CN104560756A - Method for preparing high-stress-resistance yeast strain - Google Patents

Method for preparing high-stress-resistance yeast strain Download PDF

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CN104560756A
CN104560756A CN201410535557.5A CN201410535557A CN104560756A CN 104560756 A CN104560756 A CN 104560756A CN 201410535557 A CN201410535557 A CN 201410535557A CN 104560756 A CN104560756 A CN 104560756A
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medium
yeast
albumen
nucleic acid
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CN104560756B (en
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高岚
郭勇
刘金胜
刘颖
李宝石
蔺建民
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Sinopec Research Institute of Petroleum Processing
China Petroleum and Chemical Corp
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Sinopec Research Institute of Petroleum Processing
China Petroleum and Chemical Corp
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    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/37—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from fungi
    • C07K14/39—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from fungi from yeasts
    • C07K14/395—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from fungi from yeasts from Saccharomyces

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Abstract

The invention discloses a method for preparing a high-stress-resistance yeast strain. The method comprises the following step: in a glucose culture medium, increasing the expression quantity of a protein which has nucleotide sequences as shown in SEQ ID No. 1 and is expressed in yeast cells, wherein the glucose culture medium comprises glucose and an acetate ion source. By increasing the expression quantity of the protein which has nucleotide sequences as shown in SEQ ID No. 1 and is expressed in the yeast cells, the tolerance of the yeast cells against an inhibitor is significantly enhanced and therefore, the high-stress-resistance yeast strain capable of producing ethanol by virtue of glucose under the environment of a higher-concentration inhibitor is prepared by the method disclosed by the invention.

Description

A kind of method preparing high resistance to cold and diseases yeast strain
Technical field
The present invention relates to a kind of method preparing high resistance to cold and diseases yeast strain.
Background technology
Increase because traditional fossil energy consumption is anxious, energy scarcity becomes worldwide problem, energy development in pluralism and accelerate renewable energy source exploitation and become the Research Emphasis of countries in the world, wherein, the production of alcohol fuel and be applied on Economic development and strategic security and demonstrate important meaning.The power supply diversification strategy being representative with substitute energys such as alcohol fuels has become the important directions of China's Energy policy.Mierocrystalline cellulose is very abundant and the biomass material of cheapness, can be degraded to fermentable sugar, for the production of alcohol fuel.Greatly develop biomass fuel ethanol as renewable energy source, contribute to alleviating petroleum resources shortage, improve the problems such as atmospheric environment, in stable grain-production, promote, in the benign cycle of agriculture production and consumption and Sustainable development etc., there is active effect.
Yeast saccharomyces cerevisiae (Saccharomyces cerevisiae), as unicellular eukaryote, has the advantages that easy cultivation, growth cycle are short, has been widely used in the expression of heterologous gene.Although common yeast saccharomyces cerevisiae can efficiency utilization glucose producing and ethanol, but to the inhibition in fermented liquid and the dense alcohol resistance of height (Almeida J R M et al., 2007) poor, particularly to the suppression constituent-sensitive in cellulosic hydrolysate, make yeast saccharomyces cerevisiae cannot become the dominant strain of lignocellulose alcohol production.Secondly, in cellulosic ethanol production, cellulosic material preprocessing process produces and residual degradation product can cause suppression in various degree, wherein acetic acid, furfural, phenols and SO to follow-up strain fermentation 4 2-stronger toxic action is had to yeast, the growth of the remarkable T suppression cell of meeting and leavening property thereof etc. supressor.
At present, at cellulosic ethanol production bacterial classification---the research field of yeast saccharomyces cerevisiae, mostly concentrating on various laboratories defective type bacterial classification is that research object is transformed, and lack the wild-type yeast model that can be applied to production, multiple inhibiting thing can be tolerated, there is the yeast saccharomyces cerevisiae of high resistance to cold and diseases there is stronger practicality, obviously, it is very important that the yeast strain that research can be cultivated in dextrose culture-medium obtains the saccharomyces model with above-mentioned characteristic to screening.
Summary of the invention
The object of the invention is to overcome the low defect of existing yeast strain resistance, a kind of method preparing high resistance to cold and diseases yeast strain is provided.
To achieve these goals, the invention provides and be a kind ofly prepared in dextrose culture-medium the method with high resistance to cold and diseases yeast strain, wherein, the method comprises: in dextrose culture-medium, increase the expression amount of the albumen of yeast cell to express aminoacid sequence as shown in SEQ ID NO:1, described dextrose culture-medium contains glucose and contains acetate ion source.
By increasing the expression amount of the albumen of yeast cell to express aminoacid sequence as shown in SEQ ID NO:1, the tolerance of yeast cell to inhibition obviously strengthens, and therefore, has been obtained the yeast strain had compared with high resistance to cold and diseases by method of the present invention.
Other features and advantages of the present invention are described in detail in embodiment part subsequently.
Embodiment
Below the specific embodiment of the present invention is described in detail.Should be understood that, embodiment described herein, only for instruction and explanation of the present invention, is not limited to the present invention.
In the present invention, when not doing contrary explanation, the term " resistance " used refers to that microorganism is in the growth departed from the environment under its optimal growth condition and fermentation capacity, such as higher or lower temperature, higher or lower pH, higher or lower osmotic pressure, higher feedback inhibition, the environment such as the existence of toxic substance.
The method preparing high resistance to cold and diseases yeast strain provided by the invention, it is characterized in that, the method comprises: in dextrose culture-medium, increase the expression amount of the albumen of yeast cell to express aminoacid sequence as shown in SEQ ID NO:1, described dextrose culture-medium contains glucose and contains acetate ion source.
mstngespapegagdsrdpsgflseiigapvtvklnsgivykgdlqsvdgymnialerckevaegrvirnwgdafvrgnnvtyisadna(SEQ ID NO:1)
In the present invention, described substratum can be the various yeast culture medium that with the addition of glucose, and under preferable case, described dextrose culture-medium contains the glucose of 5-100g/L (preferred 10-80g/L).
In described dextrose culture-medium, with the content meter of acetate moiety, the content containing acetate ion source is preferably 0.5-60g/L, is more preferably 2-40g/L.
In the present invention, can be the various compound that acetate ion can be provided containing acetate ion source, as water miscible acetic acid and/or acetate, be preferably at least one in acetic acid, sodium acetate, potassium acetate and ammonium acetate.
According to the preferred embodiment of the present invention, the aldehyde (as furfural) that described dextrose culture-medium can also contain muriate (can at least one in sodium-chlor, Repone K and ammonium chloride), vitriol (can be sodium sulfate and/or potassium sulfate), carbonatoms are 2-8 and carbonatoms are at least one in the phenol (as phenol) of 6-16.More preferably, described dextrose culture-medium is also containing the muriate of 25-95g/L (most preferably being 20-60g/L), the vitriol of 0.5-10g/L (most preferably being 4-6g/L), the carbonatoms of 0.1-1g/L (the most preferably being 0.1-0.5g/L) at least one that to be the aldehyde of 2-8 and the carbonatoms of 0.1-1g/L (most preferably being 0.1-0.5g/L) be in the phenol of 6-16.
In order to obtain the better yeast strain of resistance, further preferably, the Na of described dextrose culture-medium also containing 1-6g/L 2sO 4, at least one in the phenol of KCl, 0.1-0.5g/L of NaCl, 10-30g/L of 20-60g/L and the furfural of 0.1-0.5g/L.Most preferably, the Na of described dextrose culture-medium also containing 4-6g/L 2sO 4, 35-40g/L NaCl, 20-30g/L KCl at least one (being preferably no more than at most two kinds); Or, the furfural of described dextrose culture-medium also containing 0.15-0.3g/L; Or, the phenol of described dextrose culture-medium also containing 0.2-0.4g/L.
In the present invention, described dextrose culture-medium can also contain the peptone of 5-20g/L and the yeast extract of 2-8g/L, thinks that bacterial strain provides nitrogenous source and somatomedin etc.
A preferred embodiment of the invention, dextrose culture-medium of the present invention contains component A and B component, and wherein, described component A is the glucose of 10-80g/L and the acetic acid of 1-3g/L, and described B component is the Na of 4-6g/L 2sO 4, 35-40g/L the NH of KCl, 20-40g/L of NaCl, 20-30g/L 4one or both in the KAc of NaAc and 10-30g/L of Ac, 30-40g/L.
In the present invention, the various modes can commonly used by this area increase the expression amount of the albumen of yeast cell to express aminoacid sequence as shown in SEQ ID NO:1.Such as, the expression vector of the nucleic acid being inserted with the albumen of express amino acid sequence as shown in SEQ ID NO:1 can be transformed in yeast cell, thus increase the expression amount of described albumen.Therefore, the present invention to the special requirement of described yeast cell, as long as can the albumen of express amino acid sequence as shown in SEQ ID NO:1.Preferably, described yeast cell has the nucleic acid of base sequence as shown in SEQ ID NO:2, that is, the base sequence of the coding nucleic acid of the albumen of aminoacid sequence as shown in SEQID NO:1 is as shown in SEQ ID NO:2.Now, preferably use and be inserted with the nucleic acid of base sequence as shown in SEQ ID NO:2 and the expression vector transformed yeast cell with strong promoter, to increase the expression amount of the albumen of yeast cell to express aminoacid sequence as shown in SEQ ID NO:1.
gccaccatgtccggaaaagcttctacagagggtagcgttactacggagtttctctctgatatcattggtaagacagtgaacgtcaaacttgcctcgggtttactctacagcggaagattggaatccattgatggttttatgaatgttgcactatcgagtgccactgaacactacgagagtaataacaataagcttctaaataagttcaatagtgatgtctttttgaggggcacgcaggtcatgtatatcagtgaacaaaaaatatag(SEQ ID NO:2)
Wherein, described strong promoter is preferably Padh1 promotor, Ppgk promotor or Pfbp promotor.More preferably, described strong promoter is Padh1 promotor.
Wherein, described expression vector also can make any expression vector of this expression of nucleic acid for being inserted with the nucleic acid of base sequence as shown in SEQ ID NO:2, such as, and plasmid or phage.Preferably, described expression vector for inserting the nucleic acid of base sequence as shown in SEQ ID NO:2 and the expression vector obtained between the BamH1 restriction enzyme site and XhoI restriction enzyme site of plasmid pAUR123.
Wherein, to the terminator in described expression vector, there is no particular limitation, and those skilled in the art can select suitable terminator to build the expression vector can expressing the nucleic acid of base sequence as shown in SEQ ID NO:2 easily.
According to another kind of preferred implementation of the present invention, described yeast cell has the nucleic acid of base sequence as shown in SEQID NO:3, that is, the base sequence of the coding nucleic acid of the albumen of aminoacid sequence as shown in SEQ ID NO:1 is as shown in SEQ ID NO:3.Now, preferably use and be inserted with the nucleic acid of base sequence as shown in SEQ ID NO:3 and the expression vector transformed yeast cell with promotor, to increase the expression amount of the albumen of yeast cell to express aminoacid sequence as shown in SEQ ID NO:1.
gccaccatgtccggaaaagcctctacagagggtagcgttactacggagtttctctctgatatcattggtaagacagtgaacgtcaaacttgcctcgggtttactctacagcggaagattggaatccattgatggttttatgaatgttgcactatcgagtgccactgaacactacgagagtaataacaataagcttctaaataagttcaatagtgatgtctttttgaggggcacgcaggtcatgtatatcagtgaacaaaaaatatag(SEQ ID NO:3)
Wherein, described promotor is preferably Padh1 promotor, Ppgk promotor or Pfbp promotor.More preferably, described promotor is Padh1 promotor.
Wherein, described expression vector also can make any expression vector of this expression of nucleic acid for being inserted with the nucleic acid of base sequence as shown in SEQ ID NO:3, such as, and plasmid or phage.Preferably, described expression vector for inserting the nucleic acid of base sequence as shown in SEQ ID NO:3 and the expression vector obtained between the BamH1 restriction enzyme site and XhoI restriction enzyme site of plasmid pAUR123.
Wherein, to the terminator in described expression vector, there is no particular limitation, and those skilled in the art can select suitable terminator to build the expression vector can expressing the nucleic acid of base sequence as shown in SEQ ID NO:3 easily.
In the present invention, the mode increasing the expression amount of the albumen of yeast cell to express aminoacid sequence as shown in SEQ ID NO:1 can be the mode of conventional culturing yeast cell, at being preferably included in 28-33 DEG C, culturing yeast cell 24-72 hour in dextrose culture-medium.
Below will be described the present invention by embodiment.Experiment in following examples and detection method, if no special instructions, be ordinary method; Institute's use instrument, if no special instructions, is conventional sense and operating instrument; Experiment material used, if no special instructions, is and purchases available from routine biochemistry Reagent Company; Quantitative test in following examples, all arranges and repeats experiment for three times, results averaged.The substratum adopted in embodiment etc. are as follows:
The yeast strain used in embodiment---yeast saccharomyces cerevisiae (Saccharomyces cerevisiae) RIPP-0 is deposited in China Committee for Culture Collection of Microorganisms's common micro-organisms center on September 24th, 2013 and (is abbreviated as CGMCC, address: No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City, Institute of Microorganism, Academia Sinica, postcode: 100101), deposit number is CGMCC No.8202.
YEPD substratum: glucose 10g/L, peptone 10g/L, yeast extract 5g/L.
Screening culture medium: glucose 20g/L, peptone 10g/L, yeast extract 5g/L, agar powder 20g/L, acetic acid 3g/L, stress factors (Na 2sO 4, NaCl, KCl, NH 4ac, NaAc etc.) 5-40g/L, natural pH, is down flat plate for subsequent use.
Acid seed culture medium: glucose 20g/L, peptone 10g/L, yeast extract 5g/L, acetic acid 1-3g/L, pH5.0.
Glucose fermentation substratum: glucose 80g/L, peptone 5g/L, yeast extract 3g/L, CaCl 20.25g/L, MgCl 20.25g/L, KH 2pO 42.5g/L, pH 5.5, adds separately the stress factors such as acetic acid, furfural (suppression composition) as required as the glucose fermentation substratum containing suppression composition.
The composition of fermented liquid adopts high effective liquid chromatography for measuring: sample is after accurate dilutions, and first through 10000r/min high speed centrifugation 10min, supernatant liquor sample introduction after 0.45 μm of filtering with microporous membrane measures.High-efficient liquid phase chromatogram condition is: Agilent 1260 liquid chromatograph, Hi-plex H (8 μm, 7.7 × 30 μm) chromatographic column, and column temperature 60 DEG C, moving phase is acetonitrile: water (80:20), flow velocity 0.5mL/min.
Sugared content × 100% in sugar utilization (%)=(in the sugared content-fermented liquid in substratum residual sugar content)/substratum.
Ethanol production (g/L) is defined as the content of ethanol in often liter of fermented liquid, and its value is higher, show the ability of strain fermentation producing and ethanol and alcohol resistance stronger.
Embodiment 1
Entrust nucleic acid lsm6-Y and lsm6 of lucky agate (GenePharma) the company synthetic alkali basic sequence in Shanghai as shown in SEQ ID NO:2 and SEQID NO:3, with reference to " Molecular Cloning: A Laboratory guide (third edition) " (Sambrook J, 2001) method in, between the BamHI restriction enzyme site lsm6-Y and lsm6 being connected to yeast saccharomyces cerevisiae plasmid pAUR123 and XhoI restriction enzyme site, form recombinant expression vector pAUR-lsm6-Y and pAUR-lsm6.
Embodiment 2
(1) the mono-bacterium colony of picking yeast saccharomyces cerevisiae RIPP-0 is in yeast YEPD substratum, 30 DEG C, 180r/min is cultured to the logarithm middle and later periods (about 16h), the centrifugal 5min collecting cell of 4000r/min, with the Tris-HCl buffer solution twice of 4 DEG C of sterilized waters and pH 7.5,10mM, add 0.6M Sorbitol Solution USP suspension cell, be diluted to concentration and be about 10 5-10 6individual cell/mL, get 0.2ml bacterium liquid, embodiment 1 is obtained recombinant expression vector pAUR-lsm6-Y with electroporated method (with reference to " Molecular Cloning: A Laboratory guide (third edition) ", Sambrook J, 2001) proceed in yeast saccharomyces cerevisiae RIPP-0 Host Strains, bacterium liquid adds 1mL YEPD substratum, at 30 DEG C, hatch 1h.
(2) step (1) is hatched the bacterium liquid after 1h to be spread evenly across containing 3g/L acetic acid and 30g/LNH 4on two kinds of composite sifting flat boards of Ac and 3g/L acetic acid and 40g/L NaAc, at 30 DEG C, cultivate 60h, obtain primary dcreening operation bacterial strain 64 strain.
(3) primary dcreening operation bacterial strain is accessed in acid seed culture medium, 30 DEG C, 200r/min cultivates after 24h, obtain that biomass is normal, the strain of microscopy normal bacterial strain 35, centrifugal collecting cell obtains bacterium mud, and the access of bacterium mud is equipped with in the 250mL triangular flask of 100mL glucose fermentation substratum (acetic acid containing 3g/L), initial cell density is about 1.0-1.2g/L (dry cell weight basis), 30 DEG C, 120r/min carries out multiple sieve.Analyze Acetate tolerance and the ethanol production of above-mentioned bacterial strains, therefrom obtain the recombinant bacterial strain that 9 strain 30 DEG C tolerates 3g/L acetic acid, ethanol production is greater than 20g/L.And then acetic acid (3g/L), furfural (0.2g/L), phenol (0.3g/L), the Na to 9 strain bacterium 2sO 4(5g/L), NaCl (40g/L), KCl (25g/L), NH 4tolerance and the ethanol production of the stress factors such as Ac (20g/L), NaAc (30g/L) are analyzed, namely this 9 strain bacterium is cultivated by the screening culture medium containing stress factors as above, final acquisition 1 strain tolerates multiple inhibiting composition and has the Wine brewing yeast strain RIPP-20 of high resistance to cold and diseases, and this yeast strain all can grow in the screening culture medium containing above-mentioned stress factors and ethanol production is high.
(4) according to method in step (1)-(3), pAUR-lsm6 is proceeded in yeast saccharomyces cerevisiae RIPP-0 Host Strains with electroporated method, final acquisition 1 strain tolerates multiple inhibiting composition and has the yeast saccharomyces cerevisiae RIPP-06 of high resistance to cold and diseases, and this yeast strain all can grow in the screening culture medium containing above-mentioned stress factors and ethanol production is high.
Testing example 1
This testing example is used for the glucose fermentation performance of yeast strain illustrating that the present invention obtains.
(1) seed liquor is prepared
Yeast saccharomyces cerevisiae RIPP-0, RIPP-06 and RIPP-20 are inoculated in acid seed culture medium (containing acetic acid 3g/L) respectively, and 30 DEG C, 200r/min shaking culture 12h, obtain first order seed nutrient solution;
After described first order seed medium centrifugal, cell is transferred in seed culture medium (not containing acetic acid), and 30 DEG C, 200r/min shaking culture 16h, obtain seed liquor.
(2) glucose fermentation producing and ethanol
Seed liquor step (1) obtained is centrifugal, collecting cell obtains bacterium mud, by bacterium mud (with dry cell weight basis, control initial cell density is 1.2g/L) access and be equipped with in the shaking flask of the 250ml of 100ml glucose fermentation substratum, 30 DEG C, 120r/min shaking culture, carry out anaerobically fermenting.Each inoculation 3 Duplicate Samples, results averaged.
After above-mentioned condition fermentation 72h, get 5ml fermented liquid, the centrifugal 10min of 10000r/min, supernatant liquor carries out fermentation broth contents mensuration after 0.45 μm of filtering with microporous membrane.Result is as shown in table 1.
Testing example 2
This testing example is used for illustrating that the yeast strain that the present invention obtains is containing the leavening property in the glucose fermentation substratum suppressing composition (1g/L acetic acid).
(1) seed liquor is prepared
With the step (1) of testing example 1.
(2) suppress composition there is bottom fermentation glucose producing and ethanol
Seed liquor step (1) obtained is centrifugal, collecting cell obtains bacterium mud, by bacterium mud (with dry cell weight basis, control initial cell density is 1.2g/L) access and be equipped with in the shaking flask of 100ml containing the 250ml of the glucose fermentation substratum of suppression composition, all the other steps are with the step (2) of testing example 1.Result is as shown in table 1.
Testing example 3
This testing example is used for illustrating that the yeast strain that the present invention obtains is containing the leavening property in the glucose fermentation substratum suppressing composition (3g/L acetic acid).
(1) seed liquor is prepared
With the step (1) of testing example 1.
(2) suppress composition there is bottom fermentation glucose producing and ethanol
Seed liquor step (1) obtained is centrifugal, collecting cell obtains bacterium mud, by bacterium mud (with dry cell weight basis, control initial cell density is 1.2g/L) access and be equipped with in the shaking flask of 100ml containing the 250ml of the glucose fermentation substratum of suppression composition, all the other steps are with the step (2) of testing example 1.Result is as shown in table 1.
Testing example 4
This testing example is used for illustrating that the yeast strain that the present invention obtains is containing the leavening property in the glucose fermentation substratum suppressing composition (1g/L acetic acid and 0.2g/L furfural).
(1) seed liquor is prepared
With the step (1) of testing example 1.
(2) suppress composition there is bottom fermentation glucose producing and ethanol
Seed liquor step (1) obtained is centrifugal, collecting cell obtains bacterium mud, by bacterium mud (with dry cell weight basis, control initial cell density is 1.2g/L) access and be equipped with in the shaking flask of 100ml containing the 250ml of the glucose fermentation substratum of suppression composition, all the other steps are with the step (2) of testing example 1.Result is as shown in table 1.
Testing example 5
This testing example is used for illustrating that the yeast strain that the present invention obtains is containing the leavening property in the glucose fermentation substratum suppressing composition (1g/L acetic acid and 0.3g/L phenol).
(1) seed liquor is prepared
With the step (1) of testing example 1.
(2) suppress composition there is bottom fermentation glucose producing and ethanol
Seed liquor step (1) obtained is centrifugal, collecting cell obtains bacterium mud, by bacterium mud (with dry cell weight basis, control initial cell density is 1.2g/L) access and be equipped with in the shaking flask of 100ml containing the 250ml of the glucose fermentation substratum of suppression composition, all the other steps are with the step (2) of testing example 1.Result is as shown in table 1.
Table 1
Testing example 6
The increase of expression amount of the albumen of aminoacid sequence as shown in SEQ ID NO:1 in the Wine brewing yeast strain that this testing example is used for verifying that embodiment 2 obtains.
According to " Biochemistry Experiment guidance " (press of Wuhan University, 2011 publish) chapter 3 record method, SDS-PAGE (SDS-PAGE) is carried out to the total protein of yeast saccharomyces cerevisiae RIPP-0, RIPP-06 and RIPP-20.Result shows, the band of aminoacid sequence albumen as shown in SEQ ID NO:1 that yeast saccharomyces cerevisiae RIPP-06 and RIPP-20 produces, obviously wider than the band (about 2-5 doubly) of this albumen of yeast saccharomyces cerevisiae RIPP-0 generation, shows that yeast saccharomyces cerevisiae RIPP-06 and RIPP-20 increases to some extent relative to the expression amount of the albumen of aminoacid sequence in yeast saccharomyces cerevisiae RIPP-0 as shown in SEQ ID NO:1.
Embodiment 3
According to the method recombinant expression vector pAUR-lsm6 transformed saccharomyces cerevisiae S288C identical with embodiment 2, (purchased from ATCC, article No. is 204508D-5 tM), obtain 1 Accharomyces cerevisiae bacterial strain RIPP-D20, measure its leavening property according to the method identical with testing example 1-5, result is as shown in table 2 below.
Table 2
Saccharomyces cerevisiae S288C is the Wine brewing yeast strain of the not resistance to acetic acid of a strain, but the result as can be seen from table 2, increased the expression amount of the albumen of yeast cell to express aminoacid sequence as shown in SEQ ID NO:1 by the preferred mode of the present invention after, it strengthens the tolerance of acetic acid in glucose fermentation substratum.
Embodiment 4
Entrust the nucleic acid lsm6-N of the sudden change of lucky agate (GenePharma) the company synthetic alkali basic sequence in Shanghai as shown in SEQ ID NO:4, with reference to " Molecular Cloning: A Laboratory guide (third edition) " (Sambrook J, 2001) method in, between BamHI restriction enzyme site lsm6-N being connected to yeast saccharomyces cerevisiae plasmid pAUR123 and XhoI restriction enzyme site, form recombinant expression vector pAUR-lsm6-N.
gccaccatgtccggaaaagcatctacagagggtagcgttactacggagtttctctctgatatcattggtaagacagtgaacgtcaaacttgcctcgggtttactctacagcggaagattggaatccattgatggttttatgaatgttgcactatcgagtgccactgaacactacgagagtaataacaataagcttctaaataagttcaatagtgatgtctttttgaggggcacgcaggtcatgtatatcagtgaacaaaaaatatag(SEQ ID NO:4)
According to the method recombinant expression vector pAUR-lsm6-N transformed yeast strain RIPP-0 identical with embodiment 2, obtain 1 Accharomyces cerevisiae bacterial strain RIPP-D11, according to the method identical with testing example 1-5, its leavening property is measured, result is as shown in table 1, relatively can find out, the expression amount adopting the present invention preferred mode to increase the albumen of yeast cell to express aminoacid sequence as shown in SEQ ID NO:1 can obtain the higher and yeast strain that ethanol fermentation performance is better of resistance.
As can be seen from the above embodiments, method of the present invention can obtain and can utilize glucose and the yeast strain had compared with high resistance to cold and diseases, and its sugar utilization and ethanol production are all higher.
More than describe the preferred embodiment of the present invention in detail; but the present invention is not limited to the detail in above-mentioned embodiment, within the scope of technical conceive of the present invention; can carry out multiple simple variant to technical scheme of the present invention, these simple variant all belong to protection scope of the present invention.
It should be noted that in addition, each concrete technical characteristic described in above-mentioned embodiment, in reconcilable situation, can be combined by any suitable mode, in order to avoid unnecessary repetition, the present invention illustrates no longer separately to various possible array mode.
In addition, also can carry out arbitrary combination between various different embodiment of the present invention, as long as it is without prejudice to thought of the present invention, it should be considered as content disclosed in this invention equally.

Claims (12)

1. prepare the method for high resistance to cold and diseases yeast strain for one kind, it is characterized in that, the method comprises: in dextrose culture-medium, increases the expression amount of the albumen of yeast cell to express aminoacid sequence as shown in SEQ ID NO:1, and described dextrose culture-medium contains glucose and contains acetate ion source.
2. method according to claim 1, wherein, in described dextrose culture-medium, the content of glucose is 5-100g/L, is preferably 10-80g/L; With the content meter of acetate moiety, the content containing acetate ion source is 0.5-60g/L, is preferably 2-40g/L; At least one in acetic acid, sodium acetate, potassium acetate and ammonium acetate is preferably containing acetate ion source.
3. method according to claim 2, wherein, described dextrose culture-medium is also containing muriate, vitriol, the carbonatoms at least one that to be the aldehyde of 2-8 and carbonatoms be in the phenol of 6-16, preferably, described dextrose culture-medium is also containing the muriate of 25-95g/L, the vitriol of 0.5-10g/L, the carbonatoms of the 0.1-1g/L at least one that to be the aldehyde of 2-8 and the carbonatoms of 0.1-1g/L be in the phenol of 6-16.
4. according to the method in claim 2 or 3, wherein, the Na of described dextrose culture-medium also containing 1-6g/L 2sO 4, at least one in the phenol of KCl, 0.1-0.5g/L of NaCl, 10-30g/L of 20-60g/L and the furfural of 0.1-0.5g/L.
5. according to the method in claim 2 or 3, wherein, described dextrose culture-medium is also containing the peptone of 5-20g/L and the yeast extract of 2-8g/L.
6. method according to claim 1, wherein, the base sequence of the coding nucleic acid of the albumen of aminoacid sequence as shown in SEQ ID NO:1 is as SEQ ID NO:2.
7. method according to claim 6, wherein, use and be inserted with the nucleic acid of base sequence as shown in SEQ IDNO:2 and the expression vector transformed yeast cell with strong promoter, to increase the expression amount of the albumen of yeast cell to express aminoacid sequence as shown in SEQ ID NO:1.
8. method according to claim 7, wherein, described strong promoter is Padh1 promotor, Ppgk promotor or Pfbp promotor.
9. method according to claim 7, wherein, described expression vector for inserting the nucleic acid of base sequence as shown in SEQ ID NO:2 and the expression vector obtained between the BamH1 restriction enzyme site and XhoI restriction enzyme site of plasmid pAUR123.
10. method according to claim 1, wherein, the base sequence of the coding nucleic acid of the albumen of aminoacid sequence as shown in SEQ ID NO:1 is as shown in SEQ ID NO:3.
11. methods according to claim 10, wherein, use and be inserted with the nucleic acid of base sequence as shown in SEQ IDNO:3 and the expression vector transformed yeast cell with promotor, to increase the expression amount of the albumen of yeast cell to express aminoacid sequence as shown in SEQ ID NO:1; Described expression vector for inserting the nucleic acid of base sequence as shown in SEQ ID NO:3 and the expression vector obtained between the BamH1 restriction enzyme site and XhoI restriction enzyme site of plasmid pAUR123.
12. methods according to claim 1, wherein, at the mode increasing the expression amount of the albumen of yeast cell to express aminoacid sequence as shown in SEQ ID NO:1 is included in 28-33 DEG C, culturing yeast cell 24-72 hour in dextrose culture-medium.
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