CN109628336A - One plant of Saccharomyces cerevisiae gene engineering bacteria for knocking out FBP1 gene and its construction method and application - Google Patents
One plant of Saccharomyces cerevisiae gene engineering bacteria for knocking out FBP1 gene and its construction method and application Download PDFInfo
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- 241000894006 Bacteria Species 0.000 title claims abstract description 47
- 101150010122 FBP1 gene Proteins 0.000 title claims abstract description 43
- 240000004808 Saccharomyces cerevisiae Species 0.000 title claims abstract description 37
- 235000014680 Saccharomyces cerevisiae Nutrition 0.000 title claims abstract description 36
- 108090000623 proteins and genes Proteins 0.000 title claims abstract description 31
- 238000010276 construction Methods 0.000 title claims abstract description 9
- 238000000855 fermentation Methods 0.000 claims abstract description 50
- 230000004151 fermentation Effects 0.000 claims abstract description 50
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims description 13
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 claims description 6
- 230000001580 bacterial effect Effects 0.000 claims description 6
- 239000008103 glucose Substances 0.000 claims description 6
- CSNNHWWHGAXBCP-UHFFFAOYSA-L Magnesium sulfate Chemical compound [Mg+2].[O-][S+2]([O-])([O-])[O-] CSNNHWWHGAXBCP-UHFFFAOYSA-L 0.000 claims description 4
- 244000253724 Saccharomyces cerevisiae S288c Species 0.000 claims description 4
- 235000004905 Saccharomyces cerevisiae S288c Nutrition 0.000 claims description 4
- PCHJSUWPFVWCPO-UHFFFAOYSA-N gold Chemical compound [Au] PCHJSUWPFVWCPO-UHFFFAOYSA-N 0.000 claims description 4
- 229910052737 gold Inorganic materials 0.000 claims description 4
- 239000010931 gold Substances 0.000 claims description 4
- 102100037181 Fructose-1,6-bisphosphatase 1 Human genes 0.000 claims description 3
- 101001028852 Homo sapiens Fructose-1,6-bisphosphatase 1 Proteins 0.000 claims description 3
- 239000001963 growth medium Substances 0.000 claims description 3
- 239000001888 Peptone Substances 0.000 claims description 2
- 108010080698 Peptones Proteins 0.000 claims description 2
- HCHKCACWOHOZIP-UHFFFAOYSA-N Zinc Chemical compound [Zn] HCHKCACWOHOZIP-UHFFFAOYSA-N 0.000 claims description 2
- BFNBIHQBYMNNAN-UHFFFAOYSA-N ammonium sulfate Chemical compound N.N.OS(O)(=O)=O BFNBIHQBYMNNAN-UHFFFAOYSA-N 0.000 claims description 2
- 229910052921 ammonium sulfate Inorganic materials 0.000 claims description 2
- 235000011130 ammonium sulphate Nutrition 0.000 claims description 2
- 229940041514 candida albicans extract Drugs 0.000 claims description 2
- 229910052943 magnesium sulfate Inorganic materials 0.000 claims description 2
- 235000019341 magnesium sulphate Nutrition 0.000 claims description 2
- 239000002773 nucleotide Substances 0.000 claims description 2
- 125000003729 nucleotide group Chemical group 0.000 claims description 2
- 235000019319 peptone Nutrition 0.000 claims description 2
- 238000012216 screening Methods 0.000 claims description 2
- 239000012138 yeast extract Substances 0.000 claims description 2
- 229910052725 zinc Inorganic materials 0.000 claims description 2
- 239000011701 zinc Substances 0.000 claims description 2
- QAOWNCQODCNURD-UHFFFAOYSA-N Sulfuric acid Chemical compound OS(O)(=O)=O QAOWNCQODCNURD-UHFFFAOYSA-N 0.000 claims 2
- XEEYBQQBJWHFJM-UHFFFAOYSA-N iron Substances [Fe] XEEYBQQBJWHFJM-UHFFFAOYSA-N 0.000 claims 1
- 229910052742 iron Inorganic materials 0.000 claims 1
- 229910000402 monopotassium phosphate Inorganic materials 0.000 claims 1
- 235000019796 monopotassium phosphate Nutrition 0.000 claims 1
- PJNZPQUBCPKICU-UHFFFAOYSA-N phosphoric acid;potassium Chemical compound [K].OP(O)(O)=O PJNZPQUBCPKICU-UHFFFAOYSA-N 0.000 claims 1
- 238000003209 gene knockout Methods 0.000 abstract description 5
- 238000006243 chemical reaction Methods 0.000 abstract description 3
- 239000003550 marker Substances 0.000 abstract description 3
- 230000003311 flocculating effect Effects 0.000 abstract description 2
- 238000000034 method Methods 0.000 abstract description 2
- 238000010353 genetic engineering Methods 0.000 abstract 3
- 238000004519 manufacturing process Methods 0.000 abstract 1
- 230000002906 microbiologic effect Effects 0.000 abstract 1
- 108020004414 DNA Proteins 0.000 description 13
- PEDCQBHIVMGVHV-UHFFFAOYSA-N Glycerine Chemical compound OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 description 9
- 239000002609 medium Substances 0.000 description 9
- 210000001822 immobilized cell Anatomy 0.000 description 7
- 101100503236 Mus musculus Folr1 gene Proteins 0.000 description 6
- 235000019441 ethanol Nutrition 0.000 description 6
- 239000012530 fluid Substances 0.000 description 6
- 235000015097 nutrients Nutrition 0.000 description 6
- 210000004027 cell Anatomy 0.000 description 5
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- 230000015572 biosynthetic process Effects 0.000 description 4
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- 238000000246 agarose gel electrophoresis Methods 0.000 description 3
- 238000005119 centrifugation Methods 0.000 description 3
- 230000001954 sterilising effect Effects 0.000 description 3
- 238000012546 transfer Methods 0.000 description 3
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 3
- QTBSBXVTEAMEQO-UHFFFAOYSA-N Acetic acid Chemical compound CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 description 2
- 229920000742 Cotton Polymers 0.000 description 2
- 230000004913 activation Effects 0.000 description 2
- 230000008901 benefit Effects 0.000 description 2
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- 238000012512 characterization method Methods 0.000 description 2
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- 238000005516 engineering process Methods 0.000 description 2
- 238000000605 extraction Methods 0.000 description 2
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- 238000012239 gene modification Methods 0.000 description 2
- 230000005017 genetic modification Effects 0.000 description 2
- 235000013617 genetically modified food Nutrition 0.000 description 2
- 230000004110 gluconeogenesis Effects 0.000 description 2
- 238000009630 liquid culture Methods 0.000 description 2
- 239000000463 material Substances 0.000 description 2
- 238000012856 packing Methods 0.000 description 2
- 238000011144 upstream manufacturing Methods 0.000 description 2
- FBPFZTCFMRRESA-FSIIMWSLSA-N D-Glucitol Natural products OC[C@H](O)[C@H](O)[C@@H](O)[C@H](O)CO FBPFZTCFMRRESA-FSIIMWSLSA-N 0.000 description 1
- 102000053602 DNA Human genes 0.000 description 1
- 108010037362 Extracellular Matrix Proteins Proteins 0.000 description 1
- 102000010834 Extracellular Matrix Proteins Human genes 0.000 description 1
- 238000012408 PCR amplification Methods 0.000 description 1
- ZLMJMSJWJFRBEC-UHFFFAOYSA-N Potassium Chemical compound [K] ZLMJMSJWJFRBEC-UHFFFAOYSA-N 0.000 description 1
- 229960000583 acetic acid Drugs 0.000 description 1
- 238000004458 analytical method Methods 0.000 description 1
- 238000013459 approach Methods 0.000 description 1
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- 230000001276 controlling effect Effects 0.000 description 1
- 239000013078 crystal Substances 0.000 description 1
- SEGLCEQVOFDUPX-UHFFFAOYSA-N di-(2-ethylhexyl)phosphoric acid Chemical compound CCCCC(CC)COP(O)(=O)OCC(CC)CCCC SEGLCEQVOFDUPX-UHFFFAOYSA-N 0.000 description 1
- 238000004090 dissolution Methods 0.000 description 1
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- 238000005189 flocculation Methods 0.000 description 1
- 230000016615 flocculation Effects 0.000 description 1
- 230000035784 germination Effects 0.000 description 1
- 239000012362 glacial acetic acid Substances 0.000 description 1
- 238000003987 high-resolution gas chromatography Methods 0.000 description 1
- 239000001257 hydrogen Substances 0.000 description 1
- 229910052739 hydrogen Inorganic materials 0.000 description 1
- 125000004435 hydrogen atom Chemical class [H]* 0.000 description 1
- 239000005457 ice water Substances 0.000 description 1
- 230000002779 inactivation Effects 0.000 description 1
- SURQXAFEQWPFPV-UHFFFAOYSA-L iron(2+) sulfate heptahydrate Chemical compound O.O.O.O.O.O.O.[Fe+2].[O-]S([O-])(=O)=O SURQXAFEQWPFPV-UHFFFAOYSA-L 0.000 description 1
- 229910000359 iron(II) sulfate Inorganic materials 0.000 description 1
- 101150044508 key gene Proteins 0.000 description 1
- 230000031700 light absorption Effects 0.000 description 1
- 230000000813 microbial effect Effects 0.000 description 1
- 238000002156 mixing Methods 0.000 description 1
- 239000013612 plasmid Substances 0.000 description 1
- 231100000572 poisoning Toxicity 0.000 description 1
- 230000000607 poisoning effect Effects 0.000 description 1
- 230000008092 positive effect Effects 0.000 description 1
- 239000011591 potassium Substances 0.000 description 1
- 229910052700 potassium Inorganic materials 0.000 description 1
- 238000004321 preservation Methods 0.000 description 1
- 230000001105 regulatory effect Effects 0.000 description 1
- 230000028327 secretion Effects 0.000 description 1
- 230000035939 shock Effects 0.000 description 1
- 239000000600 sorbitol Substances 0.000 description 1
- 238000002798 spectrophotometry method Methods 0.000 description 1
- 230000006641 stabilisation Effects 0.000 description 1
- 238000011105 stabilization Methods 0.000 description 1
- 239000008223 sterile water Substances 0.000 description 1
- 239000000758 substrate Substances 0.000 description 1
- 150000008163 sugars Chemical class 0.000 description 1
- 239000008399 tap water Substances 0.000 description 1
- 235000020679 tap water Nutrition 0.000 description 1
- 230000009466 transformation Effects 0.000 description 1
- 210000005253 yeast cell Anatomy 0.000 description 1
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- 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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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
- C12N15/80—Vectors or expression systems specially adapted for eukaryotic hosts for fungi
- C12N15/81—Vectors or expression systems specially adapted for eukaryotic hosts for fungi for yeasts
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P7/00—Preparation of oxygen-containing organic compounds
- C12P7/02—Preparation of oxygen-containing organic compounds containing a hydroxy group
- C12P7/04—Preparation of oxygen-containing organic compounds containing a hydroxy group acyclic
- C12P7/06—Ethanol, i.e. non-beverage
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- 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 the Saccharomyces cerevisiae gene engineering bacterias that one plant knocks out FBP1 gene, belong to microbiological genetic engineering field.This method utilizes golden load element resistance marker AurR gene constructed FBP1 gene knockout component and successful conversion saccharomyces cerevisiae yeast strain.The invention also discloses the construction methods of genetic engineering bacterium.The present invention further discloses application of the genetic engineering bacterium in immobilization fermentation production model.The present invention weakens saccharomyces cerevisiae flocculating properties in free fermentation obviously, and the biofilm formed in immobilization fermentation is reduced, and adhesion reduces, and free cell increased significantly.
Description
Technical field
The invention belongs to gene engineering technology fields, and in particular to the genes of brewing yeast engineering of one plant of knockout FBP1 gene
Bacterium and its construction method and application.
Background technique
Biomembrane is a kind of bio aggregate that the extracellular matrix by microbial cell and its secretion collectively constitutes, solely with it
Special microcolony form and high environmental resistance and be valued by people.The industry important as one kind of biomembrane is answered
With plastidogenetic biomembrane during immobilization fermentation, immobilized cell shows higher bottom to fermenting and producing after forming a film
Object tolerance and faster fermentation efficiency.The serialization of fermentation process and greatly improving for fermentation efficiency.It is conventional under usual conditions
Free cell fermentation 50g glucose take around or so 8 hours, and our immobilized cell only needs 6 hours just
Ethyl alcohol can be converted the sugars into.With the increase of batch fermentation, immobilization fermentation system can eventually arrive at a best stabilized shape
State, immobilized cell at this time can persistently be carried out the fermentation of tens batches with the stabilization fermentation period of 4 hours, presented
The outstanding fermentation efficiency of immobilized cell and good fermenting stability.Under the conditions of investigating sugar at the beginning of high concentration, immobilized cell
In the fermentation performance experiment of free cell, it has been found that it is resistance to that immobilized cell possesses the higher substrate/product of specific ionization cell
By property.In general, the glucose more than 200g/L will generate apparent murder by poisoning to yeast cells, and immobilized cell can
To keep higher cell viability and fermenting property in the glucose environment for being more than 300g/L.Therefore how fixation is rationally adjusted
Change the important ring for being formed into immobilization fermentation of biofilm in fermentation.Fermentation list of the biofilm as immobilization fermentation
Member industrially has been demonstrated with positive effect.During immobilization fermentation, number, the thickness of biofilm all can shadows
Ring the effect of its immobilization fermentation.Formation biofilm amount is very few, and fermentation advantage is unobvious, and formation biofilm amount is excessive, can hinder mass transfer
Conduction, to influence fermenting speed.
Summary of the invention
The technical problem to be solved by the present invention is to provide the Saccharomyces cerevisiae gene engineering bacteria of one plant of knockout FBP1 gene, needle
Feature in and immobilization fermentation strong to original Wine brewing yeast strain flocculating properties more than formation biofilm amount, orientation weaken its flocculation
Formation biofilm amount in feature and immobilization fermentation.
In order to solve the above technical problems, the present invention adopts the following technical scheme:
The Saccharomyces cerevisiae gene engineering bacteria of one plant of knockout FBP1 gene, FBP1 gene inactivation in the bacterial strain.FBP1 gene is
The key gene for regulating and controlling gluconeogenesis approach analyzes the gluconeogenesis way in the three phases that biofilm is formed by transcript profile data
Diameter is all obviously enriched with, so choosing Fbp1 gene as transformation object.
Wherein, the saccharomyces cerevisiae is Saccharomyces cerevisiae S288c.
Wherein, after knocking out FBP1 gene, FBP1 gene order is as shown in SEQ ID NO.2, before the FBP1 gene knockout
Gene order as shown in SEQ ID NO.3.
The construction method of the Saccharomyces cerevisiae gene engineering bacteria of above-mentioned knockout FBP1 gene, comprising the following steps:
(1) segment transformed saccharomyces cerevisiae bacterial strain, the nucleotide sequence such as SEQ ID NO.1 institute for knocking out segment will be knocked out
Show；
(2) positive transformant is obtained using the YPD Screening of Media containing 40 μ g/mL gold load elements, obtains knocking out FBP1
The Saccharomyces cerevisiae gene engineering bacteria of gene.
Preferably, the Wine brewing yeast strain is Saccharomyces cerevisiae S288c.
The Saccharomyces cerevisiae gene engineering bacteria of above-mentioned knockout FBP1 gene is applied as fermentation strain in protection of the invention
Within the scope of.
Saccharomyces cerevisiae gene engineering bacteria the applying of the invention in alcohol prepared by fermenting of above-mentioned knockout FBP1 gene
Within protection scope.
The utility model has the advantages that
The invention discloses a kind of Saccharomyces cerevisiae gene engineering bacteria of one plant of knockout FBP1 gene and its construction method with answer
With.Since biofilm is formed excessively original strain S288c in immobilization fermentation, mass transfer conduction is hindered, leads to fermentation week
Phase accordingly lengthens, and the Saccharomyces cerevisiae gene engineering bacteria for knocking out FBP1 gene compares original Wine brewing yeast strain and wads a quilt with cotton in free fermentation
Solidifying characteristic obviously weakens, and the biofilm formed in immobilization fermentation is reduced, and adhesion reduces, and free cell increases, and reinforces
Mass transfer conduction, reduces fermentation period.
Detailed description of the invention
Fig. 1 is Fbp1 gene upstream and downstream homology arm, AurR gene amplification fragment and HO clpp gene in the embodiment of the present invention 1
Except the agarose gel electrophoresis figure of component, wherein M:DNA molecular weight marker, 1:Fbp1-kanMX (2766bp), 2:Fbp1L
(540bp), 3:AurR gene amplification fragment (1788bp), 4:Fbp1R (514bp).
Fig. 2 is that the PCR of Fbp1 gene delection transformant in the embodiment of the present invention 1 identifies electrophoretogram, wherein M:DNA molecule
Measure marker；W is virgin control group, and 1,2 be respectively two positive transformants (2468bp), and 3 be negative transformants.
Fig. 3 is that original bacteria S288c (W) and the degerming of Fbp1 clpp gene (△ Fbp1) are trained in 96 orifice plates in the embodiment of the present invention 2
It supports 3 days orifice plate bottoms and adsorbs thallus figure.
Fig. 4 is in original bacteria S288c (W) in the embodiment of the present invention 3 and the degerming of Fbp1 clpp gene (△ Fbp1) free fermentation
The different characterizations of shaking flask.
Fig. 5 is the difference of original bacteria S288c and shaking flask in the fermentation of Fbp1 gene knockout bacteria immobilization in the embodiment of the present invention 3
Characterization.
Fig. 6 is original bacteria S288c in the embodiment of the present invention 3 and the degerming of Fbp1 clpp gene in free fermentation and immobilization fermentation
Fermentation residual sugar data.
Fig. 7 is original bacteria S288c in the embodiment of the present invention 3 and the degerming of Fbp1 clpp gene in free fermentation and immobilization fermentation
Tunning ethyl alcohol data.
Specific embodiment
The building of embodiment 1:FBP1 gene knockout Wine brewing yeast strain.
The primer used in following embodiment is as follows:
AACGCTCTACCAACTGAGC (SEQ ID NO:4, FBP1-up-F)
ATTCTGGGCCTCCATGTCCGTCTGTAATTGCACTACTTGT (SEQ ID NO:5, FBP1-up-R)
AAGTAGTGCAATTACAGACGGACATGGAGGCCCAGAATAC (SEQ ID NO:6, AurR-F)
ACTGTGACTTGCCAATATGGCAGTATAGCGACCAGCATTC (SEQ ID NO:7, AurR-F)
ATGCTGGTCGCTATACTGCCATATTGGCAAGTCACAGTAG (SEQ ID NO:8, FBP1-down-F)
GCGAATTCATGTAGATCGCG (SEQ ID NO:9, FBP1-down-F)
ATTCTTAGTAGTCGCGGTCG (SEQ ID NO:10, YZ-F)
CAATGATGTGCAAGAACCCT (SEQ ID NO:11, YZ-R)
One, FBP1 knocks out the building of component.
(1) it expands to obtain upstream and downstream homology arm amplified fragments (the upper homology arm expansion of yeast FBP1 gene using regular-PCR
Increase primer sequence FBP1-up-F, FBP1-up-R as shown in SEQ ID NO:4 and SEQ ID NO:5；Lower homology arm amplimer
Sequence FBP1-down-F, FBP1-down-R is as shown in SEQ ID NO:7 and SEQ ID NO:8) and PYX212-AurR plasmid
In AurR gene amplification fragment (AurR gene magnification primer sequence AurR-F, AurR-R such as SEQ ID NO:6 and SEQ ID
Shown in NO:7), pass through agarose gel electrophoresis, three segment of gel extraction.
(2) specific expansion is carried out to above three segment by primer (as shown in SEQ ID NO:4 and SEQ ID NO:9)
Increase, by agarose gel electrophoresis, gel extraction obtains saccharomyces cerevisiae FBP1 gene knockout component.
Two, prepared by Wine brewing yeast strain competence.
(1) picking Wine brewing yeast strain is inoculated with YPD fluid nutrient medium, is incubated overnight in 30 DEG C of 200r/min, must activate kind
Sub- liquid；
(2) according to the inoculative proportion of volume ratio 10%, seed liquor is forwarded to the fresh YPD fluid nutrient medium of 100mL, in
30 DEG C of 200r/min continue to cultivate to bacterium solution OD600 between 0.8~1.2；
(3) 30min is pre-chilled in ice-water bath, and 4 DEG C of 5000r/min centrifugation 5min of low-temperature and high-speed centrifuge collect thallus；
(4) thallus is resuspended with 10mL4 DEG C of sterile water, 4 DEG C of 5000r/min centrifugation 5min of low-temperature and high-speed centrifuge collect bacterium
Body is repeated twice；Thallus, low-temperature and high-speed centrifuge 5000r/min, 4 DEG C of centrifugations is resuspended with 10mL4 DEG C of 1M sorbitol solution
5min collects thallus in triplicate；
(5) thallus, the packing of 90 μ L, mono- pipe is resuspended in 1mL1M sorbierite.
Three, Wine brewing yeast strain sense conversion and the identification of transformant.
(1) bacterium solution after packing obtained by step 2 is taken into a pipe, 10 μ L is added and convert segment, electric shock cup is transferred to after mixing；
(2) 5min is placed on ice；
(3) 1.5kv shocks by electricity, and 1mLYPD culture medium is added by electricity and turns liquid wash-off, cultivates 2h in 30 DEG C of 200r/min；
(4) it is coated on the YPD culture medium flat plate containing 40 μ g/mL gold load elements, is grown in 30 DEG C of culture to bacterium colonies；
(5) picking transformant and genome is extracted as template, use verifying primer (such as SEQ ID NO:7 and SEQ ID
Shown in NO:8) PCR amplification is carried out to identify positive transformant that FBP1 gene is knocked；
(6) the YPD fluid nutrient medium that positive transformant S288c-FBP1 access 5mL contains 40 μ g/mL gold load elements is selected
Middle activation for 24 hours, mixes, -80 DEG C of preservations with 30% glycerol 1:1 of sterilizing.
Embodiment 2:
(1) it respectively takes 100 μ L glycerol bacterium S288c (bacterium germination out) and S288c-FBP1 (the knockout bacterium that the present invention constructs) to be added to go out
It is incubated overnight, activates in the 5mL YPD fluid nutrient medium of bacterium；
(2) according to the inoculative proportion of volume ratio 10%, the bacterium solution of step (1) is forwarded to the YPD Liquid Culture of 100mL
Base continues to cultivate to bacterium solution OD600 between 0.8~1.2 in 30 DEG C of 200r/min；
(3) it takes 2mL bacterium solution to measure light absorption value at OD600, bacterium solution is diluted with the YPD fluid nutrient medium of sterilizing, so that dilute
Releasing rear bacterium solution OD600 is 0.01；
(4) take 200 μ L bacterium solutions that 96 orifice plates are added, LB liquid medium compares, and 37 DEG C of cultures are for 24 hours；
(5) 96 orifice plate bacterium solutions are poured out, is buffered 3 times, is patted dry with pure water；
(6) it takes 1% crystal violet solution, 200 μ L that 96 orifice plates are added, dyes 10min, tap water is rinsed, dried；
(7) it after taking 33% glacial acetic acid, 200 μ L that the dissolution of 96 orifice plates is added, gently vibrates, OD600 measures biomembrane yield, takes
Average value: in 96 orifice plate cultures, OD value is that in 96 orifice plate cultures, OD value is 2.2~2.4, S288c-FBP1 for 24 hours to S288c for 24 hours
1.4~1.6.Experimental result is as shown in figure 3, the saccharomyces cerevisiae for as can be seen from Figure 3 knocking out FBP1 gene goes biofilm bright
It is aobvious to reduce.
Embodiment 3:
(1) 100 μ L glycerol bacterium S288c and S288c-FBP1 is respectively taken to be added in the 5mL YPD fluid nutrient medium of sterilizing overnight
Culture, activation；
(2) according to the inoculative proportion of volume ratio 10%, the bacterium solution of step (1) is forwarded to the YPD Liquid Culture of 100mL
Base continues to cultivate to bacterium solution OD600 between 0.8~1.2 in 30 DEG C of 200r/min；
(3) seed liquor of step (2) is forwarded in the fermentation medium of 100mL, is divided into free fermentation and is sent out with immobilization
Two kinds of ferment, cotton fibre material is added as immobilization material in immobilization fermentation, ferments in 35 DEG C of 200r/min, after glucose exhausts
Reaction terminates, and using spectrophotometric determination day part fermentation liquid residual sugar amount, high resolution gas chromatography instrument measures alcohol in fermentation liquid
Content；
Wherein, fermentative medium formula is as follows: 6% glucose, 0.4% peptone, 0.4% ammonium sulfate, 0.3% di(2-ethylhexyl)phosphate
Hydrogen potassium, 0.3% yeast extract, 0.05% magnesium sulfate, 0.005% green vitriol, 0.005% Zinc vitriol.
(4) it can be seen that free fermentation and immobilization fermentation do not have significant change in fermentation period comparison by Fermentation Data,
Original strain S288c and genetic modification bacterial strain S288c-FBP1 fermentation period are respectively 36h and 28h, and fermentation period shortens about 8h；
The analysis of product ethanol yield data, in fermentation termination, original strain S288c and genetic modification bacterial strain S288c-FBP1 ethyl alcohol are produced
Amount is about 20g/L and 23g/L, and output increased is about 3g/L.
Sequence table
<110>Nanjing University of Technology
<120>one plants of Saccharomyces cerevisiae gene engineering bacterias for knocking out FBP1 gene and its construction method and application
<160> 11
<170> SIPOSequenceListing 1.0
<210> 1
<211> 2766
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 1
aacgctctac caactgagct aacaaggatg agttcttcga attttccagt ctaagataga 60
caacccatca aactgcatgg tcccgggcta acttctgctc tcttttccgg acggatggaa 120
tcgccgcttt tgaattcacc tccggggtat tattattatt cttagtagtc gcggtcgtgc 180
ggacacccgg agttatgcgg gcccgaaagc tcattatgta gtaaagctag gtaatgttaa 240
gggcgtaaga gccaacgcaa ggcagcaata gcctggtatt cccacatatc aagaaagctt 300
aaaaagttga gacagggaat ttgaaggcga agattgccga actggccaat acccactact 360
ttttttttgg tttgcttggt ttcttcctgt cgcttgccaa cttgtggcat cttccccaca 420
ctatattata aggatcgtcc tatgtatagg caatattatc catttcactc gctaacaaat 480
gtacgtatat atatggagca acaagtagtg caattacaga cggacatgga ggcccagaat 540
accctccttg acagtcttga cgtgcgcagc tcaggggcat gatgtgactg tcgcccgtac 600
atttagccca tacatcccca tgtataatca tttgcatcca tacattttga tggccgcacg 660
gcgcgaagca aaaattacgg ctcctcgctg cagacctgcg agcagggaaa cgctcccctc 720
acagacgcgt tgaattgtcc ccacgccgcg cccctgtaga gaaatataaa aggttaggat 780
ttgccactga ggttcttctt tcatatactt ccttttaaaa tcttgctagg atacagttct 840
cacatcacat ccgaacataa acaaccatgg caaacccttt ttcgagatgg tttctatcag 900
agagacctcc aaactgccat gtagccgatt tagaaacaag tttagatccc catcaaacgt 960
tgttgaaggt gcaaaaatac aaacccgctt taagcgactg ggtgcattac atcttcttgg 1020
gatccatcat gctgtttgtg ttcattacta atcccgcacc ttggatcttc aagatccttt 1080
tttattgttt cttgggcact ttattcatca ttccagctac gtcacagttt ttcttcaatg 1140
ccttgcccat cctaacatgg gtggcgctgt atttcacttc atcgtacttt ccagatgacc 1200
gcaggcctcc tattactgtc aaagtgttac cagcggtgga aacaatttta tacggcgaca 1260
atttaagtga tattcttgca acatcgacga attccttttt ggacatttta gcatggttac 1320
cgtacggact atttcattat ggggccccat ttgtcgttgc tgccatctta ttcgtatttg 1380
gtccaccaac tgttttgcaa ggttatgctt ttgcatttgg ttatatgaac ctgtttggtg 1440
ttatcatgca aaatgtcttt ccagccgctc ccccatggta taaaattctc tatggattgc 1500
aatcagccaa ctatgatatg catggctcgc ctggtggatt agctagaatt gataagctac 1560
tcggtattaa tatgtatact acatgttttt caaattcctc cgtcattttc ggtgcttttc 1620
cttcactgca ttccgggtgt gctactatgg aagccctgtt tttctgttat tgttttccaa 1680
aattgaagcc cttgtttatt gcttatgttt gctggttatg gtggtcaact atgtatctga 1740
cacaccatta ttttgtagac cttatggcag gttctgtgct gtcatacgtt attttccagt 1800
acacaaagta cacacattta ccaattgtag atacatctct tttttgcaga tggtcataca 1860
cttcaattga gaaatacgat atatcaaaga gtgatccatt ggctgcagat tcaaacgata 1920
tcgaaagtgt ccctttgtcc aacttggaac ttgactttga tcttaatatg actgatgaac 1980
ccagtgtaag cccttcgtta tttgatggat ctacttctgt ttctcgttcg tccgccacgt 2040
ctataacgtc actaggtgta aagagggctt aaactgacaa taaaaagatt cttgttttca 2100
agaacttgtc atttgtatag tttttttata ttgtagttgt tctattttaa tcaaatgtta 2160
gcgtgattta tatttttttt cgcctcgaca tcatctgccc agatgcgaag ttaagtgcgc 2220
agaaagtaat atcatgcgtc aatcgtatgt gaatgctggt cgctatactg ccatattggc 2280
aagtcacagt agttcaatga tcgccttctt ttcttatttt ctttgttctg tactttagta 2340
cgcgaaaaaa aaaaatctgt atatgtcctt atatatatat atatttatat atatatatgt 2400
gtatgtatgt gtaccgtaag cattactcct tctaataatg aaaattctta ggaaaagaga 2460
aaggaagtag cgaatggaat gggatggaag ttttaaagaa cattagaatt tatcctttgt 2520
caaacttcat cacatcaacc aagaactata taaacctacc aaatgaatta agaaacctaa 2580
ttagtgaaga gcaggagagt aaactagggt tcttgcacat cattgaaagt gattttaaac 2640
cttcggtagc gctgcaaaag ttggtgaatt gtactacggg ggacgaaaag atcctaatca 2700
tagatatagt atcaatatgg tcccaacaaa agcaaagaca gcatggcgcg atctacatga 2760
attcgc 2766
<210> 2
<211> 1771
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 2
ggacatggag gcccagaata ccctccttga cagtcttgac gtgcgcagct caggggcatg 60
atgtgactgt cgcccgtaca tttagcccat acatccccat gtataatcat ttgcatccat 120
acattttgat ggccgcacgg cgcgaagcaa aaattacggc tcctcgctgc agacctgcga 180
gcagggaaac gctcccctca cagacgcgtt gaattgtccc cacgccgcgc ccctgtagag 240
aaatataaaa ggttaggatt tgccactgag gttcttcttt catatacttc cttttaaaat 300
cttgctagga tacagttctc acatcacatc cgaacataaa caaccatggc aaaccctttt 360
tcgagatggt ttctatcaga gagacctcca aactgccatg tagccgattt agaaacaagt 420
ttagatcccc atcaaacgtt gttgaaggtg caaaaataca aacccgcttt aagcgactgg 480
gtgcattaca tcttcttggg atccatcatg ctgtttgtgt tcattactaa tcccgcacct 540
tggatcttca agatcctttt ttattgtttc ttgggcactt tattcatcat tccagctacg 600
tcacagtttt tcttcaatgc cttgcccatc ctaacatggg tggcgctgta tttcacttca 660
tcgtactttc cagatgaccg caggcctcct attactgtca aagtgttacc agcggtggaa 720
acaattttat acggcgacaa tttaagtgat attcttgcaa catcgacgaa ttcctttttg 780
gacattttag catggttacc gtacggacta tttcattatg gggccccatt tgtcgttgct 840
gccatcttat tcgtatttgg tccaccaact gttttgcaag gttatgcttt tgcatttggt 900
tatatgaacc tgtttggtgt tatcatgcaa aatgtctttc cagccgctcc cccatggtat 960
aaaattctct atggattgca atcagccaac tatgatatgc atggctcgcc tggtggatta 1020
gctagaattg ataagctact cggtattaat atgtatacta catgtttttc aaattcctcc 1080
gtcattttcg gtgcttttcc ttcactgcat tccgggtgtg ctactatgga agccctgttt 1140
ttctgttatt gttttccaaa attgaagccc ttgtttattg cttatgtttg ctggttatgg 1200
tggtcaacta tgtatctgac acaccattat tttgtagacc ttatggcagg ttctgtgctg 1260
tcatacgtta ttttccagta cacaaagtac acacatttac caattgtaga tacatctctt 1320
ttttgcagat ggtcatacac ttcaattgag aaatacgata tatcaaagag tgatccattg 1380
gctgcagatt caaacgatat cgaaagtgtc cctttgtcca acttggaact tgactttgat 1440
cttaatatga ctgatgaacc cagtgtaagc ccttcgttat ttgatggatc tacttctgtt 1500
tctcgttcgt ccgccacgtc tataacgtca ctaggtgtaa agagggctta aactgacaat 1560
aaaaagattc ttgttttcaa gaacttgtca tttgtatagt ttttttatat tgtagttgtt 1620
ctattttaat caaatgttag cgtgatttat attttttttc gcctcgacat catctgccca 1680
gatgcgaagt taagtgcgca gaaagtaata tcatgcgtca atcgtatgtg aatgctggtc 1740
gctatactgc catattggca agtcacagta g 1771
<210> 3
<211> 1047
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 3
atgccaactc tagtaaatgg accaagaaga gactctaccg aagggtttga taccgatatc 60
atcactcttc ctagattcat aatcgagcac cagaagcaat ttaagaacgc tactggtgat 120
ttcacattag tactgaatgc cttgcaattc gcgttcaaat ttgtatctca caccatcaga 180
cgtgctgaat tggttaactt ggttgggtta gcaggcgctt ccaacttcac tggtgaccag 240
caaaagaagt tggacgttct aggtgatgaa atatttatca atgccatgag ggctagtggg 300
atcatcaagg tccttgtatc tgaagaacag gaagacttga tcgtttttcc cacaaacacg 360
ggctcatacg cagtgtgttg tgatcctatt gatggctcct caaatttgga cgccggtgtc 420
tccgttggaa ctatcgcgtc tatattcaga ctgctaccag actcatcagg tactataaac 480
gacgtactga gatgtggtaa agaaatggta gccgcttgct atgccatgta cggatcctct 540
acgcatctag tattgacatt gggtgatgga gttgatgggt ttaccttaga cacaaacttg 600
ggcgaattca tcttgactca tcctaactta agaattccgc ctcaaaaggc catctactca 660
attaatgaag gtaacaccct ctactggaac gagactataa gaacatttat tgagaaagtc 720
aaacaacccc aagcagacaa caacaacaag cctttctcgg ctaggtatgt tggatccatg 780
gttgctgatg ttcacaggac gtttctttac ggtggccttt tcgcataccc ttgcgacaag 840
aagagcccca acggaaaact gaggttgctt tatgaggcct tcccaatggc tttcttaatg 900
gaacaagcag ggggaaaagc ggtcaacgat cgcggagaga gaatcttgga tttggtgcca 960
agtcatatcc atgacaaatc ttctatttgg ttgggttctt caggtgaaat tgacaaattt 1020
ttagaccata ttggcaagtc acagtag 1047
<210> 4
<211> 19
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 4
aacgctctac caactgagc 19
<210> 5
<211> 40
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 5
attctgggcc tccatgtccg tctgtaattg cactacttgt 40
<210> 6
<211> 40
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 6
aagtagtgca attacagacg gacatggagg cccagaatac 40
<210> 7
<211> 40
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 7
actgtgactt gccaatatgg cagtatagcg accagcattc 40
<210> 8
<211> 40
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 8
atgctggtcg ctatactgcc atattggcaa gtcacagtag 40
<210> 9
<211> 20
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 9
gcgaattcat gtagatcgcg 20
<210> 10
<211> 20
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 10
attcttagta gtcgcggtcg 20
<210> 11
<211> 20
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 11
caatgatgtg caagaaccct 20
Claims (8)
1. the Saccharomyces cerevisiae gene engineering bacteria of one plant of knockout FBP1 gene, which is characterized in that FBP1 gene inactivates in the bacterial strain.
2. knocking out the Saccharomyces cerevisiae gene engineering bacteria of FBP1 gene according to claim 1, which is characterized in that the wine brewing ferment
Mother is Saccharomyces cerevisiae S288c.
3. knocking out the Saccharomyces cerevisiae gene engineering bacteria of FBP1 gene according to claim 1, which is characterized in that knock out FBP1 base
Because after, FBP1 gene order is as shown in SEQ ID NO.2.
4. knocking out the construction method of the Saccharomyces cerevisiae gene engineering bacteria of FBP1 gene described in claim 1, which is characterized in that including
Following steps:
(1) segment transformed saccharomyces cerevisiae bacterial strain will be knocked out, the nucleotide sequence for knocking out segment is as shown in SEQ ID NO.1；
(2) positive transformant is obtained using the YPD Screening of Media containing 40 μ g/mL gold load elements, obtains knocking out FBP1 gene
Saccharomyces cerevisiae gene engineering bacteria.
5. according to claim 4 except the construction method of the Saccharomyces cerevisiae gene engineering bacteria of FBP1 gene, which is characterized in that institute
Stating Wine brewing yeast strain is Saccharomyces cerevisiae S288c.
6. knocking out application of the Saccharomyces cerevisiae gene engineering bacteria of FBP1 gene as fermentation strain described in claim 1.
7. knocking out application of the Saccharomyces cerevisiae gene engineering bacteria of FBP1 gene in alcohol prepared by fermenting described in claim 1.
8. application according to claim 7, which is characterized in that the culture medium of alcohol fermentation is as follows: 60g/L glucose, 4g/
L peptone, 4g/L ammonium sulfate, 3g/L potassium dihydrogen phosphate, 3g/L yeast extract, 0.5g/L magnesium sulfate, seven hydrated sulfuric acid of 0.05g/L are sub-
Iron, 0.05g/L Zinc vitriol.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112877229A (en) * | 2021-01-25 | 2021-06-01 | 南京工业大学 | Sok 2-knocked-out saccharomyces cerevisiae genetically engineered bacterium and construction method and application thereof |
| CN110747138B (en) * | 2019-11-06 | 2021-06-25 | 南京工业大学 | Saccharomyces cerevisiae gene engineering bacterium and construction method and application thereof |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109136254A (en) * | 2018-07-10 | 2019-01-04 | 天津科技大学 | A kind of seamless gene knockout method of efficient saccharomyces cerevisiae and its application |
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2019
- 2019-01-17 CN CN201910043699.2A patent/CN109628336A/en active Pending
Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109136254A (en) * | 2018-07-10 | 2019-01-04 | 天津科技大学 | A kind of seamless gene knockout method of efficient saccharomyces cerevisiae and its application |
Non-Patent Citations (3)
| Title |
|---|
| ANA KITANOVIC ET AL.: "Fructose-1,6-bisphosphatase mediates cellular responses to DNA damage and aging in Saccharomyces cerevisiae", 《MUTATION RESEARCH》 * |
| JUAN J. MERCADO AND JUANA M. GANCEDO: "Regulatory regions in the yeast FRPl and PCKl genes", 《FEBS》 * |
| VIDYA R. VELAGAPUDI ET AL.: "Metabolic screening of Saccharomyces cerevisiae single knockout strains reveals unexpected mobilization of metabolic potential", 《PROCESS BIOCHEMISTRY》 * |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110747138B (en) * | 2019-11-06 | 2021-06-25 | 南京工业大学 | Saccharomyces cerevisiae gene engineering bacterium and construction method and application thereof |
| CN112877229A (en) * | 2021-01-25 | 2021-06-01 | 南京工业大学 | Sok 2-knocked-out saccharomyces cerevisiae genetically engineered bacterium and construction method and application thereof |
| CN112877229B (en) * | 2021-01-25 | 2023-08-25 | 南京工业大学 | Saccharomyces cerevisiae genetically engineered bacterium for knocking out Sok2, construction method and application thereof |
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Application publication date: 20190416 |