CN105255952A - Method for improving ethyl alcohol production efficiency through INO2 gene overexpression - Google Patents
Method for improving ethyl alcohol production efficiency through INO2 gene overexpression Download PDFInfo
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- CN105255952A CN105255952A CN201510714158.XA CN201510714158A CN105255952A CN 105255952 A CN105255952 A CN 105255952A CN 201510714158 A CN201510714158 A CN 201510714158A CN 105255952 A CN105255952 A CN 105255952A
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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
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- Y02E—REDUCTION OF GREENHOUSE GAS [GHG] EMISSIONS, RELATED TO ENERGY GENERATION, TRANSMISSION OR DISTRIBUTION
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Abstract
The invention discloses a method for improving ethyl alcohol production efficiency through INO2 gene overexpression, and belongs to the technical field of biological energy development. According to the method, an engineering strain obtained by performing INO2 overexpression in a strain with single ino4 gene deletion is applied to fermentation production of ethyl alcohol; compared with a wild no-load contrast strain, when fermentation is performed for 52 h, the biomass is increased by 186.6%, the ethyl alcohol yield is increased by 204.1%, and the ethyl alcohol-thallus yield is increased by 5.6%. Thus, the method has great industrial application value and broad industrial application prospects, and meanwhile important information is provided for constructing excellent saccharomyces cerevisiae genetic engineering strains capable of achieving a high ethyl alcohol yield.
Description
Technical field
The present invention relates to a kind of method being improved Alcohol Production efficiency by overexpression INO2 gene, especially the yeast saccharomyces cerevisiae applying transcription factor single-gene disappearance and overexpression improves alcohol to a method for thalline yield, belongs to bioenergy development technique field.
Background technology
Fossil energy (coal, oil, Sweet natural gas etc.) is the topmost energy on our times, and along with the development of countries in the world economic society, the mankind increase progressively day by day to the demand of the energy, but the reserves of fossil energy are limited; Meanwhile, the global warming that the greenhouse gases that fossil energy burning produces cause and the problem brought, annoying people always.Therefore, find and develop the focus that reproducible new forms of energy have become whole world concern.Fuel alcohol has the advantages such as clean, safety, environmental friendliness and raw material are renewable, and be considered to the ideal substitute of petroleum resources, market potential is huge.At present, use comparatively widely in states such as Europe, the U.S. and Brazil.
Production by Microorganism Fermentation fuel alcohol is the focus of current research, and yeast saccharomyces cerevisiae is that bacterial strain is produced in desirable zymamsis, has: can grow rapidly and produce ethanol on simple substratum; Stronger adaptability should be had by environment (as high osmotic pressure, high temperature and ethanol are poisoned) to non-life; Genetic modification is easy to operate; Cheap substrate can be utilized, the characteristics such as fermentation costs is lower.Utilize thick mash fermentation to produce the technology of ethanol and within 10 years, obtain very large progress in the past, but and the developed country such as U.S. compare and still there is very large gap, the subject matter of existence is: the high osmotic pressure suppression that the high concentration substrate of earlier fermentation causes; The murder by poisoning that the high concentration ethanol in fermentation later stage causes suppresses; The restraining effect that in fermenting process, uppity high-temperature causes.The existence of these problems becomes the principal element of restriction thick mash fermentation technology production efficiency raising.
At present, research yeast saccharomyces cerevisiae (Saccharomycescerevisae) being tolerated to high osmotic pressure, alcohol in high concentration and high temperature has achieved a lot of progress, but relevant tolerance mechanism is not yet studied clear.Therefore, want to improve S.cerevisiae by the transformation means of rationality very difficult to the tolerance of adverse circumstance.Although traditional strain improvement means (as Natural Selection, selection by mutation) have achieved certain effect at raising bacterial strain in stress tolerance, exist workload large, there is the shortcomings such as randomness.In recent years, along with the fast development of molecular biology and omics technology, for the relation of broad scale research genes of brewing yeast and ethanol fermentation provides condition.At present, bibliographical information is not had can to improve the production efficiency of alcohol by overexpression INO2.
Summary of the invention
First technical problem that the present invention will solve is to provide a kind of method improving Alcohol Production efficiency, and described method utilizes ino4 genetically deficient and the Yeast engineering bacteria fermentative production alcohol of overexpression INO2 gene.
In one embodiment of the invention, described engineering bacteria is saccharomyces cerevisiae engineered yeast.
In one embodiment of the invention, the nucleotide sequence of described INO4 gene is as shown in SEQIDNO.1.
In one embodiment of the invention, the nucleotide sequence of described INO2 gene is as shown in SEQIDNO.2.
In one embodiment of the invention, the aminoacid sequence of described INO4 genes encoding is as shown in SEQIDNO.3.
In one embodiment of the invention, the aminoacid sequence of described INO2 genes encoding is as shown in SEQIDNO.4.
In one embodiment of the invention, described method is process LAN INO2 gene in ino4 single-gene gene-deleted strain.
In one embodiment of the invention, described process LAN is to expression plasmid pHAC181 (JiangL by INO2 gene fragment clone, 2004) obtain restructuring high-expression plasmid pHAC181-INO2 on, then recombinant plasmid transformed is expressed in ino4 single-gene gene-deleted strain.
In one embodiment of the invention, described expression plasmid pHAC181 inserts 3 histidine-taggedly to obtain in SphI and the EcoRV site of YCplac181, refer to document: AnalysesoftheeffectsofRck2pmutantsonPbs2p
dD-inducedtoxicityinSaccharomycescervisiaeidentifyaMAPkinas edockingmotif, andunexpectedfunctionalinactivationduetoacidicsubstituti onofT379, MolGenGenomics, 2004,271:208 – 219.
In one embodiment of the invention, described ino4 single-gene gene-deleted strain, purchased from Invotrogen company, is numbered Sc04163923_s1.
In one embodiment of the invention, described fermentative production is forwarded to fermention medium after being activated by Yeast engineering bacteria, makes to inoculate kind of an OD for daughter bacteria liquid post-fermentation and culture base
600for 0.4-0.5, in 28-30 DEG C, 200-220r/min cultivates after 7-8 hour and transfers quiescent culture to, quiescent culture 50-52h.
In one embodiment of the invention, described fermentative production is forwarded to fermention medium after being activated by Yeast engineering bacteria, makes to inoculate kind of an OD for daughter bacteria liquid post-fermentation and culture base
600be 0.5, in 30 DEG C, 220r/min transfers quiescent culture to after cultivating 8 hours, continue to cultivate 52h.
In one embodiment of the invention, described fermention medium contains glucose 100g/L, ammonium sulfate 7.5g/L, potassium primary phosphate 3.5g/L, magnesium sulfate heptahydrate 0.75g/L, yeast extract 0.2g/L, Histidine 0.02g/L, uridylic 0.02g/L, leucine 0.1g/L.
In one embodiment of the invention, the preferred YPD substratum of described activation.YPD solid medium is rule, cultivates 2d with primary activation bacterial classification in 30 DEG C, then be forwarded in YPD liquid nutrient medium, 30 DEG C, 220r/min shaking table cultivates 23h to saturated.
Second technical problem that the present invention will solve be to provide a kind of Alcohol Production efficiency improve saccharomyces cerevisiae engineered yeast, described saccharomyces cerevisiae engineered yeast lacked encoding amino acid sequence as the ino4 gene of SEQIDNO.3 and process LAN encoding amino acid sequence as the INO2 gene of SEQIDNO.4.
Described saccharomyces cerevisiae engineered yeast is process LAN INO2 gene in the yeast saccharomyces cerevisiae of ino4 single-gene disappearance.
Beneficial effect of the present invention:
The engineering strain fermentative production alcohol that the present invention is applied in overexpression INO2 in ino4 single-gene deletion mycopremna and obtains, compared to the unloaded control strain of wild-type, fermentation proceeds to 52h, biomass improves 186.6%, alcohol output improves 204.1%, alcohol improves 5.6% to thalline yield, has good industrial application value and prospect, and the excellent Saccharomyces cerevisiae gene engineering bacteria strain simultaneously for building High-alcohol-yield provides important information.
Accompanying drawing explanation
Fig. 1: Fungal biodiversity comparison diagram; Wherein ino4+INO2 representative overexpression INO2 and engineering strain of obtaining in ino4 single-gene deletion mycopremna, WT+V representative is containing unloaded wild type control strain;
Fig. 2: alcohol output comparison diagram; Wherein ino4+INO2 representative overexpression INO2 and engineering strain of obtaining in ino4 single-gene deletion mycopremna, WT+V representative is containing unloaded wild type control strain;
Fig. 3: spirit yield comparison diagram; Wherein ino4+INO2 representative overexpression INO2 and engineering strain of obtaining in ino4 single-gene deletion mycopremna, WT+V representative is containing unloaded wild type control strain.
Embodiment
The method of high-performance liquid chromatogram determination ethanol content: in fermented liquid, the mensuration of ethanol content adopts high performance liquid chromatograph (HPLC) to detect.Fermentation liquor process and supernatant liquor are after 0.22 μm of filtering with microporous membrane, and utilize RID (differential refraction detector) to detect, liquid-phase chromatography method is as follows: L.Chromatographic column: ShodexSH1011 sugar post organic acid post; Column temperature: 50 DEG C;
Moving phase: 0.0275% (v/v) dilute sulphuric acid, through 0.22 μm of membrane filtration and degasification; Flow velocity: 1mL/min; Detection time: 17min; Sample size: 20 μ L.
Alcohol is to the method for calculation of thalline yield: calculation of yield formula is as follows:
In formula, y is ethanol production, and p is ethanol concn, and X is dry cell weight x=0.2Z, Z is OD value.
The composition of YPD substratum: glucose 2%, yeast extract 1%, peptone 2%, the deionization water capacity, pH nature, autoclaving (115 DEG C, 20min).
The composition (g/L) of fermention medium: glucose 100, ammonium sulfate 7.5, potassium primary phosphate 3.5, magnesium sulfate heptahydrate 0.75, yeast extract 0.2, Histidine 0.02, uridylic 0.02, leucine 0.1, deionized water constant volume, pH nature, autoclaving (115 DEG C, 20min).
Embodiment 1: the structure of Saccharomyces cerevisiae gene engineering bacteria
With purchased from Invotrogen company, the ino4 single-gene deletion mycopremna YOL108C being numbered Sc04163923_s1 is starting strain, process LAN is by building high-expression plasmid pHAC181-INO2, concrete grammar is for (to comprise promoter sequence and ORF frame by INO2 gene fragment, do not comprise terminator codon), then this fragment is cloned on high-expression plasmid pHAC181, finally DNA sequencing is carried out to correct recon, authentication sequence is not undergone mutation, finally obtain restructuring high-expression plasmid pHAC181-INO2, by Li-acetate method, plasmid pHAC181-INO2 is imported ino4 single-gene gene-deleted strain again, thus obtain INO2 gene high expression bacterial strain.
Embodiment 2: yeast saccharomyces cerevisiae zymamsis
According to the compound method of solid medium, prepare YPD flat board and YPD liquid nutrient medium in advance.
The Wine brewing yeast strain YOL108C of Saccharomyces cerevisiae gene engineering bacteria of the present invention, wild type strain BY4743BG (BY4743 containing G418 resistant gene) and ino4 single-gene disappearance is activated at the flat lining out of YPD; Ino4 single-gene gene-deleted strain YOL108C utilizes G418 resistant gene to replace gene INO4 and obtains in BY4743BG background bacterium; (http://clones.lifetechnologies.com/cloneinfo.php? clone=yeast).YPD flat board is put into 30 DEG C of constant incubators and cultivate 2d.In super clean bench work, get a large single colony inoculation in the YPD liquid nutrient medium of 30mL, 30 DEG C from the bacterial strain flat board of each activation, 220r/min shaking table cultivates 23h to saturated.Cultivate after 23 hours, in EP pipe, add 50 μ L through incubated overnight bacterial strain bacterium liquid, dilute 20 times, survey OD
600value.According to the OD recorded
600value, calculates institute's seed addition bacterium liquid measure, makes the final OD of the fermention medium after inoculating kind of daughter bacteria liquid
600value is 0.5, and adds on super clean bench and calculate the kind daughter bacteria liquid measure of gained in the fermention medium of 100mL.
Fermention medium is put into 30 DEG C, the shaking table of 220r/min is cultivated, and transfers quiescent culture to after 8 hours.
At 32h and 52h fermentation time point, get the bacterium liquid of 1000 μ L in the EP pipe of 1.5mL, centrifugal 4min in the whizzer of 12000rpm, centrifugal good after respectively to the supernatant liquor shifting 600 μ L in the EP pipe of two groups of 1.5mL, one group add 600 μ L trichoroacetic acid(TCA)s be kept in the refrigerator of 4 DEG C preserve spend the night, within second day, measure ethanol concn by liquid phase.Another group does not add trichoroacetic acid(TCA), and the refrigerator being placed in-20 DEG C saves backup.
Meanwhile, at 32h and 52h fermentation time point, get 3 group of 50 μ L zymocyte liquid sample, in the EP pipe of 1.5mL, dilute 20 times, measure OD value, record data, for calculating Fungal biodiversity.
To be kept at first group of supernatant samples in 4 DEG C of refrigerators, the centrifugal 2min of 12000rpm under room temperature, gets the supernatant liquid filtering of 600 μ L, and filtered liquid is transferred in liquid phase sample bottle, measures ethanol concn.The sample surveying liquid phase can not be arranged at once, be saved in the refrigerator of-20 DEG C.
By liquid phase sample to be measured, upper liquid phase machine, copy test data, data are made Excel table, and make OD, alcohol output, ethanol production figure, as Figure 1-3, apply Saccharomyces cerevisiae gene engineering bacteria strain fermentative production alcohol of the present invention, compared to wild type control strain (the wild type strain BY4743BG containing unloaded pHAC181, represent with WT+V), fermentation proceeds to 52h, and biomass improves 186.6%, alcohol output improves 204.1%, and alcohol improves 5.6% to thalline yield.In addition, the present invention also finds, disappearance INO2 or INO4 gene can cause Alcohol Production efficiency to reduce separately.
Although the present invention with preferred embodiment openly as above; but it is also not used to limit the present invention, any person skilled in the art, without departing from the spirit and scope of the present invention; all can do various changes and modification, what therefore protection scope of the present invention should define with claims is as the criterion.
Claims (10)
1. improve a method for Alcohol Production efficiency, it is characterized in that, described method utilizes ino4 genetically deficient and the Yeast engineering bacteria fermentative production alcohol of overexpression INO2 gene.
2. method according to claim 1, is characterized in that, the aminoacid sequence of described ino4 genes encoding is as shown in SEQIDNO.3.
3. method according to claim 1, is characterized in that, the aminoacid sequence of described INO2 genes encoding is as shown in SEQIDNO.4.
4. method according to claim 1 and 2, is characterized in that, described method is process LAN INO2 gene in the yeast saccharomyces cerevisiae of ino4 single-gene disappearance.
5. method according to claim 1, is characterized in that, described fermentative production is forwarded to fermention medium after being activated by Yeast engineering bacteria, makes to inoculate kind of an OD for daughter bacteria liquid post-fermentation and culture base
600for 0.4-0.5, in 28-30 DEG C, 200-220r/min cultivates after 7-8 hour and transfers quiescent culture to, quiescent culture 50-52h.
6. method according to claim 5, is characterized in that, described fermentative production is forwarded to fermention medium after being activated by Yeast engineering bacteria, makes to inoculate kind of an OD for daughter bacteria liquid post-fermentation and culture base
600be 0.5, in 30 DEG C, 220r/min transfers quiescent culture to after cultivating 8 hours, continue to cultivate 52h.
7. the method according to claim 5 or 6, it is characterized in that, described fermention medium contains glucose 100g/L, ammonium sulfate 7.5g/L, potassium primary phosphate 3.5g/L, magnesium sulfate heptahydrate 0.75g/L, yeast extract 0.2g/L, Histidine 0.02g/L, uridylic 0.02g/L, leucine 0.1g/L.
8. a saccharomyces cerevisiae engineered yeast, is characterized in that, described saccharomyces cerevisiae engineered yeast lacked encoding amino acid sequence as the ino4 gene of SEQIDNO.3 and process LAN encoding amino acid sequence as the INO2 gene of SEQIDNO.4.
9. saccharomyces cerevisiae engineered yeast according to claim 8, is characterized in that, described saccharomyces cerevisiae engineered yeast is process LAN INO2 gene in the yeast saccharomyces cerevisiae of ino4 single-gene disappearance.
10. the application of saccharomyces cerevisiae engineered yeast described in claim 8 or 9 in Alcohol Production.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP3399041A1 (en) * | 2017-05-02 | 2018-11-07 | Intelligent Synthetic Biology Center | Enhancement of ginsenoside production by improvement of yeast cell organelle |
| 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 |
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| CN104099377A (en) * | 2014-07-16 | 2014-10-15 | 江南大学 | Method for improving alcohol production efficiency |
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| CN104099377A (en) * | 2014-07-16 | 2014-10-15 | 江南大学 | Method for improving alcohol production efficiency |
Non-Patent Citations (3)
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| BINGZHI LI ET AL.: "Transcriptome analysis of differential responses of diploid and haploid yeast to ethanol stress", 《JOURNAL OF BIOTECHNOLOGY》 * |
| BRENDA S. LOEWY ET AL.: "The IN02 and IN04 Loci of Saccharomyces cerevisiae Are Pleiotropic Regulatory Genes", 《MOLECULAR AND CELLULAR BIOLOGY》 * |
| 刘石雪等: "酿酒酵母乙醇耐受性的研究进展", 《中国生物工程杂志》 * |
Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP3399041A1 (en) * | 2017-05-02 | 2018-11-07 | Intelligent Synthetic Biology Center | Enhancement of ginsenoside production by improvement of yeast cell organelle |
| CN108795786A (en) * | 2017-05-02 | 2018-11-13 | 智能合成生物中心 | Ginsenoside yield is improved by improveing yeast cells device |
| JP2018186801A (en) * | 2017-05-02 | 2018-11-29 | インテリジェント シンセティック バイオロジー センター | Increasing production of ginsenoside by improvement of organelle of yeast |
| US11186616B2 (en) | 2017-05-02 | 2021-11-30 | Korea Research Institute Of Chemical Technology | Increased production of ginsenosides through yeast cell organelle improvement |
| CN108795786B (en) * | 2017-05-02 | 2023-03-14 | 韩国化学研究院 | Improving ginsenoside yield by improving yeast organelle |
| 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 |
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