CN104560756B - A method of preparing high resistance to cold and diseases yeast strain - Google Patents
A method of preparing high resistance to cold and diseases yeast strain Download PDFInfo
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Abstract
The invention discloses a kind of methods for preparing high resistance to cold and diseases yeast strain, wherein, this method comprises: increasing the expression quantity of yeast cell to express amino acid sequence albumen as shown in SEQ ID NO:1 in dextrose culture-medium, the dextrose culture-medium contains glucose and source containing acetate ion.By the expression quantity for increasing yeast cell to express amino acid sequence albumen as shown in SEQ ID NO:1, the tolerance of mortifier is remarkably reinforced in yeast cells, therefore, the high resistance to cold and diseases yeast strain that glucose producing and ethanol can be utilized in the higher environment of mortifier concentration has been made by means of the present invention.
Description
Technical field
The present invention relates to a kind of methods for preparing high resistance to cold and diseases yeast strain.
Background technique
Since traditional fossil energy dosage suddenly increases, energy shortages has become worldwide problem, and energy diversification develops and adds
Fast renewable energy exploitation has become the Research Emphasis of countries in the world, wherein the production of alcohol fuel and applies in economic development
With important meaning is shown on strategic security.It is had become by the energy supply diversification strategy of representative of alternative energy sources such as alcohol fuels
For the important directions of China's energy policy.Cellulose is very abundant and cheap biomass material, can be degraded to send out
Ferment sugar, the production for alcohol fuel.Biomass fuel ethyl alcohol is greatly developed as renewable energy, helps to alleviate petroleum
The problems such as shortage of resources, improvement atmospheric environment, is stablizing the benign cycle of grain-production, promotion agricultural production and consumption and can hold
Supervention exhibition etc. has positive effect.
Saccharomyces cerevisiae (Saccharomyces cerevisiae) is used as unicellular eukaryote, has easily culture, growth
Period short feature, is widely used to the expression of heterologous gene.Although common saccharomyces cerevisiae can be produced efficiently using glucose
Ethyl alcohol, but in fermentation liquid mortifier and highly concentrated alcohol resistance (Almeida J R M et al., 2007) it is poor, especially
It is to make saccharomyces cerevisiae that can not become the dominant bacteria of lignocellulosic ethyl alcohol production the inhibition constituent-sensitive in cellulosic hydrolysate
Strain.Secondly, cellulosic material preprocessing process generates and remaining degradation product can send out subsequent bacterial strain in cellulosic ethanol production
Ferment causes different degrees of inhibition, wherein acetic acid, furfural, phenols and SO4 2-Etc. inhibiting factors have stronger toxic action to yeast,
Growth and its fermenting property of cell can be significantly inhibited.
Currently, in cellulosic ethanol production strain --- the research field of saccharomyces cerevisiae is mostly focused on various experiments
Room deficiency strain is transformed for research object, and lacks the wild-type yeast model that can be applied to production, is resistant to
Multiple inhibiting object, the saccharomyces cerevisiae with high resistance to cold and diseases have stronger practicability, it is clear that research can be in dextrose culture-medium
It is particularly important that the yeast strain of middle culture obtains saccharomyces model with the above characteristics to screening.
Summary of the invention
The defect that the purpose of the present invention is overcome existing yeast strain resistance low provides and a kind of prepares high resistance to cold and diseases ferment
The method of mother strains.
To achieve the goals above, the present invention provides a kind of prepare has high resistance to cold and diseases saccharomycete in dextrose culture-medium
The method of strain, wherein this method comprises: increasing yeast cell to express amino acid sequence such as SEQ ID in dextrose culture-medium
The expression quantity of albumen shown in NO:1, the dextrose culture-medium contain glucose and source containing acetate ion.
By increasing the expression quantity of yeast cell to express amino acid sequence albumen as shown in SEQ ID NO:1, yeast is thin
The tolerance of mortifier is remarkably reinforced in born of the same parents, therefore, has been made by means of the present invention with the saccharomycete compared with high resistance to cold and diseases
Strain.
Other features and advantages of the present invention will the following detailed description will be given in the detailed implementation section.
Specific embodiment
Detailed description of the preferred embodiments below.It should be understood that described herein specific
Embodiment is merely to illustrate and explain the present invention, and is not intended to restrict the invention.
In the present invention, in the absence of explanation to the contrary, the term " resistance " used refers to that microorganism is being deviateed
The growth and fermentability in environment under its best growing condition, such as higher or lower temperature, higher or lower pH,
Higher or lower osmotic pressure, higher feedback inhibition, the environment such as presence of noxious material.
The method provided by the invention for preparing high resistance to cold and diseases yeast strain, which is characterized in that this method comprises: in glucose
In culture medium, increase the expression quantity of yeast cell to express amino acid sequence albumen as shown in SEQ ID NO:1, the glucose
Culture medium contains glucose and source containing acetate ion.
mstngespapegagdsrdpsgflseiigapvtvklnsgivykgdlqsvdgymnialerckevaegrvirnwgdafvr
gnnvt yisadna(SEQ ID NO:1)
In the present invention, the culture medium can be the various yeast culture mediums for being added to glucose, described under preferable case
Dextrose culture-medium contains the glucose of 5-100g/L (preferably 10-80g/L).
In the dextrose culture-medium, with the content meter of acetate, the content in the source containing acetate ion is preferably 0.5-
60g/L, more preferably 2-40g/L.
In the present invention, source containing acetate ion can be the various compounds for being capable of providing acetate ion, such as water-soluble
Acetic acid and/or acetate, preferably at least one of acetic acid, sodium acetate, potassium acetate and ammonium acetate.
According to the preferred embodiment of the present invention, the dextrose culture-medium can also (can be chlorination containing chloride
At least one of sodium, potassium chloride and ammonium chloride), sulfate (can for sodium sulphate and/or potassium sulfate), carbon atom number 2-8
Aldehyde (such as furfural) and carbon atom number be 6-16 at least one of phenol (such as phenol).It is highly preferred that the glucose culture
Base also contain the chloride of 25-95g/L (most preferably 20-60g/L), 0.5-10g/L (most preferably 4-6g/L) sulfate,
The carbon atom number of 0.1-1g/L (most preferably 0.1-0.5g/L) is the aldehyde and 0.1-1g/L (most preferably 0.1-0.5g/L) of 2-8
Carbon atom number be 6-16 at least one of phenol.
In order to obtain resistance more preferably yeast strain, it is further preferred that the dextrose culture-medium also contains 1-6g/
The Na of L2SO4, 20-60g/L NaCl, 10-30g/L the phenol of KCl, 0.1-0.5g/L and the furfural of 0.1-0.5g/L in
It is at least one.Most preferably, the dextrose culture-medium is also containing the Na of 4-6g/L2SO4, 35-40g/L NaCl, 20-30g/L
At least one of KCl (being preferably up to no more than two kinds)；Alternatively, the dextrose culture-medium also contains 0.15-0.3g/L
Furfural；Alternatively, the dextrose culture-medium is also containing the phenol of 0.2-0.4g/L.
In the present invention, the dextrose culture-medium can also the peptone containing 5-20g/L and 2-8g/L extraction from yeast
Object, to provide nitrogen source and growth factor etc. for bacterial strain.
A preferred embodiment of the invention, dextrose culture-medium of the invention contain component A and B component,
In, the component A is the glucose of 10-80g/L and the acetic acid of 1-3g/L, and the B component is the Na of 4-6g/L2SO4、35-40g/
The NH of KCl, 20-40g/L of NaCl, 20-30g/L of L4One of KAc of NaAc and 10-30g/L of Ac, 30-40g/L or
Two kinds.
In the present invention, yeast cell to express amino acid sequence such as SEQ can be increased by various modes commonly used in the art
The expression quantity of albumen shown in ID NO:1.For example, can will be inserted with express amino acid sequence as shown in SEQ ID NO:1
The expression vector of the nucleic acid of albumen is transformed into yeast cells, to increase the expression quantity of the albumen.Therefore, the present invention is to institute
It states yeast cells not require particularly, as long as being capable of express amino acid sequence albumen as shown in SEQ ID NO:1.It is excellent
Selection of land, the yeast cells have base sequence nucleic acid as shown in SEQ ID NO:2, that is, amino acid sequence such as SEQ ID
The base sequence of the code nucleic acid of albumen shown in NO:1 is as shown in SEQ ID NO:2.At this point, it is preferable to use inserted with base sequence
The nucleic acid as shown in SEQ ID NO:2 and the expression vector transformed yeast cell with strong promoter are arranged, to increase yeast cells
The expression quantity of express amino acid sequence albumen as shown in SEQ ID NO:1.
gccaccatgtccggaaaagcttctacagagggtagcgttactacggagtttctctctgatatcattggtaagacagt
gaacgtcaaacttgcctcgggtttactctacagcggaagattggaatccattgatggttttatgaatgttgcactat
cgagtgccactgaacactacgagagtaataacaataagcttctaaataagttcaatagtgatgtctttttgaggggc
acgcaggtcatgtatatcagtgaacaaaaaatatag(SEQ ID NO:2)
Wherein, the strong promoter is preferably Padh1 promoter, Ppgk promoter or Pfbp promoter.It is highly preferred that institute
Stating strong promoter is Padh1 promoter.
Wherein, the expression vector can be that inserted with base sequence nucleic acid as shown in SEQ ID NO:2 and can make
Any expression vector of the expression of nucleic acid, for example, plasmid or bacteriophage.Preferably, the expression vector is in plasmid pAUR123
BamH1 restriction enzyme site and XhoI restriction enzyme site between be inserted into base sequence nucleic acid as shown in SEQ ID NO:2 obtained from
Expression vector.
Wherein, to the terminator in the expression vector, there is no particular limitation, and those skilled in the art can hold very much
It changes places and selects terminator appropriate to construct the expression vector that can express base sequence nucleic acid as shown in SEQ ID NO:2.
According to another preferred method of implementation of the present invention, the yeast cells has base sequence such as SEQ ID NO:3
Shown in nucleic acid, that is, the base sequence such as SEQ ID of the code nucleic acid of amino acid sequence albumen as shown in SEQ ID NO:1
Shown in NO:3.At this point, it is preferable to use inserted with base sequence nucleic acid as shown in SEQ ID NO:3 and with the expression of promoter
Carrier transformed yeast cell, to increase the expression quantity of yeast cell to express amino acid sequence albumen as shown in SEQ ID NO:1.
gccaccatgtccggaaaagcctctacagagggtagcgttactacggagtttctctctgatatcattggtaagacagt
gaacgtcaaacttgcctcgggtttactctacagcggaagattggaatccattgatggttttatgaatgttgcactat
cgagtgccactgaacactacgagagtaataacaataagcttctaaataagttcaatagtgatgtctttttgaggggc
acgcaggtcatgtatatcagtgaacaaaaaatatag(SEQ ID NO:3)
Wherein, the promoter is preferably Padh1 promoter, Ppgk promoter or Pfbp promoter.It is highly preferred that described
Promoter is Padh1 promoter.
Wherein, the expression vector can be that inserted with base sequence nucleic acid as shown in SEQ ID NO:3 and can make
Any expression vector of the expression of nucleic acid, for example, plasmid or bacteriophage.Preferably, the expression vector is in plasmid pAUR123
BamH1 restriction enzyme site and XhoI restriction enzyme site between be inserted into base sequence nucleic acid as shown in SEQ ID NO:3 obtained from
Expression vector.
Wherein, to the terminator in the expression vector, there is no particular limitation, and those skilled in the art can hold very much
It changes places and selects terminator appropriate to construct the expression vector that can express base sequence nucleic acid as shown in SEQ ID NO:3.
In the present invention, the expression quantity for increasing yeast cell to express amino acid sequence albumen as shown in SEQ ID NO:1
Mode can be the mode of conventional culture yeasts cell, be preferably included at 28-33 DEG C, ferment is cultivated in dextrose culture-medium
Mother cell 24-72 hours.
The present invention will be described in detail by way of examples below.Experiment and detection method in following embodiment, such as
It is conventional method without specified otherwise；Used instrument is unless otherwise specified conventional detection and operation instrument；Used
Experimental material is unless otherwise specified to be commercially available from conventional biochemical reagent company；Quantitative examination in following embodiment
It tests, is respectively provided with three repeated experiments, results are averaged.Culture medium used in the examples etc. is as follows:
Yeast strain used in embodiment --- saccharomyces cerevisiae (Saccharomyces cerevisiae) RIPP-0 in
Be deposited on September 24th, 2013 China Committee for Culture Collection of Microorganisms's common micro-organisms center (be abbreviated as CGMCC,
Address: postcode: 100101) Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3, Institute of Microorganism, Academia Sinica, are protected
Hiding number is CGMCC No.8202.
YEPD culture medium: glucose 10g/L, peptone 10g/L, yeast extract 5g/L.
Screening and culturing medium: glucose 20g/L, peptone 10g/L, yeast extract 5g/L, agar powder 20g/L, acetic acid 3g/
L, stress factors (Na2SO4、NaCl、KCl、NH4Ac, NaAc etc.) 5-40g/L, natural pH, inverted plate is spare.
Acid seed culture medium: glucose 20g/L, peptone 10g/L, yeast extract 5g/L, acetic acid 1-3g/L,
pH5.0。
Glucose fermentation culture medium: glucose 80g/L, peptone 5g/L, yeast extract 3g/L, CaCl20.25g/L,
MgCl20.25g/L, KH2PO42.5g/L, pH 5.5 is individually added into the stress factors such as acetic acid, furfural (inhibiting ingredient) as needed
As the glucose fermentation culture medium containing inhibition ingredient.
The ingredient of fermentation liquid uses high effective liquid chromatography for measuring: sample is after accurate dilutions, first through 10000r/min high
Speed centrifugation 10min, supernatant sample introduction after 0.45 μm of filtering with microporous membrane measure.High-efficient liquid phase chromatogram condition are as follows: Agilent
1260 liquid chromatographs, Hi-plex H (8 μm, 7.7 × 30 μm) chromatographic column, 60 DEG C of column temperature, mobile phase is acetonitrile: water (80:
20), flow velocity 0.5mL/min.
Sugared content in sugar utilization (%)=(residual sugar content in sugared content-fermentation liquid in culture medium)/culture medium ×
100%.
Ethanol production (g/L) is defined as the content of ethyl alcohol in every liter of fermentation liquid, and value is higher, shows strain fermentation producing and ethanol
Ability and alcohol resistance it is stronger.
Embodiment 1
Shanghai Ji Ma (GenePharma) company is entrusted to synthesize base sequence such as SEQ ID NO:2 and SEQ ID NO:3 institute
The nucleic acid lsm6-Y and lsm6 shown, referring to the method in " Molecular Cloning:A Laboratory guide (third edition) " (Sambrook J, 2001),
Lsm6-Y and lsm6 are connected between the BamHI restriction enzyme site and XhoI restriction enzyme site of saccharomyces cerevisiae plasmid pAUR123, constituted
Recombinant expression carrier pAUR-lsm6-Y and pAUR-lsm6.
Embodiment 2
(1) picking saccharomyces cerevisiae RIPP-0 single bacterium drops down onto saccharomycete YEPD culture medium, and 30 DEG C, 180r/min cultivates to right
Number middle and later periods (about 16h), 4000r/min is centrifuged 5min and collects cell, with the Tris-HCl of 4 DEG C of sterile waters and pH 7.5,10mM
Buffer washes twice, and 0.6M sorbitol solution suspension cell is added, being diluted to concentration is about 105-106A cell/mL, takes
Embodiment 1 is obtained recombinant expression carrier pAUR-lsm6-Y with electroporated method (referring to " Molecular Cloning: A Laboratory by 0.2ml bacterium solution
Guide (third edition) ", Sambrook J, 2001) it is transferred in saccharomyces cerevisiae RIPP-0 host strain, 1mL YEPD culture is added in bacterium solution
Base is incubated for 1h at 30 DEG C.
(2) bacterium solution after step (1) to be incubated for 1h is spread evenly across containing 3g/L acetic acid and 30g/LNH4Ac and 3g/L
On acetic acid and two kinds of composite sifting plates of 40g/L NaAc, 60h is cultivated at 30 DEG C, obtains 64 plants of primary dcreening operation bacterial strain.
(3) primary dcreening operation bacterial strain is accessed in acid seed culture medium, 30 DEG C, after 200r/min culture for 24 hours, obtaining biomass just
Often, 35 plants of the normal bacterial strain of microscopy is collected by centrifugation cell and obtains bacterium mud, and bacterium mud access is trained equipped with 100mL glucose fermentation
In the 250mL triangular flask for supporting base (acetic acid containing 3g/L), initial cell density is about 1.0-1.2g/L (dry cell weight basis), and 30
DEG C, 120r/min carry out secondary screening.The Acetate tolerance and ethanol production of above-mentioned bacterial strains are analyzed, 9 plants of 30 DEG C of tolerance 3g/ are therefrom obtained
L acetic acid, ethanol production are greater than the recombinant bacterial strain of 20g/L.And then acetic acid (3g/L), furfural (0.2g/L), phenol to 9 plants of bacterium
(0.3g/L)、Na2SO4(5g/L)、NaCl(40g/L)、KCl(25g/L)、NH4Ac (20g/L), NaAc (30g/L) etc. stress because
The tolerance and ethanol production of son are analyzed, i.e., with this 9 plants of screening and culturing medium culture for containing stress factors as described above
Bacterium, the final Wine brewing yeast strain RIPP-20 for obtaining 1 plant of tolerance multiple inhibiting ingredient and there is high resistance to cold and diseases, the yeast strain
It can be grown in the screening and culturing medium containing above-mentioned stress factors and ethanol production is high.
(4) according to method in step (1)-(3), pAUR-lsm6 is transferred to the place saccharomyces cerevisiae RIPP-0 with electroporated method
In main bacterium, the final saccharomyces cerevisiae RIPP-06 for obtaining 1 plant of tolerance multiple inhibiting ingredient and there is high resistance to cold and diseases, the yeast strain
It can be grown in the screening and culturing medium containing above-mentioned stress factors and ethanol production is high.
Testing example 1
The glucose fermentation performance for the yeast strain that this testing example is used to illustrate that the present invention obtains.
(1) seed liquor is prepared
Saccharomyces cerevisiae RIPP-0, RIPP-06 and RIPP-20 are inoculated in acid seed culture medium (containing acetic acid 3g/L) respectively,
30 DEG C, 200r/min shaken cultivation 12h, obtain first order seed culture solution；
After the first order seed medium centrifugal, cell switching is in seed culture medium (being free of acetic acid), 30 DEG C, 200r/
Min shaken cultivation 16h, obtains seed liquor.
(2) glucose fermentation producing and ethanol
The seed liquor centrifugation that step (1) is obtained, collects cell and obtains bacterium mud, and by bacterium mud, (with dry cell weight basis, control is just
Beginning cell concentration is 1.2g/L) it accesses in the shaking flask of the 250ml equipped with 100ml glucose fermentation culture medium, 30 DEG C, 120r/min
Shaken cultivation carries out anaerobic fermentation.3 Duplicate Samples of inoculation every time, results are averaged.
Above-mentioned condition is fermented after 72h, and 5ml fermentation liquid is taken, and 10000r/min is centrifuged 10min, and supernatant is through 0.45 μm of micropore
Fermentation broth contents measurement is carried out after membrane filtration.The results are shown in Table 1.
Testing example 2
This testing example is used to illustrate that the yeast strain that the present invention obtains is containing the Portugal for inhibiting ingredient (1g/L acetic acid)
Fermenting property in grape sugar fermentation medium.
(1) seed liquor is prepared
With (1) the step of testing example 1.
(2) glucose fermentation producing and ethanol in the presence of inhibiting ingredient
The seed liquor centrifugation that step (1) is obtained, collects cell and obtains bacterium mud, and by bacterium mud, (with dry cell weight basis, control is just
Beginning cell concentration is 1.2g/L) it accesses in the shaking flask equipped with 250ml of the 100ml containing the glucose fermentation culture medium for inhibiting ingredient,
Remaining step is the same as (2) the step of testing example 1.The results are shown in Table 1.
Testing example 3
This testing example is used to illustrate that the yeast strain that the present invention obtains is containing the Portugal for inhibiting ingredient (3g/L acetic acid)
Fermenting property in grape sugar fermentation medium.
(1) seed liquor is prepared
With (1) the step of testing example 1.
(2) glucose fermentation producing and ethanol in the presence of inhibiting ingredient
The seed liquor centrifugation that step (1) is obtained, collects cell and obtains bacterium mud, and by bacterium mud, (with dry cell weight basis, control is just
Beginning cell concentration is 1.2g/L) it accesses in the shaking flask equipped with 250ml of the 100ml containing the glucose fermentation culture medium for inhibiting ingredient,
Remaining step is the same as (2) the step of testing example 1.The results are shown in Table 1.
Testing example 4
This testing example be used to illustrate the yeast strain that obtains of the present invention containing inhibit ingredient (1g/L acetic acid and
0.2g/L furfural) glucose fermentation culture medium in fermenting property.
(1) seed liquor is prepared
With (1) the step of testing example 1.
(2) glucose fermentation producing and ethanol in the presence of inhibiting ingredient
The seed liquor centrifugation that step (1) is obtained, collects cell and obtains bacterium mud, and by bacterium mud, (with dry cell weight basis, control is just
Beginning cell concentration is 1.2g/L) it accesses in the shaking flask equipped with 250ml of the 100ml containing the glucose fermentation culture medium for inhibiting ingredient,
Remaining step is the same as (2) the step of testing example 1.The results are shown in Table 1.
Testing example 5
This testing example be used to illustrate the yeast strain that obtains of the present invention containing inhibit ingredient (1g/L acetic acid and
0.3g/L phenol) glucose fermentation culture medium in fermenting property.
(1) seed liquor is prepared
With (1) the step of testing example 1.
(2) glucose fermentation producing and ethanol in the presence of inhibiting ingredient
The seed liquor centrifugation that step (1) is obtained, collects cell and obtains bacterium mud, and by bacterium mud, (with dry cell weight basis, control is just
Beginning cell concentration is 1.2g/L) it accesses in the shaking flask equipped with 250ml of the 100ml containing the glucose fermentation culture medium for inhibiting ingredient,
Remaining step is the same as (2) the step of testing example 1.The results are shown in Table 1.
Table 1
Testing example 6
This testing example is used to verify amino acid sequence such as SEQ ID NO in the Wine brewing yeast strain of the acquisition of embodiment 2:
The increase of the expression quantity of albumen shown in 1.
It is right according to the method that " Biochemistry Experiment guidance " (publishing house of Wuhan University publishes for 2011) chapter 3 is recorded
The total protein of saccharomyces cerevisiae RIPP-0, RIPP-06 and RIPP-20 carry out sodium dodecyl sulfate polyacrylamide gel electrophoresis
(SDS-PAGE).The results show that the amino acid sequence that saccharomyces cerevisiae RIPP-06 and RIPP-20 are generated is as shown in SEQ ID NO:1
The band of albumen is significantly wider than the band (about 2-5 times) of the albumen of saccharomyces cerevisiae RIPP-0 generation, shows saccharomyces cerevisiae RIPP-
06 and RIPP-20 has relative to the expression quantity of the albumen as shown in SEQ ID NO:1 of amino acid sequence in saccharomyces cerevisiae RIPP-0
Increased.
Embodiment 3
It (is purchased from according to method recombinant expression carrier pAUR-lsm6 transformed saccharomyces cerevisiae S288C same as Example 2
ATCC, article No. are204508D-5TM), obtain 1 Accharomyces cerevisiae bacterial strain RIPP-D20, according to testing example 1-
5 identical methods are measured its fermenting property, as a result as shown in table 2 below.
Table 2
Saccharomyces cerevisiae S288C is one plant of Wine brewing yeast strain intolerant to acetic acid, but it can be seen from the results in table 2 logical
Cross the expression quantity that currently preferred mode increases yeast cell to express amino acid sequence albumen as shown in SEQ ID NO:1
Afterwards, the tolerance of acetic acid is enhanced in glucose fermentation culture medium.
Embodiment 4
Entrust the nucleic acid of Shanghai Ji Ma (GenePharma) company synthesis base sequence mutation as shown in SEQ ID NO:4
Lsm6-N is connected to by lsm6-N referring to the method in " Molecular Cloning:A Laboratory guide (third edition) " (Sambrook J, 2001)
Between the BamHI restriction enzyme site and XhoI restriction enzyme site of saccharomyces cerevisiae plasmid pAUR123, recombinant expression carrier pAUR- is constituted
lsm6-N。
gccaccatgtccggaaaagcatctacagagggtagcgttactacggagtttctctctgatatcattggtaagacagt
gaacgtcaaacttgcctcgggtttactctacagcggaagattggaatccattgatggttttatgaatgttgcactat
cgagtgccactgaacactacgagagtaataacaataagcttctaaataagttcaatagtgatgtctttttgaggggc
acgcaggtcatgtatatcagtgaacaaaaaatatag(SEQ ID NO:4)
According to method same as Example 2 recombinant expression carrier pAUR-lsm6-N transformed yeast bacterial strain RIPP-0, obtain
1 Accharomyces cerevisiae bacterial strain RIPP-D11 is obtained, its fermenting property is measured according to method identical with testing example 1-5,
The results are shown in Table 1, compares as can be seen that increasing yeast cell to express amino acid sequence such as using currently preferred mode
The expression quantity of albumen shown in SEQ ID NO:1 can obtain that resistance is higher and alcohol fermentation performance more preferably yeast strain.
As can be seen from the above embodiments, method of the invention, which can obtain, can utilize glucose and have compared with high resistance to cold and diseases
Yeast strain, and its sugar utilization and ethanol production are higher.
The preferred embodiment of the present invention has been described above in detail, still, during present invention is not limited to the embodiments described above
Detail within the scope of the technical concept of the present invention can be with various simple variants of the technical solution of the present invention are made, this
A little simple variants all belong to the scope of protection of the present invention.
It is further to note that specific technical features described in the above specific embodiments, in not lance
In the case where shield, can be combined in any appropriate way, in order to avoid unnecessary repetition, the present invention to it is various can
No further explanation will be given for the combination of energy.
In addition, various embodiments of the present invention can be combined randomly, as long as it is without prejudice to originally
The thought of invention, it should also be regarded as the disclosure of the present invention.
Claims (11)
1. a kind of method for preparing yeast strain, which is characterized in that this method comprises: culture yeasts is thin in dextrose culture-medium
Born of the same parents, the transformation into yeast cell have base sequence nucleic acid as shown in SEQ ID NO:3, and the dextrose culture-medium contains grape
Sugar and source containing acetate ion.
2. according to the method described in claim 1, wherein, in the dextrose culture-medium, the content of glucose is 5-100g/L；
With the content meter of acetate, the content in the source containing acetate ion is 0.5-3g/L.
3. according to the method described in claim 1, wherein, in the dextrose culture-medium, the content of glucose is 10-80g/L；
With the content meter of acetate, the content in the source containing acetate ion is 2-3g/L.
4. method described in any one of -3 according to claim 1, wherein source containing acetate ion is acetic acid, sodium acetate, second
At least one of sour potassium and ammonium acetate.
5. method described in any one of -3 according to claim 1, wherein the dextrose culture-medium also contain chloride,
At least one of sulfate, furfural and phenol.
6. method described in any one of -3 according to claim 1, wherein the dextrose culture-medium is also containing 1-5g/L's
Na2SO4, 20-40g/L NaCl, 10-25g/L the phenol of KCl, 0.1-0.3g/L and the furfural of 0.1-0.2g/L at least
It is a kind of.
7. method described in any one of -3 according to claim 1, wherein the dextrose culture-medium also contains 5-20g/L
Peptone and 2-8g/L yeast extract.
8. according to the method described in claim 1, wherein, the method includes using inserted with base sequence such as SEQ ID NO:
Nucleic acid shown in 3 and the expression vector transformed yeast cell with promoter.
9. according to the method described in claim 8, wherein, the promoter is that Padh1 promoter, Ppgk promoter or Pfbp are opened
Mover.
10. method according to claim 8 or claim 9, wherein the expression vector is the BamH1 digestion in plasmid pAUR123
Expression vector obtained from base sequence nucleic acid as shown in SEQ ID NO:3 is inserted between site and XhoI restriction enzyme site.
11. according to the method described in claim 1, wherein, the mode of culture yeasts cell is included at 28-33 DEG C, in grape
Culture yeasts cell 24-72 hours in sugar culture-medium.
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| LSM蛋白增强木糖发酵重组酵母抗逆性能的研究;高岚等;《高校化学工程学报》;20131015;830-835 * |
| Sm-like protein enhanced tolerance of recombinant Saccharomyces cerevisiae to inhibitors in hemicellulosic hydrolysate;Lan Gao, Liming Xia;《Bioresource Technology》;20120831;504-507 * |
| The Zymomonas mobilis regulator hfq contributes to tolerance against multiple lignocellulosic pretreatment inhibitors;Shihui Yang等;《BMC Microbiology》;20100507;1-11 * |
| U6 snRNA-associated Sm-like protein LSm6;Hane,J.K.等;《UniProtKB/Swiss-Prot: Q0UWI9.2》;20080520;序列说明 * |
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