CN104099377B - A kind of method for improving Alcohol Production efficiency - Google Patents
A kind of method for improving Alcohol Production efficiency Download PDFInfo
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- CN104099377B CN104099377B CN201410340315.0A CN201410340315A CN104099377B CN 104099377 B CN104099377 B CN 104099377B CN 201410340315 A CN201410340315 A CN 201410340315A CN 104099377 B CN104099377 B CN 104099377B
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02E—REDUCTION OF GREENHOUSE GAS [GHG] EMISSIONS, RELATED TO ENERGY GENERATION, TRANSMISSION OR DISTRIBUTION
- Y02E50/00—Technologies for the production of fuel of non-fossil origin
- Y02E50/10—Biofuels, e.g. bio-diesel
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Abstract
The invention discloses a kind of methods for improving Alcohol Production efficiency, belong to bioenergy development technique field.The present invention is using the Wine brewing yeast strain of ACE2 single-genes missing and/or the Wine brewing yeast strain fermenting and producing alcohol of SWI5 single-genes missing, compared to wild mushroom, ethanol production improves 55.9% and 86.5%, alcohol improves 155.2% and 72.5% to thalline yield, with good industrial application value and prospect, while the excellent Saccharomyces cerevisiae gene engineering bacteria strain to build High-alcohol-yield provides important information.
Description
Technical field
The present invention relates to a kind of method for improving Alcohol Production efficiency, especially a kind of application transcription factor single-gene missing
Saccharomyces cerevisiae improve alcohol to the method for thalline yield, belong to bioenergy development technique field.
Background technology
Fossil energy (coal, oil, natural gas etc.) is the most important energy on our times, with the economic society in countries in the world
The development of meeting, the mankind are increasingly incremental to the demand of the energy, but the reserves of fossil energy are limited；Meanwhile fossil energy is fired
Global warming and caused problem caused by burning the greenhouse gases generated, annoying people always.Therefore, it finds and sends out
Opening up reproducible new energy has become the hot spot of whole world concern.Fuel alcohol has cleaning, safe, environmental-friendly and raw material
The advantages that renewable, it is considered to be the ideal substitute of petroleum resources, market potential are huge.At present, Europe, the U.S. and bar
Xi Deng states use relatively broadly.
Production by Microorganism Fermentation fuel alcohol is the hot spot of current research, and saccharomyces cerevisiae is ideal alcoholic fermentation production
Bacterial strain has：It can rapidly be grown on simple culture medium and produce ethyl alcohol；To non-life suitable environment (such as hyperosmosis, height
Temperature and ethyl alcohol are poisoned) there is stronger adaptability；Genetic modification is easy to operate；Can utilize cheap substrate, fermentation costs compared with
Low characteristic.Very big progress was obtained past 10 years, but flourishing with the U.S. etc. using the technology of thick mash fermentation production ethyl alcohol
Country is compared there are still very big gap, and existing main problem is：It is hypertonic caused by the high concentration substrate of earlier fermentation
System is constrained thoroughly；Murder by poisoning caused by the high concentration ethanol in later stage of fermenting inhibits；In fermentation process caused by uppity high-temperature
Inhibiting effect.The presence of these problems becomes the principal element that limitation thick mash fermentation technology production efficiency improves.
At present, for saccharomyces cerevisiae (Saccharomycescerevisae) tolerance hyperosmosis, alcohol in high concentration and height
The research of temperature has achieved many progress, but correlation tolerance mechanism not yet study it is clear.Therefore, to passing through the transformation of rationality
Means are very difficult to the tolerance of adverse circumstance to improve S.cerevisiae.Although traditional strain improvement means (as sieved naturally
Choosing, mutation breeding) in terms of bacterial strain is improved to stress tolerance achieved certain effect, but there are heavy workload, exist
The shortcomings of randomness.In recent years, it is broad scale research saccharomyces cerevisiae base with the fast development of molecular biology and omics technology
Cause and the relationship of alcohol fermentation provide condition.
Invention content
The invention solves first technical problem be to provide it is a kind of improve Alcohol Production efficiency method, be application
The Wine brewing yeast strain of ACE2 single-genes missing and/or the Wine brewing yeast strain fermenting and producing alcohol of SWI5 single-genes missing.
The nucleotide sequence of the Gene A CE2 is as shown in SEQIDNO.1.
The nucleotide sequence of the gene SWI5 is as shown in SEQIDNO.2.
The Wine brewing yeast strain of ACE2 single-genes missing is preferably purchased from the number of Invotrogen companies
Saccharomyces cerevisiae (the http of Sc04146899_s1://www.lifetechnologies.com/order/genome-
database/browse/gene-expression/keyword/YLR131CICID=search-gex-YLR131C).
The Wine brewing yeast strain of SWI5 single-genes missing is preferably purchased from the number of Invotrogen companies
Saccharomyces cerevisiae (the http of Sc04111156_s1://www.lifetechnologies.com/order/genome-
database/browse/gene-expression/keyword/YDR146CICID=search-gex-YDR146C).
The method preferably will be forwarded to fermentation medium after actication of culture so that the fermented and cultured after inoculation seed bacterium solution
The OD of base600For 0.4-0.5, switch to quiescent culture after 28-30 DEG C, 200-220r/min cultures 7-8h, continue to cultivate 50-
52h。
The OD of fermentation medium further preferably after inoculation seed bacterium solution600It is 0.5, in 30 DEG C, 220r/min cultures 8h
After switch to quiescent culture, continue cultivate 52h.
The preferred YPD culture mediums of actication of culture.It crosses on YPD solid mediums, 2d is cultivated in 30 DEG C with tentatively living
Change strain, then be forwarded in YPD fluid nutrient mediums, 30 DEG C, 220r/min shaking table cultures 23h to saturation.
The fermentation medium contains (g/L)：Glucose 100, ammonium sulfate 7.5, potassium dihydrogen phosphate 3.5, epsom salt
0.75, yeast extract 0.2, histidine 0.02, uracil 0.02, leucine 0.1, deionized water constant volume, pH natures.115℃
Sterilize 20min.
The invention solves second technical problem be to provide it is a kind of improve saccharomyces cerevisiae Alcohol Production ability method,
It is to knock out the ACE2 genes of saccharomyces cerevisiae or SWI5 genes.The nucleotide sequence such as SEQIDNO.1 of the Gene A CE2
It is shown.The nucleotide sequence of the gene SWI5 is as shown in SEQIDNO.2.
The present invention using ACE2 or SWI5 single-genes missing Wine brewing yeast strain fermenting and producing alcohol, compared to not lacking
55.9% and 86.5% has been respectively increased in the wild mushroom of said gene, ethanol production, and alcohol improves 155.2% to thalline yield
With 72.5%, there is good industrial application value and prospect, while be the excellent genes of brewing yeast work of structure High-alcohol-yield
Journey bacterial strain provides important information.
Description of the drawings
Deletion mycopremna (corresponding gene is ACE2 and SWI5) and the wild type control bacterium of 2 open genes of Fig. 1 saccharomyces cerevisiaes
The comparison figure of ethanol productions of the strain BY4743 when fermenting 32 and 52 hours, Fungal biodiversity and alcohol getting rate；Upper, middle and lower figure
It respectively is the ethanol production between bacterial strain, Fungal biodiversity and alcohol getting rate and compares figure.
Specific embodiment
The method that high performance liquid chromatography measures ethanol content：The measure of ethanol content uses high performance liquid chromatography in zymotic fluid
Instrument (HPLC) detects.Zymotic fluid is through processing and supernatant is after 0.22 μm of filtering with microporous membrane, utilizes RID (differential pulse polarograplls
Device) it is detected, liquid-phase chromatography method is as follows：Chromatographic column：BIO-RAD organic acid columns；Column temperature：35℃；
Mobile phase：0.0275% (v/v) dilute sulfuric acid, through 0.22 μm of membrane filtration and degasification；Flow velocity：0.6mL/min；Detection
Time：25min；Sample size：20μL.
Ethyl alcohol is to the computational methods of thalline yield：Calculation of yield formula is as follows：
Y is alcohol yied in formula, and p is concentration of alcohol, and x is OD values.
The composition of YPD culture mediums：Glucose 2%, yeast extract 1%, peptone 2%, the deionization water capacity, pH naturally,
High pressure sterilization (115 DEG C, 20min).
The composition (g/L) of fermentation medium：Glucose 100, ammonium sulfate 7.5, potassium dihydrogen phosphate 3.5, epsom salt
0.75, yeast extract 0.2, histidine 0.02, uracil 0.02, leucine 0.1, deionized water constant volume, pH is naturally, high pressure is gone out
Bacterium (115 DEG C, 20min).
1 Ethanol in Saccharomyces cerevisiae of embodiment ferments
According to the preparation method of solid medium, YPD tablets and YPD fluid nutrient mediums are prepared in advance.
In the flat lining out activated strains wild-type strain BY4743 (http of YPD://clones.lifetechnologie
s.com/cloneinfo.phpClone=yeast), the Wine brewing yeast strain YLR131C (http of ACE2 single-genes missing://
www.lifetechnologies.com/order/genome-database/browse/gene-expression/
keyword/YLR131CICID=search-gex-YLR131C), the Wine brewing yeast strain YDR146C of SWI5 single-genes missing
(http://www.lifetechnologies.com/order/genome-database/browse/gene-
expression/keyword/YDR146CICID=search-gex-YDR146C).YPD tablets are put into 30 DEG C of constant temperature trainings
Support case culture 2d.It works in super-clean bench, a big single bacterium colony is taken to be inoculated in the YPD of 30mL from the bacterial strain flat board of each activation
In fluid nutrient medium, 30 DEG C, 220r/min shaking table cultures 23h to saturation.After culture 23 hours, 50 μ L processes are added in EP pipes
Bacterial strain bacterium solution is incubated overnight, dilutes 20 times, surveys OD600Value.According to the OD measured600Value calculates institute's seed addition bacterium solution amount so that connect
The final OD of fermentation medium after various daughter bacteria liquid600It is 0.5 to be worth, and the seed bacterium solution obtained by calculating is added on super-clean bench
It measures in the fermentation medium of 100mL.
Fermentation medium is put into 30 DEG C, the shaking table culture of 220r/min switchs to quiescent culture after 8 hours.
In 32h and 52h fermentation times point, the bacterium solution of 1000 μ L is taken in the EP pipes of 1.5mL, in the centrifuge of 12000rpm
Middle centrifugation 4min shifts the supernatant of 600 μ L into the EP pipes of two groups of 1.5mL respectively after centrifugation is good, one group adds 600 μ L, tri- chloroethenes
Acid is stored in 4 DEG C of refrigerator and preserves overnight, passes through liquid phase measurement concentration of alcohol within second day.Another group is not added with trichloroacetic acid, puts
It is saved backup in -20 DEG C of refrigerator.
Meanwhile in 32h and 52h fermentation times point, 3 group of 50 μ L zymocyte liquid sample is taken, dilutes 20 in the EP pipes of 1.5mL
Times, OD values are measured, data are recorded, for calculating Fungal biodiversity.
The first group of supernatant samples that will be stored in 4 DEG C of refrigerators, 12000rpm centrifugations 2min, takes 600 μ L's at room temperature
Supernatant liquid filtering, filtered fluid are transferred in liquid phase sample bottle, measure concentration of alcohol.The sample of survey liquid phase cannot be arranged at once, protected
It is stored in -20 DEG C of refrigerator.
By liquid phase sample to be measured, upper liquid phase machine copies test data, data is made Excel tables, and make OD, second
Alcohol yield, alcohol yied figure, as shown in Figure 1, the Wine brewing yeast strain fermentation producing wine lacked using ACE2 or SWI5 single-genes
Essence, compared to wild mushroom, 55.9% and 86.5% has been respectively increased in ethanol production, and alcohol improves 155.2% to thalline yield
With 72.5%.
Although the present invention has been described by way of example and in terms of the preferred embodiments, it is not limited to the present invention, any to be familiar with this skill
The people of art without departing from the spirit and scope of the present invention, can do various change and modification, therefore the protection model of the present invention
Enclosing be subject to what claims were defined.
Claims (3)
- A kind of 1. method for improving Alcohol Production efficiency, which is characterized in that be the Wine brewing yeast strain lacked with ACE2 single-genes Or the Wine brewing yeast strain fermenting and producing alcohol of SWI5 single-genes missing；YPD fluid nutrient mediums are forwarded to prepare seed liquor after the bacterial strain is activated, and seed liquor is seeded to fermentation medium, So that it is inoculated with the OD of fermentation medium after seed bacterium solution600For 0.4-0.5, in 28-30oC, it is small to cultivate 7-8 by 200-220 r/min When after switch to quiescent culture, continue to cultivate 50-52 h；The formula of the fermentation medium is：100 g/L of glucose, 7.5 g/L of ammonium sulfate, 3.5 g/L of potassium dihydrogen phosphate, seven 0.75 g/L of water magnesium sulfate, 0.2 g/L of yeast extract, histidine 0.02g/L, uracil 0.02g/L, leucine 0.1g/L；The nucleotide sequence of the ACE2 is as shown in SEQ ID NO.1；The nucleotide sequence of the SWI5 is as shown in SEQ ID NO.2.
- 2. according to the method described in claim 1, it is characterized in that, it is forwarded to YPD fluid nutrient mediums after the bacterial strain is activated To prepare seed liquor, seed liquor is seeded to fermentation medium so that the OD of fermentation medium after inoculation seed bacterium solution600For 0.5, in 30oC, 220 r/min switch to quiescent culture after cultivating 8 hours, continue to cultivate 52 h.
- 3. according to the method described in claim 1, it is characterized in that, in 30 on YPD solid mediumsoC cultivates 2 d with preliminary Activated strains, then be forwarded in YPD fluid nutrient mediums, 30oC, 220 r/min cultivate 23 h and obtain seed liquor to saturation.
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| CN104099377B true CN104099377B (en) | 2018-07-06 |
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Families Citing this family (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105255952B (en) * | 2015-10-28 | 2018-10-16 | 江南大学 | A method of Alcohol Production efficiency is improved by overexpression INO2 genes |
| CN105255951B (en) * | 2015-10-28 | 2018-10-16 | 江南大学 | A method of Alcohol Production efficiency is improved by overexpression HAC1 genes |
| CN109666705A (en) * | 2019-01-07 | 2019-04-23 | 山东理工大学 | Chromatin remodeling factors gene is in the application for improving fermentation alcohol yield in S. cervisiae |
| CN109913381B (en) * | 2019-04-03 | 2021-05-04 | 山东理工大学 | Method for improving fermentation ethanol yield by regulating cell cycle transcription factor |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US6949356B1 (en) * | 1999-10-20 | 2005-09-27 | Microbia, Inc. | Methods for improving secondary metabolite production in fungi |
| US7314974B2 (en) * | 2002-02-21 | 2008-01-01 | Monsanto Technology, Llc | Expression of microbial proteins in plants for production of plants with improved properties |
-
2014
- 2014-07-16 CN CN201410340315.0A patent/CN104099377B/en active Active
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US6949356B1 (en) * | 1999-10-20 | 2005-09-27 | Microbia, Inc. | Methods for improving secondary metabolite production in fungi |
| US7314974B2 (en) * | 2002-02-21 | 2008-01-01 | Monsanto Technology, Llc | Expression of microbial proteins in plants for production of plants with improved properties |
Non-Patent Citations (2)
| Title |
|---|
| ACE2, an Activator of Yeast Metallothionein Expression Which Is Homologous to SWIS;GERALDINE BUTLER et al.;《MOLECULAR AND CELLULAR BIOLOGY》;19910131;第11卷(第1期);476-485 * |
| Different Consequences of ACE2 and SWI5 Gene Disruptions for Virulence of Pathogenic and Nonpathogenic Yeasts;Donna M. MacCallum et al.;《INFECTION AND IMMUNITY》;20060930;第74卷(第9期);5244–5248 * |
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