CN105255951B - A method of Alcohol Production efficiency is improved by overexpression HAC1 genes - Google Patents
A method of Alcohol Production efficiency is improved by overexpression HAC1 genes Download PDFInfo
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- CN105255951B CN105255951B CN201510713933.XA CN201510713933A CN105255951B CN 105255951 B CN105255951 B CN 105255951B CN 201510713933 A CN201510713933 A CN 201510713933A CN 105255951 B CN105255951 B CN 105255951B
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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 improving Alcohol Production efficiency by overexpression HAC1 genes, belong to bioenergy development technique field.The present invention is overexpression HAC1 and the engineered strain fermenting and producing alcohol that obtains in ace2 single-gene deletion mycopremnas, and compared to wild type zero load control strain, ethanol production improves 200.8%, and alcohol improves 138.9% to thalline yield;Compared to ace2 single-gene deletion mycopremnas, ethanol production improves 13.1%;Important information is provided with good industrial application value and foreground, while for the excellent Saccharomyces cerevisiae gene engineering bacteria strain for building High-alcohol-yield.
Description
Technical field
The present invention relates to a kind of method improving Alcohol Production efficiency by overexpression HAC1 genes, especially one kind is answered
Alcohol is improved to the method for thalline yield with the saccharomyces cerevisiae of transcription factor single-gene missing and overexpression, belongs 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 always people.Therefore, it finds and sends out
Open up the hot spot that reproducible new energy has become 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.Currently, 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:Ethyl alcohol can be rapidly grown and produced on simple culture medium;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.The technology that ethyl alcohol is produced using thick mash fermentation obtained prodigious progress past 10 years, but flourishing with the U.S. etc.
Country is compared to prodigious gap, existing main problem is still had: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.
Currently, for saccharomyces cerevisiae (Saccharomyces cerevisae) 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 improving bacterial strain 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, the method
It is the efficiency that overexpression HAC1 genes improve saccharomycetes to make fermentation production alcohol in ace2 gene-deleted strains.
In one embodiment of the invention, the engineering bacteria is saccharomyces cerevisiae engineered yeast.
In one embodiment of the invention, the nucleotide sequence of the ACE2 genes is as shown in SEQ ID NO.1.
In one embodiment of the invention, the nucleotide sequence of the HAC1 genes is as shown in SEQ ID NO.2.
In one embodiment of the invention, the amino acid sequence of the ACE2 gene codes such as SEQ ID NO.3 institutes
Show.
In one embodiment of the invention, the method is overexpressed in the saccharomyces cerevisiae that ace2 single-genes lack
HAC1 genes.
In one embodiment of the invention, the saccharomyces cerevisiae of the ace2 single-genes missing is public purchased from Invotrogen
Department, number Sc04146899_s1.
In one embodiment of the invention, described be overexpressed is by HAC1 gene fragment clones to expression plasmid
Obtain recombination high-expression plasmid pHAC181-HAC1 on pHAC181 (Jiang L, 2004), then by recombinant plasmid transformed to ace2
It is expressed in single-gene gene-deleted strain.
In one embodiment of the invention, the expression plasmid pHAC181 be YCplac181 SphI and
What 3 histidine tags obtained be inserted into the sites EcoRV, refers to document:Analyses of the effects of Rck2p
mutants on Pbs2pDD-induced toxicity in Saccharomyces cervisiae identify a MAP
kinase docking motif,and unexpected functional inactivation due to acidic
substitution of T379,Mol Gen Genomics,2004,271:208–219.
In one embodiment of the invention, the fermenting and producing is that fermentation training is forwarded to after activating Yeast engineering bacteria
Support base so that the OD of fermentation medium after inoculation seed bacterium solution600For 0.4-0.5,7- is cultivated in 28-30 DEG C, 200-220r/min
Switch to stationary culture, stationary culture 50-52h after 8 hours.
In one embodiment of the invention, the fermenting and producing is that fermentation training is forwarded to after activating Yeast engineering bacteria
Support base so that the OD of fermentation medium after inoculation seed bacterium solution600It is 0.5, switchs to after 30 DEG C, 220r/min are cultivated 8 hours quiet
Culture is set, continues to cultivate 52h.
In one embodiment of the invention, the fermentation medium contain glucose 100g/L, ammonium sulfate 7.5g/L,
Potassium dihydrogen phosphate 3.5g/L, epsom salt 0.75g/L, yeast extract 0.2g/L, histidine 0.02g/L, uracil
0.02g/L, leucine 0.1g/L.
In one embodiment of the invention, the preferred YPD culture mediums of activation.It crosses on YPD solid mediums,
2d are cultivated in 30 DEG C with primary activation strain, then are forwarded in YPD fluid nutrient mediums, 30 DEG C, 220r/min shaking table cultures 23h extremely
Saturation.
The invention solves second technical problem be to provide the saccharomyces cerevisiae engineering that a kind of Alcohol Production efficiency improves
Bacterium, the saccharomyces cerevisiae engineered yeast have lacked the ace2 genes of encoding amino acid sequence such as SEQ ID NO.3 and have been overexpressed nucleosides
The HAC1 genes of acid sequence such as SEQ ID NO.2.
The saccharomyces cerevisiae engineered yeast is the overexpression HAC1 genes in the saccharomyces cerevisiae that ace2 single-genes lack.
Beneficial effects of the present invention:
The work that the present invention is obtained using the overexpression HAC1 genes in the Wine brewing yeast strain that ace2 single-genes lack
Journey yeast strain ferments produce alcohol, and compared to wild type zero load control strain, when fermentation proceeds to 52h, ethanol production improves
200.8%, alcohol improves 138.9% to thalline yield;Compared to the bacterial strain that ace2 single-genes missing only occurs, ethyl alcohol production
Amount improves 13.1%.The present invention has good industrial application value and foreground, while being the excellent wine of structure High-alcohol-yield
Brewer yeast engineering strain provides important information.
Description of the drawings
Fig. 1:Fungal biodiversity compares figure;Wherein ace2+HAC1 represents the overexpression in ace2 single-gene deletion mycopremnas
HAC1 and the engineered strain obtained, WT+V are represented containing unloaded wild type control strain;
Fig. 2:Ethanol production compares figure;Wherein ace2+HAC1 represents the overexpression in ace2 single-gene deletion mycopremnas
HAC1 and the engineered strain obtained, WT+V are represented containing unloaded wild type control strain;
Fig. 3:Alcohol getting rate compares figure;Wherein ace2+HAC1 represents the overexpression in ace2 single-gene deletion mycopremnas
HAC1 and the engineered strain obtained, WT+V are represented containing unloaded wild type control strain.
Specific implementation mode
The method that high performance liquid chromatography measures ethanol content:The measurement 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:Shodex SH1011 sugar column organic acid columns;Column temperature:50℃;Flowing
Phase:0.0275% (v/v) dilute sulfuric acid, through 0.22 μm of membrane filtration and degasification;Flow velocity:1mL/min;Detection time:17min;Into
Sample amount:20μL.
Computational methods of the ethyl alcohol to thalline yield:Calculation of yield formula is as follows:
Y is alcohol yied in formula, and p is concentration of alcohol, and X is dry cell weight x=0.2Z, and Z 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).
Embodiment 1:The structure of Saccharomyces cerevisiae gene engineering bacteria
To be purchased from Invotrogen companies, ace2 single-gene deletion mycopremnas YLR131C that number is Sc04146899_s1
(http://www.lifetechnologies.com/order/genome-database/browse/gene-
expression/keyword/YLR 131CICID=search-gex-YLR131C it is) starting strain, overexpression is to pass through
Build high-expression plasmid pHAC181-HAC1, specific method be by HAC1 genetic fragments (comprising 865bp promoter sequences and its
ORF frames do not include terminator codon, total 1850bp), then this segment is cloned on high-expression plasmid pHAC181, finally
DNA sequencing is carried out to correct recon, verification sequence does not mutate, and finally obtains recombination high-expression plasmid pHAC181-
HAC1, then plasmid pHAC181-HAC1 is imported by ace2 single-gene gene-deleted strains by Li-acetate method, it is high to obtain HAC1 genes
Express bacterial strain.
Embodiment 2:Ethanol in Saccharomyces cerevisiae ferments
According to the preparation method of solid medium, YPD tablets and YPD fluid nutrient mediums are prepared in advance.
Saccharomyces cerevisiae gene engineering bacteria, wild-type strain BG (resistances containing G418 in the flat lining out activation present invention of YPD
The BY4743 of gene) (http://clones.lifetechnologies.com/cloneinfo.phpClone=yeast)
(ace2 single-gene gene-deleted strains are utilized in BY4743 background bacterium with the Wine brewing yeast strain YLR131C of ace2 single-genes missing
G418 resistant genes replace what ACE2 genes obtained).YPD tablets are put into 30 DEG C of constant incubator culture 2d.In super-clean bench work
Make, from taking a big single bacterium colony to be inoculated in the YPD fluid nutrient mediums of 30mL in the bacterial strain flat board of each activation, 30 DEG C,
220r/min shaking table cultures 23h is extremely saturated.After cultures 23 hours, 50 μ L are added in EP pipes and are incubated overnight bacterial strain bacterium solution,
OD is surveyed in 20 times of dilution600Value.According to the OD measured600Value calculates institute's seed addition bacterium solution amount so that the hair after inoculation seed bacterium solution
The final OD of ferment culture medium600Value is 0.5, and the seed bacterium solution amount obtained by calculating is added on super-clean bench and is trained in the fermentation of 100mL
It supports in base.
Fermentation medium is put into 30 DEG C, the shaking table culture of 220r/min switchs to stationary culture after 8 hours.
In 32h and 52h fermentation time points, take the bacterium solution of 1000 μ L 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, sets
It is saved backup in -20 DEG C of refrigerator.
Meanwhile in 32h and 52h fermentation time points, 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, liquid phase machine copies test data, data is made Excel tables, and make OD, second
Alcohol yield, alcohol yied figure apply the overexpression HAC1 in the Wine brewing yeast strain of ace2 gene delections as shown in Figs. 1-3
Gene and obtain Engineering Yeast strain fermentation production alcohol, (contain empty plasmid compared to wild type zero load control strain
The wild-type strain BG of pHAC181, is indicated with WT+V), when fermentation proceeds to 52h, ethanol production improves 200.8%, alcohol
138.9% is improved to thalline yield;Compared to ace2 single-gene deletion mycopremnas, ethanol production improves 13.1%.In addition, this
Invention also found, individually lack the Yeast engineering bacteria of HAC1 genes, and Alcohol Production efficiency is to reduce.
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 can do various change and modification, therefore the protection model of the present invention without departing from the spirit and scope of the present invention
Enclosing be subject to what claims were defined.
Claims (8)
1. a kind of method improving Alcohol Production efficiency, which is characterized in that the method is to utilize ace2 gene delections and excess
Express the Yeast engineering bacteria fermenting and producing alcohol of HAC1 genes;The amino acid sequence of the ace2 gene codes such as SEQ ID
Shown in NO.3;The nucleotide sequence of the HAC1 genes is as shown in SEQ ID NO.2.
2. according to any method of claim 1, which is characterized in that the method is the wine brewing lacked in ace2 single-genes
HAC1 genes are overexpressed in yeast.
3. according to the method described in claim 1, it is characterized in that, the fermenting and producing is transferred after activating Yeast engineering bacteria
To fermentation medium so that the OD of fermentation medium after inoculation seed bacterium solution600For 0.4-0.5, in 28-30 DEG C, 200-220r/
Min switchs to stationary culture, stationary culture 50-52h after cultivating 7-8 hours.
4. according to the method described in claim 3, it is characterized in that, the fermenting and producing is transferred after activating Yeast engineering bacteria
To fermentation medium so that the OD of fermentation medium after inoculation seed bacterium solution600It is 0.5, is cultivated 8 hours in 30 DEG C, 220r/min
After switch to stationary culture, continue cultivate 52h.
5. method according to claim 3 or 4, which is characterized in that the fermentation medium contains glucose 100g/L, sulphur
Sour ammonium 7.5g/L, potassium dihydrogen phosphate 3.5g/L, epsom salt 0.75g/L, yeast extract 0.2g/L, histidine 0.02g/L,
Uracil 0.02g/L, leucine 0.1g/L.
6. a kind of saccharomyces cerevisiae engineered yeast, which is characterized in that the saccharomyces cerevisiae engineered yeast has lacked encoding amino acid sequence such as
The ace2 genes of SEQ ID NO.3 and the HAC1 genes for being overexpressed nucleotide sequence such as SEQ ID NO.2.
7. saccharomyces cerevisiae engineered yeast according to claim 6, which is characterized in that the saccharomyces cerevisiae engineered yeast is in ace2
It is overexpressed HAC1 genes in the saccharomyces cerevisiae of single-gene missing.
8. application of the saccharomyces cerevisiae engineered yeast of claim 6 or 7 in terms of Alcohol Production.
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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 |
CN110484572B (en) * | 2019-08-30 | 2021-04-06 | 浙江工业大学 | Method for increasing yield of saccharomyces cerevisiae nerolidol |
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Mechanisms of ethanol tolerance in Saccharomyces cerevisiae;Menggen Ma et al.;《Appl Microbiol Biotechnol》;20100531;第87卷;第830页左栏第3段 * |
利用功能基因组学方法研究酿酒酵母乙醇生物合成的调控机理;徐国强等;《微生物学杂志》;20131231;第33卷(第6期);19-07 * |
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