L MgSO4·7H2O, 0.125mmol / L Ca(NO3)2·4H2O, 0.25mmol / LFe-citrate, 46mmol / LH3BO3, 9μmol / L MnCl2·4H2O, 0.8μmol / LZnSO4·7H2O, 0.3μmol / LCuSO4·5H2O, 0.35μmol / L(NH4)6Mo7O 24·4H2O; total RNA was extracted using the Trizol UNIQ-10 column total RNA extraction kit from Shanghai Bioengineering Co., Ltd.; cDNA was synthesized using the primeScriptTMⅡ first-strand cDNA synthesis kit from Shanghai Bioengineering Co., Ltd.; gel recovery used the NucleoSpin Gel and PCR Clean-Up gel recovery kit from TAKARA; plasmid DNA was extracted using the SanPrep column plasmid DNA small-scale extraction kit from Shanghai Bioengineering Co., Ltd.; PCR kit produced by TAKARA was used for PCR amplification, In- HD Cloning Kit is used to connect the gene fragment to the linearized plasmid. The cerevisiae yeast is a uracil auxotrophic cerevisiae INVSc1 strain.

[0042] SC-URA medium, PEG3350, and 10×TE buffer were purchased from Coolaber; lithium acetate, salmon sperm DNA, and DMSO were purchased from Beijing Solebow. The primers used for PCR amplification are shown in Table 1.

[0043] Table 1 Primer sequences

[0044]

[0045] (1) Material preparation: Select seeds of Lycium barbarum with full grains, sterilize and germinate them, plant them in vermiculite and add nutrient solution. Change the nutrient solution every 3 days and place them in a place with a day and night temperature of (24±2)℃ / (18±2)℃ and a light exposure time of 16 h·d. -1 , the light intensity is about 600 μmol·m -2 ·s -1 , in an environment with a relative humidity of about 50%, when the seedlings grow to 3 weeks old, water the seedlings with 1 / 2 Hoagland nutrient solution containing 80 mmol / LD-sorbitol for 24 hours, take 100 mg of leaves and quick-freeze them in liquid nitrogen, and refer to the instructions of the total RNA extraction kit and the cDNA synthesis kit to extract the total RNA of the leaves and then synthesize the first-chain cDNA.

[0046] (2) Using the above cDNA as a template, clone the LbWIN1 open reading frame with BamH I and Kpn I restriction sites at both ends and the LbCER1 open reading frame with BamH I and Kpn I restriction sites at both ends by PCR. Prepare a 1.2% agarose gel and electrophoresed the PCR products. The LbWIN1 fragment size should be 631 bp and the LbCER1 gene fragment size should be 1901 bp. Quickly cut the target fragment with the correct band position under ultraviolet light, use the SanPrep column DNA gel recovery kit to recover the target fragment, and store it at -20°C for later use. Use the SanPrep column plasmid DNA small-scale extraction kit to extract the target plasmid from Escherichia coli containing pESC-URA-GAL1-GAL10.

[0047] (3) Construction of pESC-URA-GAL1:LbWIN1 plasmid. The pESC-URA-GAL1-GAL10 plasmid was double-digested with BamH I and Kpn I, and the digestion system was 30°C for 3h. The plasmid was digested with BamH I and Kpn I respectively, and the undigested plasmid was used as a control. After agarose gel electrophoresis, the 6544bp large fragment band was recovered using the SanPrep column DNA gel recovery kit as linearized plasmid 1. The PCR-amplified LbWIN1 open reading frame and the linearized plasmid 1 were connected together using In-Fusion HD Enzyme Premix to obtain the recombinant pESC-URA-GAL1:LbWIN1 plasmid, which was then transformed into competent Escherichia coli DH5α cells.

[0048] (4) Construction of pESC-URA-GAL10:LbCER1 plasmid. The pESC-URA-GAL1-GAL10 plasmid was double-digested with NotI and SpeI, and the digestion system was 30°C for 3h. The plasmid was digested with NotI and SpeI respectively, and the undigested plasmid was used as a control. After agarose gel electrophoresis, the 6544bp large fragment band was recovered using the SanPrep column DNA gel recovery kit as linearized plasmid 2. The PCR-amplified LbCER1 open reading frame and the linearized plasmid 2 were connected together using In-Fusion HD Enzyme Premix enzyme to obtain the recombinant pESC-URA-GAL10:LbCER1 plasmid, which was then transformed into competent Escherichia coli DH5α cells.

[0049] (5) Construction of pESC-URA-GAL1:LbWIN1-GAL10:LbCER1 plasmid. The pESC-URA-GAL1:LbCER1 plasmid was double-digested with BamH I and Kpn I, and the digestion system was 30°C for 1 h. After agarose gel electrophoresis, the 8425 bp large fragment band was recovered using the SanPrep column DNA gel recovery kit as linearized plasmid 3; HD CloningPlus homologous recombination enzyme connects linearized plasmid 3 to LbWIN1 open reading frame. Use primers WIN1-F and WIN1-R to verify the fragment size of bacterial liquid PCR, and the fragment size should be 600bp; use primers CER1-F and CER1-R to verify the fragment size of bacterial liquid PCR, and the fragment size should be 1881bp. Sequencing verifies the correctness of plasmid construction, and the correctly constructed pESC-URA-GAL1:LbWIN1-GAL10:LbCER1 plasmid is transformed into competent E. coli DH5α cells.

[0050] (6) Preparation of competent yeast cells. Inoculate Saccharomyces cerevisiae on YPD solid medium and incubate at 30°C for 2 days; pick a single colony and inoculate it in 5 mL YPD liquid medium and culture it at 30°C overnight; pipette an appropriate amount of yeast liquid into 50 mL YPD liquid medium and culture it at 30°C and 170 r / min for 3 h, so that the OD600 value of the liquid is 0.4; centrifuge the liquid at 3000 r / min for 5 min and discard the supernatant; add 30 mL of sterile water to the yeast precipitate to suspend the yeast cells, centrifuge at 3000 r / min for 5 min and discard the supernatant; add 1.5 mL of TE / LiAc solution to a centrifuge tube and slowly suspend the yeast to obtain competent yeast cells, and pipette 100 μL from each tube into each 1.5 mL EP tube.

[0051] (7) Lithium acetate method to transform competent yeast cells. Take 0.2 μg of plasmid DNA in 100 μL of competent yeast cells and add 15 μL of salmon sperm DNA (10 mg mL -1, boiled for 5 minutes in advance, cooled on an ice box for later use), repeatedly blown and mixed with a pipette; draw 400μL of PEG / LiAc solution into the EP tube, mix the solution with a vortexer for 10 seconds, and culture on a shaker at 30℃ for 40 minutes; add 70μL DMSO to the above solution, mix it upside down; heat shock it in a 42℃ water bath for 15 minutes, and quickly put it in an ice box for 2 minutes; centrifuge at 12000r / min at room temperature for 30 seconds, remove the supernatant, and then add 100μL 1×TE buffer to each EP tube. After mixing, draw the yeast solution and evenly spread it on the SC-Ura nutrient-deficient solid medium with a coating rod, and culture it upside down at 30℃ for 3 days to grow a single colony. Saccharomyces cerevisiae transformed with the empty plasmid pESC-URA-GAL1-GAL10 was used as a control.

[0052] (8) Screening of recombinant yeast. The Saccharomyces cerevisiae INVSc1 strain was cultured on SC-Ura nutrient-deficient solid medium, and the colonies formed were the recombinant yeasts that were successfully transformed.

[0053] (9) Salt, alkali and osmotic stress tolerance test of recombinant yeast. The successfully transformed recombinant yeast was inoculated on yeast culture medium with a bacterial loop and cultured at 30°C for 48 h; a single colony was inoculated on yeast culture medium at 30°C and 200 r / min for 24 h; the OD value of the yeast was adjusted using yeast liquid culture medium. 600 to 0.4; the yeast cells were inoculated into yeast culture medium at a ratio of 1:100, and cultured at 30°C and 200 r / min for 24 h; the OD of empty yeast and recombinant yeast was adjusted with yeast liquid culture medium 600 To 2.

[0054] The salt stress method is as follows: 1 mL of each of the above yeast cells is taken, centrifuged at 5000 r / min for 1 min to collect the cells, and the supernatant is discarded. 1 mL of 5M NaCl solution is added, and the cells are suspended and stressed at 30°C and 200 r / min for 1 h.

[0055] The alkaline stress method is as follows: 1 mL of each of the above yeast cells is taken, centrifuged at 5000 r / min for 1 min to collect the cells, and the supernatant is discarded. 1 mL of 8% NaHCO3 solution is added, and the cells are suspended and stressed at 30°C and 200 r / min for 1 h.

[0056] Osmotic stress included PEG6000 stress and sorbitol stress;

[0057] The PEG6000 stress method is as follows: 1 mL of each of the above yeast cells is taken, centrifuged at 5000 r / min for 1 min to collect the cells, and the supernatant is discarded. 1 mL of 40% PEG6000 solution is added, the cells are suspended, and then placed in a 30°C shaker and stressed at 200 r / min for 1 h.

[0058] The sorbitol stress method is as follows: 1 mL of each of the above yeast cells is taken, centrifuged at 5000 r / min for 1 min to collect the cells, and the supernatant is discarded. A 5M sorbitol solution is added, the cells are suspended, and then placed in a 30°C shaker and stressed at 200 r / min for 1 h.

[0059] Dilute the above treated bacterial solutions by 10 1 , 10 2 , 10 3 , 10 4 , 10 5 2 μL of the bacterial suspension was spotted on yeast culture medium and cultured at 30°C for 48 h. The pESC-URA strain of Saccharomyces cerevisiae (wild strain INVSc1 of Saccharomyces cerevisiae), pESC-URA-GAL1:LbWIN1 strain of Saccharomyces cerevisiae, pESC-URA-GAL10:LbCER1 strain of Saccharomyces cerevisiae, and

[0060] The growth status of the pESC-URA-GAL1:LbWIN1--GAL10:LbCER1 strain is as follows:

[0061] (1) Saccharomyces cerevisiae wild strain INVSc1 was treated with 5 M NaCl for 1 h and diluted to 10 -4 The yeast transformed with LbCER1 showed no growth when diluted to 10 -4 Poor growth, 10 -5 The yeast transformed with LbWIN1 showed no growth when diluted to 10 -3 Poor growth, 10 -4 However, yeast transformed with LbWIN1 and LbCER1 did not grow when diluted to 10 -5 It can still grow normally and form obvious colonies when the LbWIN1 and LbCER1 genes are simultaneously introduced into the yeast Saccharomyces cerevisiae, which has enhanced tolerance to salt stress.

[0062] (2) Saccharomyces cerevisiae wild strain INVSc1 was treated with 5 M sorbitol for 1 h and diluted to 10 -4 When the yeast was diluted to 10 -3 The yeast transformed with LbWIN1 showed no growth when diluted to 10 -3 However, yeast transformed with LbWIN1 and LbCER1 genes did not grow when diluted to 10-3 When it grows vigorously, it forms colonies. -4 This indicates that the tolerance of Saccharomyces cerevisiae transformed with LbWIN1 and LbCER1 to osmotic stress is enhanced.

[0063] (3) After the wild strain INVSc1 of Saccharomyces cerevisiae was treated with 8% NaHCO3 for 1 h, it was diluted to 10 -4 The yeast transformed with LbCER1 showed no growth when diluted to 10 -3 Poor growth, 10 -4 The yeast transformed with LbWIN1 showed no growth when diluted to 10 -4 However, yeast transformed with LbWIN1 and LbCER1 genes showed no growth when diluted to 10 -5 It can still grow normally and form obvious colonies when LbWIN1 and LbCER1 are introduced into the yeast, indicating that the tolerance of Saccharomyces cerevisiae to alkaline stress is enhanced.

[0064] (4) After the wild-type yeast INVSc1 was treated with 40% PEG6000 for 1 h, it was diluted to 10 -4 When the yeast was diluted to 10 -4 Poor growth, 10 -5 The yeast transformed with LbWIN1 showed no growth when diluted to 10 -3 However, yeast transformed with LbWIN1 and LbCER1 genes did not grow when diluted to 10 -5 It can still grow normally and form obvious colonies when LbWIN1 and LbCER1 are introduced into the yeast, indicating that the tolerance of Saccharomyces cerevisiae to osmotic stress is enhanced.

Claims

1. A method for improving the salt, alkali and osmotic tolerance of brewer's yeast, characterized in that: The method comprises the following steps: co-expressing LbWIN1 and LbCER1 genes of Lycium barbarum in the INVSc1 strain of Saccharomyces cerevisiae, wherein the yeast expression plasmid for transgenic is pESC-URA-GAL1-GAL10.

2. A method for improving the salt, alkali and osmotic tolerance of brewer's yeast according to claim 1, characterized in that: The method comprises the following steps: pESC-URA-GAL1:LbWIN1-GAL10:LbCER1 plasmid construction; Preparation of competent yeast cells; Lithium acetate method was used to transform competent yeast cells; Recombinant yeast screening; Testing of the tolerance of recombinant yeast to salt, alkali and osmotic stress; Evaluation of the tolerance of recombinant yeast to salt, alkali and osmotic stress.

3. A method for improving the salt, alkali and osmotic tolerance of brewer's yeast according to claim 2, characterized in that: The pESC-URA-GAL1:LbWIN1-GAL10:LbCER1 plasmid construction method comprises the following steps: (1) Extraction of pESC-URA-GAL1-GAL10 plasmid: The LbWIN1 open reading frame with BamH I and Kpn I restriction sites at both ends and the LbCER1 open reading frame with BamH I and Kpn I restriction sites at both ends were amplified by PCR, and the target bands were recovered; the pESC-URA-GAL1-GAL10 plasmid was extracted from the Escherichia coli culture medium; (2) Construction of pESC-URA-GAL1:LbWIN1 plasmid: The pESC-URA-GAL1-GAL10 plasmid in step (1) was double-digested with BamH I and Kpn I, and a large fragment of 6544 bp was recovered after complete digestion to obtain a linearized plasmid 1; the LbWIN1 open reading frame amplified by PCR in step (1) was ligated with the linearized plasmid 1 to obtain a recombinant pESC-URA-GAL1:LbWIN1 plasmid, and the plasmid was transformed into competent Escherichia coli DH5α cells; (3) Construction of pESC-URA-GAL10:LbCER1 plasmid: The pESC-URA-GAL1-GAL10 plasmid in step (1) was double-digested with NotI and SpeI, and a large fragment of 6544 bp was recovered after complete digestion to obtain a linearized plasmid 2; the LbCER1 open reading frame amplified by PCR in step (1) was connected with the linearized plasmid 2, so that LbCER1 was inserted behind the GAL10 promoter of the linearized plasmid 2 to obtain the pESC-URA-GAL10:LbCER1 plasmid, and the plasmid was transformed into competent Escherichia coli DH5α cells; (4) Construction of pESC-URA-GAL1:LbWIN1-GAL10:LbCER1 plasmid: The pESC-URA-GAL10:LbCER1 plasmid was double-digested with BamH I and Kpn I, and the 8425 bp large fragment was recovered to obtain a linearized plasmid 3; the LbWIN1 open reading frame obtained by PCR amplification in step (1) was connected with the linearized plasmid 3, so that LbWIN1 was inserted behind the GAL1 promoter of the linearized plasmid 3, and the pESC-URA-GAL1:LbWIN1-GAL10:LbCER1 plasmid was successfully constructed, and transformed into competent Escherichia coli DH5α cells.

4. A method for improving the salt, alkali and osmotic tolerance of brewer's yeast as claimed in claim 3, characterized in that: The preparation method of the competent yeast cells is as follows: A single colony of Saccharomyces cerevisiae was picked from the YPD solid medium, inoculated into the YPD liquid medium, and cultured overnight to activate the Saccharomyces cerevisiae; the activated bacterial solution was inoculated into the YPD liquid medium, and cultured with shaking until the OD600 value of the bacterial solution was 0.4-0.6; the yeast cells were washed by centrifugation, and then the yeast cells were slowly resuspended with TE / LiAc solution to obtain competent yeast cells.

5. A method for improving the salt, alkali and osmotic tolerance of brewer's yeast as claimed in claim 4, characterized in that: The culture condition of the single colony of Saccharomyces cerevisiae in the YPD liquid culture medium is inverted culture at 30° C. for 2 to 4 days.

6. A method for improving the salt, alkali and osmotic tolerance of brewer's yeast according to claim 5, characterized in that: The method for transforming competent yeast cells using the lithium acetate method is as follows: In 100 μL competent yeast cells, take 0.2 μg of plasmid DNA, add 15 μL salmon sperm DNA and 400 μL PEG / LiAc solution, mix well, add 70 μL DMSO, heat shock in a water bath, add 100 μL 1×TE buffer, evenly spread on yeast culture medium, and culture upside down at 30°C for 3 to 4 days until a single colony grows; Saccharomyces cerevisiae transformed with the empty plasmid pESC-URA-GAL1-GAL10 was used as a control.

7. A method for improving the salt, alkali and osmotic tolerance of brewer's yeast according to claim 6, characterized in that: The water bath heat shock temperature is 40-45°C, and the heat shock time is 10-20 minutes.

8. A method for improving the salt, alkali and osmotic tolerance of brewer's yeast according to claim 7, characterized in that: The recombinant yeast screening process is as follows: culturing the Saccharomyces cerevisiae INVSc1 strain on the SC-Ura nutritional deficiency solid culture medium, and the colonies formed are the recombinant Saccharomyces cerevisiae that have been successfully transformed.

9. A method for improving the salt, alkali and osmotic tolerance of Saccharomyces cerevisiae according to any one of claims 1 to 8, characterized in that: The detection of the tolerance of recombinant yeast to salt, alkali and osmotic stress specifically includes the following steps: (1) The successfully transformed recombinant yeasts were inoculated on yeast culture medium with a bacterial loop and cultured at 30°C for 48 h; a single colony was inoculated on yeast culture medium at 30°C and 200 r / min for 24 h; the yeast cell OD was adjusted with yeast liquid culture medium 600 The value was adjusted to 0.4, and then inoculated into yeast culture medium at a ratio of 1:100, cultured at 30°C and 200 r / min for 24 h, and the recombinant yeast cells were collected by centrifugation; the OD of the Saccharomyces cerevisiae transformed with the empty plasmid pESC-URA-GAL1-GAL10 and the recombinant yeast were adjusted with yeast liquid culture medium 600 Value to 2; (2) Salt, alkali and osmotic stress: Salt stress: resuspend the recombinant yeast cells in 2-8 M NaCl solution and treat for 1 h; Alkaline stress: resuspend the recombinant yeast cells in 4-10% NaHCO3 solution and treat for 1 hour; Osmotic stress included PEG6000 stress and sorbitol stress; PEG6000 stress: resuspend the recombinant yeast cells in 20-60% PEG6000 solution and treat for 1 h; Sorbitol stress: resuspend the recombinant yeast cells in 2-8 M sorbitol solution and treat for 1 h; (3) The bacterial solution treated with stress in step (2) is gradiently diluted and inoculated on a yeast culture medium for cultivation, and the colony growth conditions of the recombinant yeast strain and the wild yeast strain are compared.

10. A method for improving the salt, alkali and osmotic tolerance of brewer's yeast according to claim 9, characterized in that: The evaluation method of the tolerance of recombinant yeast to salt, alkali and osmotic stress is as follows: When Saccharomyces cerevisiae transformed with the empty plasmid pESC-URA-GAL1-GAL10 did not grow on yeast culture medium at a certain dilution concentration after stress treatment, the recombinant Saccharomyces cerevisiae after stress treatment grew colonies, even at high dilutions, indicating that the recombinant Saccharomyces cerevisiae had enhanced tolerance to stress.

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